HHS Public Access Author manuscript Author Manuscript
Cell Rep. Author manuscript; available in PMC 2016 May 31. Published in final edited form as: Cell Rep. 2016 May 24; 15(8): 1624–1633. doi:10.1016/j.celrep.2016.04.054.
Centromeric transcription regulates Aurora-B localization and activation Michael D. Blower1,2,3 1Department
of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114
2Department
of Genetics, Harvard Medical School, Boston, MA 02115
Author Manuscript
Summary
Author Manuscript
Centromeric transcription is widely conserved, however it is not clear what role centromere transcription plays during mitosis. Here I find that centromeres are transcribed in Xenopus egg extracts into a long noncoding RNA (lncRNA; cen-RNA) that localizes to mitotic centromeres, chromatin, and spindles. Cen-RNAs bind to the Chromosomal Passenger Complex (CPC) in vitro and in vivo. Blocking transcription or antisense inhibition of cen-RNA leads to a reduction of CPC localization to the inner centromere and misregulation of CPC component Aurora-B activation, independently of known centromere recruitment pathways. Additionally, transcription is required for normal bipolar attachment of kinetochores to the mitotic spindle, consistent with a role for cenRNA in CPC regulation. This work demonstrates that cen-RNAs promote normal kinetochore function through regulation of the localization and activation of the CPC and confirm that lncRNAs are components of the centromere.
Graphical Abstract
Author Manuscript
3
To whom correspondence should be addressed: ; Email: [email protected] Author Contributions M.B. conceptualization, investigation, formal analysis, writing, visualization, funding acquisition.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Blower
Page 2
Author Manuscript Author Manuscript
Introduction
Author Manuscript
The centromere is an important chromosomal region that nucleates the formation of the kinetochore, which mediates attachment of each chromosome to the mitotic spindle during mitosis. Recent work in Drosophila, and human and mouse tissue culture cells have indicated that centromeric repeats are transcribed into long noncoding RNAs (lncRNA) and that actively elongating RNA polymerase II is localized to the centromere/kinetochore (Chan et al., 2012; Ideue et al., 2014; Liu et al., 2015; Quenet and Dalal, 2014; Rosic et al., 2014; Wong et al., 2007). Centromeric lncRNAs (cen-RNA) associate with Cenp-A, -C, HJURP, SgoI, and Aurora-B and are required for normal kinetochore assembly. However, it is not clear how cen-RNAs promote kinetochore assembly or if these RNAs are directly associated with any components of the centromere and kinetochore. During mitosis the CPC, composed of Aurora-B, Incenp, Dasra-a/Borealin, and Survivin, localizes to the chromosome arms, inner centromeres and mitotic spindle where it phosphorylates a variety of substrates to promote successful completion of mitosis (Carmena et al., 2012). One of the most important functions of the CPC requires localization to the
Author Manuscript
inner centromere region where it serves as a sensor of normal bipolar attachment to the spindle (Lampson and Cheeseman, 2011). Work in several organisms has demonstrated that the CPC associates with spindle-enriched RNAs (Jambhekar et al., 2014) and with cenRNAs (Ferri et al., 2009; Ideue et al., 2014). In addition, the CPC binds directly to RNA in vitro and RNA binding is required for normal CPC localization to the inner centromere (Jambhekar et al., 2014). However, it was not clear if the association of the CPC with RNA required transcription or how centromeric transcription promotes accurate mitosis. I have tested the hypothesis that centromeric transcription is required for the normal localization and function of the CPC during mitosis. I find that cen-RNAs are a regulator of
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 3
Author Manuscript
the localization and activation of the CPC during mitosis and are required for normal kinetochore:microtubule attachments.
Results Centromeric repeats are transcribed in Xenopus egg extracts To determine if centromeres are transcribed in Xenopus egg extracts I stained replicated sperm nuclei for RNA polymerase II phosphorylated at Ser 2, which is indicative of elongating polymerase, and Bub1 to mark the kinetochores. Similar to results in Drosophila and humans (Chan et al., 2012; Rosic et al., 2014), I found that elongating RNA pol II was enriched at mitotic kinetochores and inner centromere regions (Figure 1A). Therefore, centromeres are a site of active transcription in mitotic Xenopus egg extracts.
Author Manuscript Author Manuscript
Xenopus laevis is a tetraploid organism with two sets of paralogous chromosomes. Previous work on Xenopus Cenp-A identified a repetitive DNA sequence, termed Frog Centromeric Repeat 1 (fcr1) that associates with Cenp-A, and maps to the centromeres of approximately half of the chromosomes (Edwards and Murray, 2005). To determine if centromeric transcription of fcr1 repeats produces a stable lncRNA associated with mitotic spindles and chromosomes, I tested for the presence fcr1-containing transcripts by RT-PCR. I found clear evidence for an fcr1-containing transcript of ~170 nt that copurified with mitotic spindles and chromosomes (Figure 1B). In some extracts the fcr1 transcript detected was greater that two repeats, but the predominant PCR product was of a single repeat. Importantly, the fcr1 lncRNAs were only detected in samples that included reverse transcriptase, similar to a spindle-associated mRNA, Xl19006 (Figure 1B). Additionally, I found that the fcr1 RNA was enriched on mitotic chromosome preparations compared to total extract, suggesting that the fcr1 RNA is retained on chromosomes (Figure 1B). I conclude that centromeres in Xenopus produce a lncRNA that is associated with mitotic chromosomes and spindles.
Author Manuscript
The association of fcr1 RNA with mitotic spindles and chromosomes suggests that this cenRNA could play an active role in mitosis. I considered two potential models for the action of cen-RNAs: first, the cen-RNA is transcribed at the centromere and remains associated with the centromeric DNA as a nascent transcript (cis-acting model), second, the cen-RNA is transcribed at the centromere, but is processed and released from the centromere (transacting model). The cis model predicts that fcr1 RNA will only be found at centromeres that contain fcr1 DNA, while the trans model predicts that the fcr1 RNA will be found at all the centromeres and perhaps other places throughout the mitotic spindle and chromosomes. To distinguish between these two models I performed combined immunofluorescence for Bub1 and RNA FISH for fcr1 using strand-specific RNA FISH probes. I found that fcr1 RNA localized to the centromere regions of the chromosomes, but was also distributed broadly throughout the mitotic chromosomes (Figure 1C and insets). The fcr1 FISH signal for both sense and antisense transcripts was significantly higher than observed in samples that did not contain FISH probe (Figure 1C and Supplemental Figure 1A). Additionally, I found that the fcr1 antisense transcript localized in bright punctate foci to the kinetochores and inner centromeres of ~40% of the centromeres, while fcr1 sense transcript exhibited diffuse localization and was rarely enriched at centromeres (Figure 1C–D and Supplemental Figure 1B). Additionally, I found that the accumulation of fcr1 antisense RNA at the centromere Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 4
Author Manuscript
was dependent on transcription as treatment of extracts with the transcription initiation inhibitor triptolide resulted in a ~65% decrease in FISH signal (Figure 1E). These results demonstrate that fcr1 antisense RNA is actively transcribed in Xenopus egg extracts and that the transcript is concentrated in centromeric chromatin.
Author Manuscript
To determine if fcr1 cen-RNA is localized to the site of transcription I labeled centromeric DNA using catalytically inactive Cas9 and an sgRNA targeting the fcr1 repeat sequence, which has recently been shown to efficiently label specific DNA sequences in Xenopus egg extract (Lane et al., 2015). I found that dCas9-FLAG localized to ~half of the centromeres (Figure 1F–G, Supplemental Figure1C). I performed combined immunofluorescence for dCas9, Bub1 and FISH for fcr1 AS RNA on chromosome spreads. I found that fcr1 AS RNA was present at many centromeres labeled by dCas9, but was also present at unlabeled centromeres. Furthermore, many dCas9-labeled centromeres did not contain detectable fcr1 AS RNA (Figure 1FG, Supplemental Figure 1C). These results demonstrate that fcr1 antisense RNA is likely processed and released from the centromere and is free to diffuse between centromeres, similar to results observed in Drosophila where a centromeric RNA from the X chromosome is present at centromeres on chromosomes in addition to the X (Rosic et al., 2014). The fact that fcr1 AS RNA is not detected at all centromeres suggests that other RNAs (such as those from the paralogous centromeres) could be present at centromeres lacking fcr1 AS RNA or that my RNA FISH detection method is less than 100% efficient. Importantly, work in Drosophila has shown that the SAT III RNA transcribed from the X chromosome is also not consistently detected at the centromeres of all chromosomes (Rosic et al., 2014). Aurora-B interacts directly with chromatin-localized RNAs
Author Manuscript Author Manuscript
Our previous work demonstrated that the CPC could bind directly to RNA in vitro (Jambhekar et al., 2014) but it has not been demonstrated that the CPC interacts directly with RNA in vivo or which subunits of the CPC interact with RNA in vivo. To determine if the CPC interacts directly with RNA on microtubules I used a recently developed approach based on UV-crosslinking to identify proteins that interact directly with mRNA (Castello et al., 2012; Castello et al., 2013). I stabilized microtubules with taxol and purified microtubules and associated proteins by centrifugation through a glycerol cushion. I then released microtubule-associated proteins using a mild salt elution and treated the associated proteins with UV light to induce protein:RNA crosslinks (Figure 2A). UV light is a zerolength crosslinker that creates covalent bonds between bases in nucleic acid and primarily aromatic amino acid residues in proteins (Darnell, 2010; Singh et al., 2013). I purified polyA containing RNAs under denaturing conditions using a LNA oligo-dT, and analyzed associated proteins by Western blot. Consistent with our in vitro data, I found that Aurora-B and Dasra-A bound directly to RNA in Xenopus egg extracts and that Incenp, Survivin, and XMAP215 did not directly bind to RNA. Control purifications demonstrated the specificity of the interaction between Aurora-B and Dasra-A with RNA, as none of the CPC proteins copurified with poly-A RNA in the absence of UV crosslinking (Figure 2A–B and Supplemental Figure 2). This result demonstrates that the CPC interacts directly with RNA bases in Xenopus egg extracts through Aurora-B and Dasra-A.
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 5
Author Manuscript Author Manuscript
To determine where the CPC interacts with RNA in mitotic spindles and chromosomes, I used the proximity ligation assay (PLA) (Soderberg et al., 2006). The PLA can detect molecular interactions that occur within ~50 nm in fixed cells. To detect the interaction of Aurora-B with RNA I performed the PLA with Aurora-B antibodies and the BWR4 monoclonal antibody, which recognizes all RNA regardless of sequence and has no affinity for DNA (Eilat and Fischel, 1991). I performed Aurora-B:RNA PLA reactions on intact metaphase spindles where I had marked Aurora-B localization using GFP-Aurora-B. In intact spindles GFP-Aurora-B localized to inner centromeres, throughout the chromatin, and along spindle microtubules (Kelly et al., 2007). In contrast to the broad distribution of GFPAurora-B, I found that the majority of Aurora-B:RNA PLA signal was detected on mitotic chromosomes (Figure 2C). A fraction of the Aurora-B:RNA PLA signal was found at inner centromere regions, but was also distributed throughout the chromatin (Figure 2C insets). I conclude that Aurora-B primarily interacts with RNA on mitotic chromatin and inner centromere regions, consistent with the localization of fcr1 cen-RNA. To determine if the CPC interacts with fcr1 RNA I performed Aurora-B immunoprecipitations from extracts containing replicated sperm nuclei. I found that AuroraB IPs contained the fcr1 cen-RNA and that control IPs did not contain fcr1 cen-RNA, similar to an Aurora-B associated mRNA, Xl19006. In addition, fcr1 cen-RNA was only detected in reactions that included reverse transcriptase, demonstrating that the PCR signal comes from the interaction of the CPC with fcr1 RNA and not genomic DNA (Figure 2D).
Author Manuscript
Our previous work demonstrated that the CPC could interact directly with several different mRNAs in vitro (Jambhekar et al., 2014). To determine if Aurora-B could directly interact with fcr1 cen-RNA in vitro and if Aurora-B exhibited sequence-specific RNA binding properties, I used EMSA to examine the interaction of untagged Aurora-B with five lengthmatched transcripts: sense and antisense fcr1, sense and antisense α-satellite, and a sequence from the MCS of pCR2.1. Similar to our previous results I found that Aurora-B interacted directly with all five transcripts tested (Figure 2EF and Supplemental Figure 3). However, I found that Aurora-B exhibited different affinities for each of the different RNAs. Aurora-B bound with highest affinity to fcr1 sense, antisense, and α-satellite sense transcripts and with a lower affinity to α-satellite antisense and pCR2.1 MCS. At most, Aurora-B exhibited a 3.3-fold higher affinity for cen-RNA compared to a nonspecific transcript. These results demonstrate that Aurora-B has modest sequence-specific RNA-binding activity, but that it is also a relatively promiscuous RNA-binding protein. Transcription is required for normal Aurora-B localization and activation
Author Manuscript
To determine if transcription of cen-RNA is important for CPC localization I inhibited transcription in Xenopus egg extracts using the initiation inhibitor triptolide (Bensaude, 2011), and monitored the localization of GFP-Aurora-B. Localization of GFP-Aurora-B to the inner centromere was reduced by ~50% in transcription inhibited extracts while the localization of Bub1 to kinetochores was unaffected (Figure 3A,D). Transcriptional inhibition also resulted in a significant reduction (~30%) of endogenous Aurora-B to the inner centromere (Figure 3B,D). To determine if transcription regulates Aurora-B activation I stained for Aurora-B phosphorylated on the T-loop, which is indicative of basal-level
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 6
Author Manuscript
Aurora-B activation (Sessa et al., 2005). Surprisingly, I found that transcriptional inhibition resulted in an increase in the intensity of pAurora-B at the inner centromere region (Figure 3C,D). However, I found that the spatial pattern of enrichment of pAurora-B at the inner centromere was disrupted in triptolide-treated extracts (Figure 3C,E). pAurora-B was significantly less enriched at the inner centromere region compared to bulk chromatin in triptolide-treated extracts demonstrating that the spatial pattern of Aurora-B activation is also disrupted in the absence of transcription. I conclude that active transcription is required for normal localization of Aurora-B to the inner centromere and for the enrichment of Aurora-B activation at the inner centromere region relative to non-centromeric chromatin.
Author Manuscript
Concentration of the CPC at the inner centromere is driven by two different histone modifications. First, phosphorylation of H2A T120 by Bub1 serves as a binding site for Sgo1, which recruits Dasra-A. Second, phosphorylation of H3 T3 by Haspin creates a binding site for Survivin (Kawashima et al., 2007; Kelly et al., 2010; Wang et al., 2010; Yamagishi et al., 2010). To determine if transcription acts upstream of known localization pathways for the CPC I stained mitotic chromosome spreads for GFP-Aurora-B, H2ApT120, H3pT3, and Bub1. I found that transcriptional inhibition did not affect the centromere intensity of H2ApT120 or the chromosomal levels of H3pT3 (Figure 3D,F,G). I conclude that transcription of centromeric RNAs is an additional pathway of Aurora-B recruitment and activation that acts in parallel to the known pathways of CPC recruitment to the inner centromere. Fcr1 RNA is required for normal Aurora-B localization
Author Manuscript
To determine if fcr1 RNA, or the act of transcription, is required for normal Aurora-B localization I used antisense LNA-gapmers to inhibit fcr1 RNA. I found that gapmers targeting fcr1 antisense transcript resulted in a reproducible reduction in the RNA FISH signal on chromosomes while fcr1 sense or GAPDH gapmers had no effect (Figure 4B). I examined Aurora-B localization in extracts treated with two gapmers each targeting fcr1 sense and antisense transcripts and compared these to a nonspecific human GAPDH gapmer control. I found that both gapmers targeting the fcr1 antisense transcript resulted in a consistent ~25% reduction in the localization of Aurora-B to the inner centromere (Figure 4A,C), while gapmers targeting the sense transcript or GAPDH had little effect on Aurora-B localization. Additionally, none of the gapmers had a significant effect on the localization of Bub1 to the kinetochore (Figure 4C). I conclude that fcr1 cen-RNA is required for normal Aurora-B localization.
Author Manuscript
My previous results demonstrated that fcr1 antisense RNA diffuses between centromeres and could be acting in trans to promote Aurora-B localization. To determine if fcr1 antisense RNA acts in cis or trans I examined the distribution of Aurora-B intensities at the centromere in antisense-treated extracts. The cis-acting model predicts that Aurora-B centromere intensity would exhibit a bimodal distribution with one population of affected centromeres and one unaffected population, while the trans-acting model predicts that all centromeres would be equally affected. Comparing the normalized centromere intensity of Aurora-B revealed that Aurora-B intensity exhibits a relatively normal distribution in all gapmer treated extracts with fcr1 antisense gapmer-treated exhibiting a lower intensity
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 7
Author Manuscript
normal distribution (Figure 4D,E), consistent with the prediction of the trans-acting model and the localization of fcr1 antisense RNA. I conclude that fcr1 antisense RNA is free to diffuse between centromeres and promotes normal Aurora-B localization at all centromeres. The modest reduction in Aurora-B localization in gapmer-treated extracts and the presence of fcr1 antisense RNA at ~ half of the centromeres suggests that additional transcripts may also contribute to normal Aurora-B localization.
Author Manuscript
To determine if fcr1 RNA is required for normal activation of Aurora-B at the centromere I treated egg extracts with antisense-gapmers or triptolide and examined the localization of Aurora-B phosphorylated in the activation loop. Consistent with my previous results I found that triptolide treatment resulted in an increase in phosphorylated Aurora-B at the centromere region (Figure 4FG) and a decreased enrichment of phosphorylated Aurora-B at the inner centromere region (Figure 4GH). Gapmers targeting GAPDH or the fcr1 sense transcript did not result in a change in pAurora-B at the inner centromere or enrichment of pAurora-B at the inner centromere (Figure 4FGH). In contrast, treatment of extracts with gapmers targeting the fcr1 antisense transcript resulted in a reproducible increase in AuroraB phosphorylation at centromeres, but did not change the pattern of pAurora-B enrichment at the centromere. These results demonstrated that fcr1 RNA regulates some aspects of Aurora-B phosphorylation and activation at the centromere, but that other factors, such as additional cen-RNAs or the act of transcription, contribute to the change in Aurora-B activation observed in transcription-inhibited extracts. Transcription is required for normal kinetochore:microtubule attachments and kinetochore alignment
Author Manuscript Author Manuscript
In Xenopus egg extracts depletion of Aurora-B leads to a complete loss of spindle assembly (Kelly et al., 2007; Sampath et al., 2004) while a reduction in the chromosomal and centromere level of the CPC through Haspin depletion leads to a much more subtle spindle assembly defect (Kelly et al., 2010). In most other systems the primary function of Aurora-B is to recognize incorrect kinetochore:microtubule attachments that do not generate tension between sister kinetochores (Lampson and Cheeseman, 2011), and inhibition of Aurora-B leads to many chromosomes with incorrect kinetochore:microtubule attachments (Hauf et al., 2003; Lampson et al., 2004). To determine if transcription inhibition resulted in defects in kinetochore:microtubule attachments I monitored kinetochore biorientation in intact spindles by staining for Bub1 and GFP-Aurora-B. In DMSO-treated extracts I found that ~90% of kinetochores demonstrated a clear bipolar attachment to the spindle (Figure 5AB). In contrast, in triptolide treated extracts I found that ~50% of kinetochores did not exhibit bipolar attachment and appeared to show syntelic attachments to one spindle pole (Figure 5AB). To determine if the incorrect kinetochore microtubule attachments observed in triptolide-treated extracts resulted in defects in kinetochore alignment I measured the relative position of kinetochores in DMSO and triptolide-treated extracts. In DMSO treated extracts I found that the vast majority of kinetochores aligned at the spindle equator, while kinetochores in triptolide-treated extracts exhibited a much broader distribution throughout the spindle, consistent with defects achieving a bipolar spindle attachment. Taken together these data demonstrate that inhibition of transcription results in defects in localization of
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 8
Author Manuscript
Aurora-B to the inner centromere leading to defects in correcting errors in kinetochore:microtubule attachment.
Discussion
Author Manuscript
I found that transcription of repetitive centromeric DNA produces a lncRNA required for the normal localization and activation of the CPC at the inner centromere. My results suggest that a high local concentration of centromeric lncRNAs on mitotic centromeres and chromosomes is an additional pathway for CPC localization and activation that acts in parallel to known pathways of CPC recruitment. Additionally, my results suggest that the CPC is one of the major mitotic targets for regulation by cen-RNAs and confirms the observation that noncoding RNAs play an active role during mitosis (Blower et al., 2005; Du et al., 2010; Ferri et al., 2009; Ideue et al., 2014; Jambhekar et al., 2014; Quenet and Dalal, 2014; Rosic et al., 2014).
Author Manuscript
Work in a wide variety of systems has demonstrated active transcription of centromeric DNA. Chromatin marks normally associated with open euchromatic DNA are a conserved feature of centromeric chromatin (Sullivan and Karpen, 2004). In addition, activation or repression of transcription from a Human Artificial Chromosomes (HAC) alters the stability of the HAC (Nakano et al., 2008). Recent work in Drosophila demonstrated that the act of transcription, rather than the resulting RNA, is required for normal Cenp-A deposition (Chen et al., 2015). While several other studies have depleted centromeric RNAs and concluded that cen-RNA is required for various mitotic processes (Ideue et al., 2014; Liu et al., 2015; Quenet and Dalal, 2014; Rosic et al., 2014). Taken together, these studies support the hypothesis that transcription of centromeric DNA plays a role in centromere/kinetochore function through both the process of transcription and through the production of a functional cen-RNA. My observation that fcr1 RNAs localized to half the centromeres affect the localization of Aurora-B to all centromeres suggests that cen-RNAs may serve to activate or modify Aurora-B at the centromere, which then diffuses away to act at other sites, similar to a model proposed for Aurora-B activation by binding to chromatin (Kelly et al., 2007). Recent work has demonstrated that many nascent transcripts are present on mitotic chromatin, but are cleared by transcriptional elongation (Liang et al., 2015) in prophase/prometaphase, while several studies have demonstrated that elongating pol II is present at metaphase centromeres (Chan et al., 2012; Liu et al., 2015; Rosic et al., 2014). These data suggest that persistent transcription during mitosis may be an additional signal that differentiates the centromere from the remainder of the chromosome.
Author Manuscript
My observation that kinetochores do not make normal bipolar attachment to the spindle in the absence of transcription is consistent with a role for transcription in the regulation of CPC localization and Aurora-B activation. A decrease in CPC localization to the inner centromere could result in decreased phosphorylation of components of the KMN network and stabilization of incorrect kinetochore:microtubule attachments (Lampson and Cheeseman, 2011). An alternative possibility is that the increased level of active, autophosphorylated Aurora-B at the inner centromere and chromatin could result in
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 9
Author Manuscript
increased phosphorylation of MCAK and a failure to destabilize incorrect kinetochore:microtubule attachments (Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004). Both scenarios would lead to stabilization of incorrect attachments to the spindle and lead to errors in chromosome segregation observed after loss of centromeric RNAs (Chan et al., 2012; Rosic et al., 2014). Our previous work and the work of other groups have demonstrated that Aurora-B can bind to a wide variety of RNAs and that RNA binding by the CPC activates Aurora-B kinase in vitro. Promiscuous RNA-binding could facilitate recognition of many different types of chromatin bound RNAs in early mitosis, while the persistence of transcription at the centromere could be an important signal for the localization of the CPC to the inner centromere during prometaphase. Understanding the spatial and temporal interactions of Aurora-B with different types of RNAs will be an important area of future research.
Author Manuscript
Methods Xenopus egg extract methods
Xenopus egg extracts were prepared and utilized for immunofluorescence as described (Hannak and Heald, 2006). Triptolide was added to extracts at a concentration for 10μM from a 10mM stock in DMSO. RNA EMSA Untagged Aurora-B:In-box (pMB940) was expressed and purified from E. coli and used for EMSA as described (Jambhekar et al., 2014) except that radioactive RNAs were used in place of fluorescently-labeled RNAs.
Author Manuscript
UV-crosslinking
Author Manuscript
Taxol stabilized microtubules were purified from 1.2 mL CSF-arrested egg extracts by centrifugation through a glycerol cushion. Microtubule-associated proteins were eluted with XB with a final concentration of 200 mM KCl and recentrifuged for 10 minutes at 22,000 x g. The supernatant was irradiated with UV light using a Stratalinker for 10 minutes on ice while unirradiated samples were kept on ice. Samples were extracted from both irradiated and control extracts for western blots of the input fraction. Extracts were then adjusted to 0.1% Sarkosyl 4 mM EDTA and heated at 65°C for 10 minutes then placed on ice for 5 minutes. Poly-A RNAs were captured using 300 pmol of LNA dT (Exiquon) conjugated to 150 μL of MyOne Streptavidin Dynabeads (Life Technologies). Bound proteins and RNAs were eluted by the addition of 50 μL of dd water and incubation at 65°C for 10 minutes. Samples from the supernatants were analyzed by western blot. RNA FISH Fcr1 RNAs were detected on mitotic chromosome spreads using strand-specific oligonucleotide probes from Stellaris.
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 10
LNA Antisense inhibition
Author Manuscript
Fcr1 RNAs were targeted for destruction using Antisene-LAN Gapmers from Exiqon. Gapmers were added to extracts at a final concentration of 100nM. See Supplemental Information for detailed descriptions of all methods.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Author Manuscript
I would like to acknowledge Judith Sharp for comments on the manuscript and members of the Blower lab for useful suggestions. I thank Alexi Arnaoutouv, Hiro Funabiki, Ming Tsai, and Dan Eilat for gifts of antibodies. This work was supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (M.D.B) and (NIH) RO1 (RO1GM086434).
References
Author Manuscript Author Manuscript
Andrews PD, Ovechkina Y, Morrice N, Wagenbach M, Duncan K, Wordeman L, Swedlow JR. Aurora B regulates MCAK at the mitotic centromere. Developmental cell. 2004; 6:253–268. [PubMed: 14960279] Bensaude O. Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity? Transcription. 2011; 2:103–108. [PubMed: 21922053] Blower MD, Nachury M, Heald R, Weis K. A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly. Cell. 2005; 121:223–234. [PubMed: 15851029] Carmena M, Wheelock M, Funabiki H, Earnshaw WC. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nature reviews Molecular cell biology. 2012; 13:789– 803. [PubMed: 23175282] Castello A, Fischer B, Eichelbaum K, Horos R, Beckmann BM, Strein C, Davey NE, Humphreys DT, Preiss T, Steinmetz LM, et al. Insights into RNA biology from an atlas of mammalian mRNAbinding proteins. Cell. 2012; 149:1393–1406. [PubMed: 22658674] Castello A, Horos R, Strein C, Fischer B, Eichelbaum K, Steinmetz LM, Krijgsveld J, Hentze MW. System-wide identification of RNA-binding proteins by interactome capture. Nature protocols. 2013; 8:491–500. [PubMed: 23411631] Chan FL, Marshall OJ, Saffery R, Kim BW, Earle E, Choo KH, Wong LH. Active transcription and essential role of RNA polymerase II at the centromere during mitosis. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109:1979–1984. [PubMed: 22308327] Chen CC, Bowers S, Lipinszki Z, Palladino J, Trusiak S, Bettini E, Rosin L, Przewloka MR, Glover DM, O’Neill RJ, et al. Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription. Developmental cell. 2015; 34:73–84. [PubMed: 26151904] Darnell RB. HITS-CLIP: panoramic views of protein-RNA regulation in living cells. Wiley Interdiscip Rev RNA. 2010; 1:266–286. [PubMed: 21935890] Du Y, Topp CN, Dawe RK. DNA binding of centromere protein C (CENPC) is stabilized by singlestranded RNA. PLoS genetics. 2010; 6:e1000835. [PubMed: 20140237] Edwards NS, Murray AW. Identification of xenopus CENP-A and an associated centromeric DNA repeat. Molecular biology of the cell. 2005; 16:1800–1810. [PubMed: 15673610] Eilat D, Fischel R. Recurrent utilization of genetic elements in V regions of antinucleic acid antibodies from autoimmune mice. J Immunol. 1991; 147:361–368. [PubMed: 1711083] Ferri F, Bouzinba-Segard H, Velasco G, Hube F, Francastel C. Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase. Nucleic acids research. 2009; 37:5071– 5080. [PubMed: 19542185]
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 11
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Hannak E, Heald R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nature protocols. 2006; 1:2305–2314. [PubMed: 17406472] Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochoremicrotubule attachment and in maintaining the spindle assembly checkpoint. The Journal of cell biology. 2003; 161:281–294. [PubMed: 12707311] Ideue T, Cho Y, Nishimura K, Tani T. Involvement of satellite I noncoding RNA in regulation of chromosome segregation. Genes to cells : devoted to molecular & cellular mechanisms. 2014; 19:528–538. [PubMed: 24750444] Jambhekar A, Emerman AB, Schweidenback CT, Blower MD. RNA stimulates Aurora B kinase activity during mitosis. PloS one. 2014; 9:e100748. [PubMed: 24968351] Kawashima SA, Tsukahara T, Langegger M, Hauf S, Kitajima TS, Watanabe Y. Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres. Genes & development. 2007; 21:420–435. [PubMed: 17322402] Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H, Funabiki H. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science. 2010; 330:235–239. [PubMed: 20705815] Kelly AE, Sampath SC, Maniar TA, Woo EM, Chait BT, Funabiki H. Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly. Developmental cell. 2007; 12:31–43. [PubMed: 17199039] Lampson MA, Cheeseman IM. Sensing centromere tension: Aurora B and the regulation of kinetochore function. Trends Cell Biol. 2011; 21:133–140. [PubMed: 21106376] Lampson MA, Renduchitala K, Khodjakov A, Kapoor TM. Correcting improper chromosome-spindle attachments during cell division. Nature cell biology. 2004; 6:232–237. [PubMed: 14767480] Lan W, Zhang X, Kline-Smith SL, Rosasco SE, Barrett-Wilt GA, Shabanowitz J, Hunt DF, Walczak CE, Stukenberg PT. Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity. Current biology : CB. 2004; 14:273–286. [PubMed: 14972678] Lane AB, Strzelecka M, Ettinger A, Grenfell AW, Wittmann T, Heald R. Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening. Developmental cell. 2015; 34:373–378. [PubMed: 26212133] Liang K, Woodfin AR, Slaughter BD, Unruh JR, Box AC, Rickels RA, Gao X, Haug JS, Jaspersen SL, Shilatifard A. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis. Molecular cell. 2015; 60:435–445. [PubMed: 26527278] Liu H, Qu Q, Warrington R, Rice A, Cheng N, Yu H. Mitotic Transcription Installs Sgo1 at Centromeres to Coordinate Chromosome Segregation. Molecular cell. 2015; 59:426–436. [PubMed: 26190260] Nakano M, Cardinale S, Noskov VN, Gassmann R, Vagnarelli P, Kandels-Lewis S, Larionov V, Earnshaw WC, Masumoto H. Inactivation of a human kinetochore by specific targeting of chromatin modifiers. Developmental cell. 2008; 14:507–522. [PubMed: 18410728] Ohi R, Sapra T, Howard J, Mitchison TJ. Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation. Molecular biology of the cell. 2004; 15:2895–2906. [PubMed: 15064354] Quenet D, Dalal Y. A long non-coding RNA is required for targeting centromeric protein A to the human centromere. eLife. 2014; 3:e03254. [PubMed: 25117489] Rosic S, Kohler F, Erhardt S. Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division. The Journal of cell biology. 2014; 207:335–349. [PubMed: 25365994] Sampath SC, Ohi R, Leismann O, Salic A, Pozniakovski A, Funabiki H. The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell. 2004; 118:187–202. [PubMed: 15260989] Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT, Musacchio A. Mechanism of Aurora B activation by INCENP and inhibition by hesperadin. Molecular cell. 2005; 18:379–391. [PubMed: 15866179]
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 12
Author Manuscript Author Manuscript
Singh G, Ricci EP, Moore MJ. RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes. Methods. 2013 Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006; 3:995–1000. [PubMed: 17072308] Sullivan BA, Karpen GH. Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nature structural & molecular biology. 2004; 11:1076–1083. Wang F, Dai J, Daum JR, Niedzialkowska E, Banerjee B, Stukenberg PT, Gorbsky GJ, Higgins JM. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science. 2010; 330:231–235. [PubMed: 20705812] Wong LH, Brettingham-Moore KH, Chan L, Quach JM, Anderson MA, Northrop EL, Hannan R, Saffery R, Shaw ML, Williams E, et al. Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome research. 2007; 17:1146–1160. [PubMed: 17623812] Yamagishi Y, Honda T, Tanno Y, Watanabe Y. Two histone marks establish the inner centromere and chromosome bi-orientation. Science. 2010; 330:239–243. [PubMed: 20929775]
Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 13
Author Manuscript
Highlights •
Centromeres are transcribed into a centromere/chromatin associated lncRNA
•
CPC binds to centromeric RNA
•
Transcription and cen-RNA is required for normal CPC localization and activation
•
Transcription is required for normal kinetochore:microtubule attachments eTOC
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 14
Author Manuscript Author Manuscript Author Manuscript
Figure 1. Active transcription of Xenopus centromeres
Author Manuscript
A. Chromosome spreads were prepared from Xenopus egg extracts containing replicated sperm DNA and stained for Bub1 and RNA Pol II pS2. Insets were magnified 3X in Photoshop. B. RT-PCR of cen-RNA in total extract and purified chromosome or spindle preparations. Mitotic chromosomes (left gels) or spindles (right gels) were purified from different Xenopus egg extracts containing replicated sperm nuclei by centrifugation through a glycerol cushion. Fcr1 RNA and a control mRNA (Xl19006) were detected by RT-PCR C. Fcr1 RNA was detected using strand-specific FISH probes in chromosome spreads stained for Bub1 to mark the centromeres. Fcr1 antisense RNA was present at the kinetochore and inner centromere regions (inset) of approximately half the centromeres. A no probe control exhibited little fluorescence on the chromosomes. Insets are magnified 3X in Photoshop. Scale bar is 5μm. D. Quantitation of the fraction of centromeres that contain fcr1 FISH signal that is enriched 3X over the general chromatin signal. Error bars are the standard deviation of 3 samples. E. Fraction of nuclei that exhibit bright, punctate fcr1 antisense FISH signal after triptolide treatement (normalized to DMSO-treated extracts. N=at least 100 nuclei from 3 different extracts). Error bars depict the standard deviation. F. Codetection of fcr1 DNA, fcr1 RNA, and centromeres using dCas9 programmed with a sgRNA targeting the fcr1 repeat. Insets are magnified 3X in Photoshop. G. Scatterplot showing the
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 15
Author Manuscript
centromere intensities of dCas9 and fcr1 RNA FISH. Intensities are plotted from three sets of reactions. See also Supplemental Figure 1 for additional analysis of fcr1 RNA FISH experiments.
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 16
Author Manuscript Author Manuscript Author Manuscript Figure 2. Interaction of Aurora-B with fcr1 RNA
Author Manuscript
A. Schematic for experiment to identify sites of direct contact between the CPC and poly-A RNA using UV crosslinking. B. Western blot analysis of fractions from UV-crosslinking experiment. See Supplemental Figure 2 for uncropped Western blots. C. Detection of the sites of Aurora-B interaction with RNA using PLA. The sites of Aurora-B:RNA interaction were examined on spindles from Xenopus egg extracts containing replicated sperm DNA and GFP-Aurora-B to mark the inner centromere. GFP-Aurora-B signal in second panel has been exaggerated in Photoshop to highlight spindle-localized Aurora-B. Scale bar is 5μm. D. Aurora-B or control IgG IPs from extracts containing replicated sperm nuclear DNA. Fcr1
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 17
Author Manuscript
and Xl19006 RNAs were detected by RT-PCR. E. Interaction of full-length, untagged recombinant Aurora-B with fcr1 sense,antisense, α-satellite sense, antisense, and MCS from pCR2.1 RNAs examined by EMSA. F. Binding curves, Kds, and Hill coefficient for AuroraB binding to each RNA calculated from three independent experiments. Also see Supplemental Figure 3 for additional EMSA gels and additional quantification.
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 18
Author Manuscript Author Manuscript
Figure 3. Transcription regulates Aurora-B localization to the inner centromere
Author Manuscript
A–C. Localization of the antigens indicated on the left to the inner centromere in control and triptolide-treated extracts. D. Quantification of the normalized signal for the indicated antigens at the centromere region in triptolide-treated extracts. N=3 extracts for all antigens except endogenous Aurora-B where n=4. E. Enrichment of pAurora-B at the inner centromere in control and triptolide treated extracts. * indicates a p-value less than 0.05, *** indicates a p-value less than 0.005, all other p-values are not significant. P-values from D calculated using a single-sample t-test. P-value for E calculated using a Wilcox signed rank test. F–G. Images of H2A pT120 and H3 pT3 in control and triptolide-treated extracts. Scale bar is 5μm.
Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 19
Author Manuscript Author Manuscript Author Manuscript Figure 4. Fcr1 antisense RNA regulates Aurora-B localization to the inner centromere
Author Manuscript
A. Localization of Bub1 and endogenous Aurora-B in extracts treated with the indicated antisense LNA-gapmer. B. Fraction of fcr1 antisense FISH positive nuclei (normalized to GAPDH control) for each indicated antisense LNA-gapmer (n=at least 100 nuclei from 3 independent extracts). C. Quantification of centromere intensity of Bub1 and Aurora-B (normalized to GAPDH-treated extracts)(n=3 extracts). * indicates a p-value less than 0.05 by single sample t-test. D. Distribution of normalized Aurora-B centromere intensity in extracts treated with the indicated antisense LNA-gapmers. E. Boxplot and points of the normalized centromere intensity of Aurora-B for each indicated antisense LNA-gapmer. At least 100 centromeres were measured per extract from three independent extracts. All data points are combined in this plot. F. Localization of Bub1 and phosphorylated Aurora-B in Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 20
Author Manuscript
extracts treated with antisense LNA-gapmers and triptolide. G. Quantitation of normalized signal for Bub1 and pAurora-B at the inner centromere region in gapmer-treated extracts. 50–200 centromere regions were quantified for each condition from three independent extracts. Error bars are standard deviation. H. Boxplot of the enrichment of Aurora-B at the inner centromere region in gapmer and triptolide-treated extracts. For this plot all enrichment values from three replicate experiments are combined into a single plot. *** indicates a p-value less that 0.005 by Wilcox Rank Sum test. Scale bar is 5μm.
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 21
Author Manuscript Author Manuscript
Figure 5. Transcription regulates kinetochore microtubule attachment
Author Manuscript
A. Intact spindles were prepared from control or triptolide-treated extracts containing GFPAurora-B and stained for Bub1 to mark the positions of the kinetochores. Single optical sections are shown for both spindles. The majority of control kinetochores exhibit clear bipolar attachment to the spindle (arrow), while ~half of kinetochores in triptolide-treated extracts exhibit monopolar attachment to the kinetochore. Insets are magnified 3X in Photoshop. B. Quantification of kinetochore attachment in control and triptolide-treated extracts. 100–200 kinetochores were scored in each of two extracts. P-value was calculated using a Wilcox signed rank test. C. Normalized position of each kinetochore was calculated and control and triptolide-treated extracts are plotted as a density histogram. Scale bar is 5μm.
Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Cell Rep. Author manuscript; available in PMC 2016 May 31. Published in final edited form as: Cell Rep. 2016 May 24; 15(8): 1624–1633. doi:10.1016/j.celrep.2016.04.054.
Centromeric transcription regulates Aurora-B localization and activation Michael D. Blower1,2,3 1Department
of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114
2Department
of Genetics, Harvard Medical School, Boston, MA 02115
Author Manuscript
Summary
Author Manuscript
Centromeric transcription is widely conserved, however it is not clear what role centromere transcription plays during mitosis. Here I find that centromeres are transcribed in Xenopus egg extracts into a long noncoding RNA (lncRNA; cen-RNA) that localizes to mitotic centromeres, chromatin, and spindles. Cen-RNAs bind to the Chromosomal Passenger Complex (CPC) in vitro and in vivo. Blocking transcription or antisense inhibition of cen-RNA leads to a reduction of CPC localization to the inner centromere and misregulation of CPC component Aurora-B activation, independently of known centromere recruitment pathways. Additionally, transcription is required for normal bipolar attachment of kinetochores to the mitotic spindle, consistent with a role for cenRNA in CPC regulation. This work demonstrates that cen-RNAs promote normal kinetochore function through regulation of the localization and activation of the CPC and confirm that lncRNAs are components of the centromere.
Graphical Abstract
Author Manuscript
3
To whom correspondence should be addressed: ; Email: [email protected] Author Contributions M.B. conceptualization, investigation, formal analysis, writing, visualization, funding acquisition.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Blower
Page 2
Author Manuscript Author Manuscript
Introduction
Author Manuscript
The centromere is an important chromosomal region that nucleates the formation of the kinetochore, which mediates attachment of each chromosome to the mitotic spindle during mitosis. Recent work in Drosophila, and human and mouse tissue culture cells have indicated that centromeric repeats are transcribed into long noncoding RNAs (lncRNA) and that actively elongating RNA polymerase II is localized to the centromere/kinetochore (Chan et al., 2012; Ideue et al., 2014; Liu et al., 2015; Quenet and Dalal, 2014; Rosic et al., 2014; Wong et al., 2007). Centromeric lncRNAs (cen-RNA) associate with Cenp-A, -C, HJURP, SgoI, and Aurora-B and are required for normal kinetochore assembly. However, it is not clear how cen-RNAs promote kinetochore assembly or if these RNAs are directly associated with any components of the centromere and kinetochore. During mitosis the CPC, composed of Aurora-B, Incenp, Dasra-a/Borealin, and Survivin, localizes to the chromosome arms, inner centromeres and mitotic spindle where it phosphorylates a variety of substrates to promote successful completion of mitosis (Carmena et al., 2012). One of the most important functions of the CPC requires localization to the
Author Manuscript
inner centromere region where it serves as a sensor of normal bipolar attachment to the spindle (Lampson and Cheeseman, 2011). Work in several organisms has demonstrated that the CPC associates with spindle-enriched RNAs (Jambhekar et al., 2014) and with cenRNAs (Ferri et al., 2009; Ideue et al., 2014). In addition, the CPC binds directly to RNA in vitro and RNA binding is required for normal CPC localization to the inner centromere (Jambhekar et al., 2014). However, it was not clear if the association of the CPC with RNA required transcription or how centromeric transcription promotes accurate mitosis. I have tested the hypothesis that centromeric transcription is required for the normal localization and function of the CPC during mitosis. I find that cen-RNAs are a regulator of
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 3
Author Manuscript
the localization and activation of the CPC during mitosis and are required for normal kinetochore:microtubule attachments.
Results Centromeric repeats are transcribed in Xenopus egg extracts To determine if centromeres are transcribed in Xenopus egg extracts I stained replicated sperm nuclei for RNA polymerase II phosphorylated at Ser 2, which is indicative of elongating polymerase, and Bub1 to mark the kinetochores. Similar to results in Drosophila and humans (Chan et al., 2012; Rosic et al., 2014), I found that elongating RNA pol II was enriched at mitotic kinetochores and inner centromere regions (Figure 1A). Therefore, centromeres are a site of active transcription in mitotic Xenopus egg extracts.
Author Manuscript Author Manuscript
Xenopus laevis is a tetraploid organism with two sets of paralogous chromosomes. Previous work on Xenopus Cenp-A identified a repetitive DNA sequence, termed Frog Centromeric Repeat 1 (fcr1) that associates with Cenp-A, and maps to the centromeres of approximately half of the chromosomes (Edwards and Murray, 2005). To determine if centromeric transcription of fcr1 repeats produces a stable lncRNA associated with mitotic spindles and chromosomes, I tested for the presence fcr1-containing transcripts by RT-PCR. I found clear evidence for an fcr1-containing transcript of ~170 nt that copurified with mitotic spindles and chromosomes (Figure 1B). In some extracts the fcr1 transcript detected was greater that two repeats, but the predominant PCR product was of a single repeat. Importantly, the fcr1 lncRNAs were only detected in samples that included reverse transcriptase, similar to a spindle-associated mRNA, Xl19006 (Figure 1B). Additionally, I found that the fcr1 RNA was enriched on mitotic chromosome preparations compared to total extract, suggesting that the fcr1 RNA is retained on chromosomes (Figure 1B). I conclude that centromeres in Xenopus produce a lncRNA that is associated with mitotic chromosomes and spindles.
Author Manuscript
The association of fcr1 RNA with mitotic spindles and chromosomes suggests that this cenRNA could play an active role in mitosis. I considered two potential models for the action of cen-RNAs: first, the cen-RNA is transcribed at the centromere and remains associated with the centromeric DNA as a nascent transcript (cis-acting model), second, the cen-RNA is transcribed at the centromere, but is processed and released from the centromere (transacting model). The cis model predicts that fcr1 RNA will only be found at centromeres that contain fcr1 DNA, while the trans model predicts that the fcr1 RNA will be found at all the centromeres and perhaps other places throughout the mitotic spindle and chromosomes. To distinguish between these two models I performed combined immunofluorescence for Bub1 and RNA FISH for fcr1 using strand-specific RNA FISH probes. I found that fcr1 RNA localized to the centromere regions of the chromosomes, but was also distributed broadly throughout the mitotic chromosomes (Figure 1C and insets). The fcr1 FISH signal for both sense and antisense transcripts was significantly higher than observed in samples that did not contain FISH probe (Figure 1C and Supplemental Figure 1A). Additionally, I found that the fcr1 antisense transcript localized in bright punctate foci to the kinetochores and inner centromeres of ~40% of the centromeres, while fcr1 sense transcript exhibited diffuse localization and was rarely enriched at centromeres (Figure 1C–D and Supplemental Figure 1B). Additionally, I found that the accumulation of fcr1 antisense RNA at the centromere Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 4
Author Manuscript
was dependent on transcription as treatment of extracts with the transcription initiation inhibitor triptolide resulted in a ~65% decrease in FISH signal (Figure 1E). These results demonstrate that fcr1 antisense RNA is actively transcribed in Xenopus egg extracts and that the transcript is concentrated in centromeric chromatin.
Author Manuscript
To determine if fcr1 cen-RNA is localized to the site of transcription I labeled centromeric DNA using catalytically inactive Cas9 and an sgRNA targeting the fcr1 repeat sequence, which has recently been shown to efficiently label specific DNA sequences in Xenopus egg extract (Lane et al., 2015). I found that dCas9-FLAG localized to ~half of the centromeres (Figure 1F–G, Supplemental Figure1C). I performed combined immunofluorescence for dCas9, Bub1 and FISH for fcr1 AS RNA on chromosome spreads. I found that fcr1 AS RNA was present at many centromeres labeled by dCas9, but was also present at unlabeled centromeres. Furthermore, many dCas9-labeled centromeres did not contain detectable fcr1 AS RNA (Figure 1FG, Supplemental Figure 1C). These results demonstrate that fcr1 antisense RNA is likely processed and released from the centromere and is free to diffuse between centromeres, similar to results observed in Drosophila where a centromeric RNA from the X chromosome is present at centromeres on chromosomes in addition to the X (Rosic et al., 2014). The fact that fcr1 AS RNA is not detected at all centromeres suggests that other RNAs (such as those from the paralogous centromeres) could be present at centromeres lacking fcr1 AS RNA or that my RNA FISH detection method is less than 100% efficient. Importantly, work in Drosophila has shown that the SAT III RNA transcribed from the X chromosome is also not consistently detected at the centromeres of all chromosomes (Rosic et al., 2014). Aurora-B interacts directly with chromatin-localized RNAs
Author Manuscript Author Manuscript
Our previous work demonstrated that the CPC could bind directly to RNA in vitro (Jambhekar et al., 2014) but it has not been demonstrated that the CPC interacts directly with RNA in vivo or which subunits of the CPC interact with RNA in vivo. To determine if the CPC interacts directly with RNA on microtubules I used a recently developed approach based on UV-crosslinking to identify proteins that interact directly with mRNA (Castello et al., 2012; Castello et al., 2013). I stabilized microtubules with taxol and purified microtubules and associated proteins by centrifugation through a glycerol cushion. I then released microtubule-associated proteins using a mild salt elution and treated the associated proteins with UV light to induce protein:RNA crosslinks (Figure 2A). UV light is a zerolength crosslinker that creates covalent bonds between bases in nucleic acid and primarily aromatic amino acid residues in proteins (Darnell, 2010; Singh et al., 2013). I purified polyA containing RNAs under denaturing conditions using a LNA oligo-dT, and analyzed associated proteins by Western blot. Consistent with our in vitro data, I found that Aurora-B and Dasra-A bound directly to RNA in Xenopus egg extracts and that Incenp, Survivin, and XMAP215 did not directly bind to RNA. Control purifications demonstrated the specificity of the interaction between Aurora-B and Dasra-A with RNA, as none of the CPC proteins copurified with poly-A RNA in the absence of UV crosslinking (Figure 2A–B and Supplemental Figure 2). This result demonstrates that the CPC interacts directly with RNA bases in Xenopus egg extracts through Aurora-B and Dasra-A.
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 5
Author Manuscript Author Manuscript
To determine where the CPC interacts with RNA in mitotic spindles and chromosomes, I used the proximity ligation assay (PLA) (Soderberg et al., 2006). The PLA can detect molecular interactions that occur within ~50 nm in fixed cells. To detect the interaction of Aurora-B with RNA I performed the PLA with Aurora-B antibodies and the BWR4 monoclonal antibody, which recognizes all RNA regardless of sequence and has no affinity for DNA (Eilat and Fischel, 1991). I performed Aurora-B:RNA PLA reactions on intact metaphase spindles where I had marked Aurora-B localization using GFP-Aurora-B. In intact spindles GFP-Aurora-B localized to inner centromeres, throughout the chromatin, and along spindle microtubules (Kelly et al., 2007). In contrast to the broad distribution of GFPAurora-B, I found that the majority of Aurora-B:RNA PLA signal was detected on mitotic chromosomes (Figure 2C). A fraction of the Aurora-B:RNA PLA signal was found at inner centromere regions, but was also distributed throughout the chromatin (Figure 2C insets). I conclude that Aurora-B primarily interacts with RNA on mitotic chromatin and inner centromere regions, consistent with the localization of fcr1 cen-RNA. To determine if the CPC interacts with fcr1 RNA I performed Aurora-B immunoprecipitations from extracts containing replicated sperm nuclei. I found that AuroraB IPs contained the fcr1 cen-RNA and that control IPs did not contain fcr1 cen-RNA, similar to an Aurora-B associated mRNA, Xl19006. In addition, fcr1 cen-RNA was only detected in reactions that included reverse transcriptase, demonstrating that the PCR signal comes from the interaction of the CPC with fcr1 RNA and not genomic DNA (Figure 2D).
Author Manuscript
Our previous work demonstrated that the CPC could interact directly with several different mRNAs in vitro (Jambhekar et al., 2014). To determine if Aurora-B could directly interact with fcr1 cen-RNA in vitro and if Aurora-B exhibited sequence-specific RNA binding properties, I used EMSA to examine the interaction of untagged Aurora-B with five lengthmatched transcripts: sense and antisense fcr1, sense and antisense α-satellite, and a sequence from the MCS of pCR2.1. Similar to our previous results I found that Aurora-B interacted directly with all five transcripts tested (Figure 2EF and Supplemental Figure 3). However, I found that Aurora-B exhibited different affinities for each of the different RNAs. Aurora-B bound with highest affinity to fcr1 sense, antisense, and α-satellite sense transcripts and with a lower affinity to α-satellite antisense and pCR2.1 MCS. At most, Aurora-B exhibited a 3.3-fold higher affinity for cen-RNA compared to a nonspecific transcript. These results demonstrate that Aurora-B has modest sequence-specific RNA-binding activity, but that it is also a relatively promiscuous RNA-binding protein. Transcription is required for normal Aurora-B localization and activation
Author Manuscript
To determine if transcription of cen-RNA is important for CPC localization I inhibited transcription in Xenopus egg extracts using the initiation inhibitor triptolide (Bensaude, 2011), and monitored the localization of GFP-Aurora-B. Localization of GFP-Aurora-B to the inner centromere was reduced by ~50% in transcription inhibited extracts while the localization of Bub1 to kinetochores was unaffected (Figure 3A,D). Transcriptional inhibition also resulted in a significant reduction (~30%) of endogenous Aurora-B to the inner centromere (Figure 3B,D). To determine if transcription regulates Aurora-B activation I stained for Aurora-B phosphorylated on the T-loop, which is indicative of basal-level
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 6
Author Manuscript
Aurora-B activation (Sessa et al., 2005). Surprisingly, I found that transcriptional inhibition resulted in an increase in the intensity of pAurora-B at the inner centromere region (Figure 3C,D). However, I found that the spatial pattern of enrichment of pAurora-B at the inner centromere was disrupted in triptolide-treated extracts (Figure 3C,E). pAurora-B was significantly less enriched at the inner centromere region compared to bulk chromatin in triptolide-treated extracts demonstrating that the spatial pattern of Aurora-B activation is also disrupted in the absence of transcription. I conclude that active transcription is required for normal localization of Aurora-B to the inner centromere and for the enrichment of Aurora-B activation at the inner centromere region relative to non-centromeric chromatin.
Author Manuscript
Concentration of the CPC at the inner centromere is driven by two different histone modifications. First, phosphorylation of H2A T120 by Bub1 serves as a binding site for Sgo1, which recruits Dasra-A. Second, phosphorylation of H3 T3 by Haspin creates a binding site for Survivin (Kawashima et al., 2007; Kelly et al., 2010; Wang et al., 2010; Yamagishi et al., 2010). To determine if transcription acts upstream of known localization pathways for the CPC I stained mitotic chromosome spreads for GFP-Aurora-B, H2ApT120, H3pT3, and Bub1. I found that transcriptional inhibition did not affect the centromere intensity of H2ApT120 or the chromosomal levels of H3pT3 (Figure 3D,F,G). I conclude that transcription of centromeric RNAs is an additional pathway of Aurora-B recruitment and activation that acts in parallel to the known pathways of CPC recruitment to the inner centromere. Fcr1 RNA is required for normal Aurora-B localization
Author Manuscript
To determine if fcr1 RNA, or the act of transcription, is required for normal Aurora-B localization I used antisense LNA-gapmers to inhibit fcr1 RNA. I found that gapmers targeting fcr1 antisense transcript resulted in a reproducible reduction in the RNA FISH signal on chromosomes while fcr1 sense or GAPDH gapmers had no effect (Figure 4B). I examined Aurora-B localization in extracts treated with two gapmers each targeting fcr1 sense and antisense transcripts and compared these to a nonspecific human GAPDH gapmer control. I found that both gapmers targeting the fcr1 antisense transcript resulted in a consistent ~25% reduction in the localization of Aurora-B to the inner centromere (Figure 4A,C), while gapmers targeting the sense transcript or GAPDH had little effect on Aurora-B localization. Additionally, none of the gapmers had a significant effect on the localization of Bub1 to the kinetochore (Figure 4C). I conclude that fcr1 cen-RNA is required for normal Aurora-B localization.
Author Manuscript
My previous results demonstrated that fcr1 antisense RNA diffuses between centromeres and could be acting in trans to promote Aurora-B localization. To determine if fcr1 antisense RNA acts in cis or trans I examined the distribution of Aurora-B intensities at the centromere in antisense-treated extracts. The cis-acting model predicts that Aurora-B centromere intensity would exhibit a bimodal distribution with one population of affected centromeres and one unaffected population, while the trans-acting model predicts that all centromeres would be equally affected. Comparing the normalized centromere intensity of Aurora-B revealed that Aurora-B intensity exhibits a relatively normal distribution in all gapmer treated extracts with fcr1 antisense gapmer-treated exhibiting a lower intensity
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 7
Author Manuscript
normal distribution (Figure 4D,E), consistent with the prediction of the trans-acting model and the localization of fcr1 antisense RNA. I conclude that fcr1 antisense RNA is free to diffuse between centromeres and promotes normal Aurora-B localization at all centromeres. The modest reduction in Aurora-B localization in gapmer-treated extracts and the presence of fcr1 antisense RNA at ~ half of the centromeres suggests that additional transcripts may also contribute to normal Aurora-B localization.
Author Manuscript
To determine if fcr1 RNA is required for normal activation of Aurora-B at the centromere I treated egg extracts with antisense-gapmers or triptolide and examined the localization of Aurora-B phosphorylated in the activation loop. Consistent with my previous results I found that triptolide treatment resulted in an increase in phosphorylated Aurora-B at the centromere region (Figure 4FG) and a decreased enrichment of phosphorylated Aurora-B at the inner centromere region (Figure 4GH). Gapmers targeting GAPDH or the fcr1 sense transcript did not result in a change in pAurora-B at the inner centromere or enrichment of pAurora-B at the inner centromere (Figure 4FGH). In contrast, treatment of extracts with gapmers targeting the fcr1 antisense transcript resulted in a reproducible increase in AuroraB phosphorylation at centromeres, but did not change the pattern of pAurora-B enrichment at the centromere. These results demonstrated that fcr1 RNA regulates some aspects of Aurora-B phosphorylation and activation at the centromere, but that other factors, such as additional cen-RNAs or the act of transcription, contribute to the change in Aurora-B activation observed in transcription-inhibited extracts. Transcription is required for normal kinetochore:microtubule attachments and kinetochore alignment
Author Manuscript Author Manuscript
In Xenopus egg extracts depletion of Aurora-B leads to a complete loss of spindle assembly (Kelly et al., 2007; Sampath et al., 2004) while a reduction in the chromosomal and centromere level of the CPC through Haspin depletion leads to a much more subtle spindle assembly defect (Kelly et al., 2010). In most other systems the primary function of Aurora-B is to recognize incorrect kinetochore:microtubule attachments that do not generate tension between sister kinetochores (Lampson and Cheeseman, 2011), and inhibition of Aurora-B leads to many chromosomes with incorrect kinetochore:microtubule attachments (Hauf et al., 2003; Lampson et al., 2004). To determine if transcription inhibition resulted in defects in kinetochore:microtubule attachments I monitored kinetochore biorientation in intact spindles by staining for Bub1 and GFP-Aurora-B. In DMSO-treated extracts I found that ~90% of kinetochores demonstrated a clear bipolar attachment to the spindle (Figure 5AB). In contrast, in triptolide treated extracts I found that ~50% of kinetochores did not exhibit bipolar attachment and appeared to show syntelic attachments to one spindle pole (Figure 5AB). To determine if the incorrect kinetochore microtubule attachments observed in triptolide-treated extracts resulted in defects in kinetochore alignment I measured the relative position of kinetochores in DMSO and triptolide-treated extracts. In DMSO treated extracts I found that the vast majority of kinetochores aligned at the spindle equator, while kinetochores in triptolide-treated extracts exhibited a much broader distribution throughout the spindle, consistent with defects achieving a bipolar spindle attachment. Taken together these data demonstrate that inhibition of transcription results in defects in localization of
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 8
Author Manuscript
Aurora-B to the inner centromere leading to defects in correcting errors in kinetochore:microtubule attachment.
Discussion
Author Manuscript
I found that transcription of repetitive centromeric DNA produces a lncRNA required for the normal localization and activation of the CPC at the inner centromere. My results suggest that a high local concentration of centromeric lncRNAs on mitotic centromeres and chromosomes is an additional pathway for CPC localization and activation that acts in parallel to known pathways of CPC recruitment. Additionally, my results suggest that the CPC is one of the major mitotic targets for regulation by cen-RNAs and confirms the observation that noncoding RNAs play an active role during mitosis (Blower et al., 2005; Du et al., 2010; Ferri et al., 2009; Ideue et al., 2014; Jambhekar et al., 2014; Quenet and Dalal, 2014; Rosic et al., 2014).
Author Manuscript
Work in a wide variety of systems has demonstrated active transcription of centromeric DNA. Chromatin marks normally associated with open euchromatic DNA are a conserved feature of centromeric chromatin (Sullivan and Karpen, 2004). In addition, activation or repression of transcription from a Human Artificial Chromosomes (HAC) alters the stability of the HAC (Nakano et al., 2008). Recent work in Drosophila demonstrated that the act of transcription, rather than the resulting RNA, is required for normal Cenp-A deposition (Chen et al., 2015). While several other studies have depleted centromeric RNAs and concluded that cen-RNA is required for various mitotic processes (Ideue et al., 2014; Liu et al., 2015; Quenet and Dalal, 2014; Rosic et al., 2014). Taken together, these studies support the hypothesis that transcription of centromeric DNA plays a role in centromere/kinetochore function through both the process of transcription and through the production of a functional cen-RNA. My observation that fcr1 RNAs localized to half the centromeres affect the localization of Aurora-B to all centromeres suggests that cen-RNAs may serve to activate or modify Aurora-B at the centromere, which then diffuses away to act at other sites, similar to a model proposed for Aurora-B activation by binding to chromatin (Kelly et al., 2007). Recent work has demonstrated that many nascent transcripts are present on mitotic chromatin, but are cleared by transcriptional elongation (Liang et al., 2015) in prophase/prometaphase, while several studies have demonstrated that elongating pol II is present at metaphase centromeres (Chan et al., 2012; Liu et al., 2015; Rosic et al., 2014). These data suggest that persistent transcription during mitosis may be an additional signal that differentiates the centromere from the remainder of the chromosome.
Author Manuscript
My observation that kinetochores do not make normal bipolar attachment to the spindle in the absence of transcription is consistent with a role for transcription in the regulation of CPC localization and Aurora-B activation. A decrease in CPC localization to the inner centromere could result in decreased phosphorylation of components of the KMN network and stabilization of incorrect kinetochore:microtubule attachments (Lampson and Cheeseman, 2011). An alternative possibility is that the increased level of active, autophosphorylated Aurora-B at the inner centromere and chromatin could result in
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 9
Author Manuscript
increased phosphorylation of MCAK and a failure to destabilize incorrect kinetochore:microtubule attachments (Andrews et al., 2004; Lan et al., 2004; Ohi et al., 2004). Both scenarios would lead to stabilization of incorrect attachments to the spindle and lead to errors in chromosome segregation observed after loss of centromeric RNAs (Chan et al., 2012; Rosic et al., 2014). Our previous work and the work of other groups have demonstrated that Aurora-B can bind to a wide variety of RNAs and that RNA binding by the CPC activates Aurora-B kinase in vitro. Promiscuous RNA-binding could facilitate recognition of many different types of chromatin bound RNAs in early mitosis, while the persistence of transcription at the centromere could be an important signal for the localization of the CPC to the inner centromere during prometaphase. Understanding the spatial and temporal interactions of Aurora-B with different types of RNAs will be an important area of future research.
Author Manuscript
Methods Xenopus egg extract methods
Xenopus egg extracts were prepared and utilized for immunofluorescence as described (Hannak and Heald, 2006). Triptolide was added to extracts at a concentration for 10μM from a 10mM stock in DMSO. RNA EMSA Untagged Aurora-B:In-box (pMB940) was expressed and purified from E. coli and used for EMSA as described (Jambhekar et al., 2014) except that radioactive RNAs were used in place of fluorescently-labeled RNAs.
Author Manuscript
UV-crosslinking
Author Manuscript
Taxol stabilized microtubules were purified from 1.2 mL CSF-arrested egg extracts by centrifugation through a glycerol cushion. Microtubule-associated proteins were eluted with XB with a final concentration of 200 mM KCl and recentrifuged for 10 minutes at 22,000 x g. The supernatant was irradiated with UV light using a Stratalinker for 10 minutes on ice while unirradiated samples were kept on ice. Samples were extracted from both irradiated and control extracts for western blots of the input fraction. Extracts were then adjusted to 0.1% Sarkosyl 4 mM EDTA and heated at 65°C for 10 minutes then placed on ice for 5 minutes. Poly-A RNAs were captured using 300 pmol of LNA dT (Exiquon) conjugated to 150 μL of MyOne Streptavidin Dynabeads (Life Technologies). Bound proteins and RNAs were eluted by the addition of 50 μL of dd water and incubation at 65°C for 10 minutes. Samples from the supernatants were analyzed by western blot. RNA FISH Fcr1 RNAs were detected on mitotic chromosome spreads using strand-specific oligonucleotide probes from Stellaris.
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 10
LNA Antisense inhibition
Author Manuscript
Fcr1 RNAs were targeted for destruction using Antisene-LAN Gapmers from Exiqon. Gapmers were added to extracts at a final concentration of 100nM. See Supplemental Information for detailed descriptions of all methods.
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Author Manuscript
I would like to acknowledge Judith Sharp for comments on the manuscript and members of the Blower lab for useful suggestions. I thank Alexi Arnaoutouv, Hiro Funabiki, Ming Tsai, and Dan Eilat for gifts of antibodies. This work was supported by a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund (M.D.B) and (NIH) RO1 (RO1GM086434).
References
Author Manuscript Author Manuscript
Andrews PD, Ovechkina Y, Morrice N, Wagenbach M, Duncan K, Wordeman L, Swedlow JR. Aurora B regulates MCAK at the mitotic centromere. Developmental cell. 2004; 6:253–268. [PubMed: 14960279] Bensaude O. Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity? Transcription. 2011; 2:103–108. [PubMed: 21922053] Blower MD, Nachury M, Heald R, Weis K. A Rae1-containing ribonucleoprotein complex is required for mitotic spindle assembly. Cell. 2005; 121:223–234. [PubMed: 15851029] Carmena M, Wheelock M, Funabiki H, Earnshaw WC. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nature reviews Molecular cell biology. 2012; 13:789– 803. [PubMed: 23175282] Castello A, Fischer B, Eichelbaum K, Horos R, Beckmann BM, Strein C, Davey NE, Humphreys DT, Preiss T, Steinmetz LM, et al. Insights into RNA biology from an atlas of mammalian mRNAbinding proteins. Cell. 2012; 149:1393–1406. [PubMed: 22658674] Castello A, Horos R, Strein C, Fischer B, Eichelbaum K, Steinmetz LM, Krijgsveld J, Hentze MW. System-wide identification of RNA-binding proteins by interactome capture. Nature protocols. 2013; 8:491–500. [PubMed: 23411631] Chan FL, Marshall OJ, Saffery R, Kim BW, Earle E, Choo KH, Wong LH. Active transcription and essential role of RNA polymerase II at the centromere during mitosis. Proceedings of the National Academy of Sciences of the United States of America. 2012; 109:1979–1984. [PubMed: 22308327] Chen CC, Bowers S, Lipinszki Z, Palladino J, Trusiak S, Bettini E, Rosin L, Przewloka MR, Glover DM, O’Neill RJ, et al. Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription. Developmental cell. 2015; 34:73–84. [PubMed: 26151904] Darnell RB. HITS-CLIP: panoramic views of protein-RNA regulation in living cells. Wiley Interdiscip Rev RNA. 2010; 1:266–286. [PubMed: 21935890] Du Y, Topp CN, Dawe RK. DNA binding of centromere protein C (CENPC) is stabilized by singlestranded RNA. PLoS genetics. 2010; 6:e1000835. [PubMed: 20140237] Edwards NS, Murray AW. Identification of xenopus CENP-A and an associated centromeric DNA repeat. Molecular biology of the cell. 2005; 16:1800–1810. [PubMed: 15673610] Eilat D, Fischel R. Recurrent utilization of genetic elements in V regions of antinucleic acid antibodies from autoimmune mice. J Immunol. 1991; 147:361–368. [PubMed: 1711083] Ferri F, Bouzinba-Segard H, Velasco G, Hube F, Francastel C. Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase. Nucleic acids research. 2009; 37:5071– 5080. [PubMed: 19542185]
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 11
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
Hannak E, Heald R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nature protocols. 2006; 1:2305–2314. [PubMed: 17406472] Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochoremicrotubule attachment and in maintaining the spindle assembly checkpoint. The Journal of cell biology. 2003; 161:281–294. [PubMed: 12707311] Ideue T, Cho Y, Nishimura K, Tani T. Involvement of satellite I noncoding RNA in regulation of chromosome segregation. Genes to cells : devoted to molecular & cellular mechanisms. 2014; 19:528–538. [PubMed: 24750444] Jambhekar A, Emerman AB, Schweidenback CT, Blower MD. RNA stimulates Aurora B kinase activity during mitosis. PloS one. 2014; 9:e100748. [PubMed: 24968351] Kawashima SA, Tsukahara T, Langegger M, Hauf S, Kitajima TS, Watanabe Y. Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres. Genes & development. 2007; 21:420–435. [PubMed: 17322402] Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H, Funabiki H. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science. 2010; 330:235–239. [PubMed: 20705815] Kelly AE, Sampath SC, Maniar TA, Woo EM, Chait BT, Funabiki H. Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly. Developmental cell. 2007; 12:31–43. [PubMed: 17199039] Lampson MA, Cheeseman IM. Sensing centromere tension: Aurora B and the regulation of kinetochore function. Trends Cell Biol. 2011; 21:133–140. [PubMed: 21106376] Lampson MA, Renduchitala K, Khodjakov A, Kapoor TM. Correcting improper chromosome-spindle attachments during cell division. Nature cell biology. 2004; 6:232–237. [PubMed: 14767480] Lan W, Zhang X, Kline-Smith SL, Rosasco SE, Barrett-Wilt GA, Shabanowitz J, Hunt DF, Walczak CE, Stukenberg PT. Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity. Current biology : CB. 2004; 14:273–286. [PubMed: 14972678] Lane AB, Strzelecka M, Ettinger A, Grenfell AW, Wittmann T, Heald R. Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening. Developmental cell. 2015; 34:373–378. [PubMed: 26212133] Liang K, Woodfin AR, Slaughter BD, Unruh JR, Box AC, Rickels RA, Gao X, Haug JS, Jaspersen SL, Shilatifard A. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis. Molecular cell. 2015; 60:435–445. [PubMed: 26527278] Liu H, Qu Q, Warrington R, Rice A, Cheng N, Yu H. Mitotic Transcription Installs Sgo1 at Centromeres to Coordinate Chromosome Segregation. Molecular cell. 2015; 59:426–436. [PubMed: 26190260] Nakano M, Cardinale S, Noskov VN, Gassmann R, Vagnarelli P, Kandels-Lewis S, Larionov V, Earnshaw WC, Masumoto H. Inactivation of a human kinetochore by specific targeting of chromatin modifiers. Developmental cell. 2008; 14:507–522. [PubMed: 18410728] Ohi R, Sapra T, Howard J, Mitchison TJ. Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation. Molecular biology of the cell. 2004; 15:2895–2906. [PubMed: 15064354] Quenet D, Dalal Y. A long non-coding RNA is required for targeting centromeric protein A to the human centromere. eLife. 2014; 3:e03254. [PubMed: 25117489] Rosic S, Kohler F, Erhardt S. Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division. The Journal of cell biology. 2014; 207:335–349. [PubMed: 25365994] Sampath SC, Ohi R, Leismann O, Salic A, Pozniakovski A, Funabiki H. The chromosomal passenger complex is required for chromatin-induced microtubule stabilization and spindle assembly. Cell. 2004; 118:187–202. [PubMed: 15260989] Sessa F, Mapelli M, Ciferri C, Tarricone C, Areces LB, Schneider TR, Stukenberg PT, Musacchio A. Mechanism of Aurora B activation by INCENP and inhibition by hesperadin. Molecular cell. 2005; 18:379–391. [PubMed: 15866179]
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 12
Author Manuscript Author Manuscript
Singh G, Ricci EP, Moore MJ. RIPiT-Seq: A high-throughput approach for footprinting RNA:protein complexes. Methods. 2013 Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006; 3:995–1000. [PubMed: 17072308] Sullivan BA, Karpen GH. Centromeric chromatin exhibits a histone modification pattern that is distinct from both euchromatin and heterochromatin. Nature structural & molecular biology. 2004; 11:1076–1083. Wang F, Dai J, Daum JR, Niedzialkowska E, Banerjee B, Stukenberg PT, Gorbsky GJ, Higgins JM. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science. 2010; 330:231–235. [PubMed: 20705812] Wong LH, Brettingham-Moore KH, Chan L, Quach JM, Anderson MA, Northrop EL, Hannan R, Saffery R, Shaw ML, Williams E, et al. Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere. Genome research. 2007; 17:1146–1160. [PubMed: 17623812] Yamagishi Y, Honda T, Tanno Y, Watanabe Y. Two histone marks establish the inner centromere and chromosome bi-orientation. Science. 2010; 330:239–243. [PubMed: 20929775]
Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 13
Author Manuscript
Highlights •
Centromeres are transcribed into a centromere/chromatin associated lncRNA
•
CPC binds to centromeric RNA
•
Transcription and cen-RNA is required for normal CPC localization and activation
•
Transcription is required for normal kinetochore:microtubule attachments eTOC
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 14
Author Manuscript Author Manuscript Author Manuscript
Figure 1. Active transcription of Xenopus centromeres
Author Manuscript
A. Chromosome spreads were prepared from Xenopus egg extracts containing replicated sperm DNA and stained for Bub1 and RNA Pol II pS2. Insets were magnified 3X in Photoshop. B. RT-PCR of cen-RNA in total extract and purified chromosome or spindle preparations. Mitotic chromosomes (left gels) or spindles (right gels) were purified from different Xenopus egg extracts containing replicated sperm nuclei by centrifugation through a glycerol cushion. Fcr1 RNA and a control mRNA (Xl19006) were detected by RT-PCR C. Fcr1 RNA was detected using strand-specific FISH probes in chromosome spreads stained for Bub1 to mark the centromeres. Fcr1 antisense RNA was present at the kinetochore and inner centromere regions (inset) of approximately half the centromeres. A no probe control exhibited little fluorescence on the chromosomes. Insets are magnified 3X in Photoshop. Scale bar is 5μm. D. Quantitation of the fraction of centromeres that contain fcr1 FISH signal that is enriched 3X over the general chromatin signal. Error bars are the standard deviation of 3 samples. E. Fraction of nuclei that exhibit bright, punctate fcr1 antisense FISH signal after triptolide treatement (normalized to DMSO-treated extracts. N=at least 100 nuclei from 3 different extracts). Error bars depict the standard deviation. F. Codetection of fcr1 DNA, fcr1 RNA, and centromeres using dCas9 programmed with a sgRNA targeting the fcr1 repeat. Insets are magnified 3X in Photoshop. G. Scatterplot showing the
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 15
Author Manuscript
centromere intensities of dCas9 and fcr1 RNA FISH. Intensities are plotted from three sets of reactions. See also Supplemental Figure 1 for additional analysis of fcr1 RNA FISH experiments.
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 16
Author Manuscript Author Manuscript Author Manuscript Figure 2. Interaction of Aurora-B with fcr1 RNA
Author Manuscript
A. Schematic for experiment to identify sites of direct contact between the CPC and poly-A RNA using UV crosslinking. B. Western blot analysis of fractions from UV-crosslinking experiment. See Supplemental Figure 2 for uncropped Western blots. C. Detection of the sites of Aurora-B interaction with RNA using PLA. The sites of Aurora-B:RNA interaction were examined on spindles from Xenopus egg extracts containing replicated sperm DNA and GFP-Aurora-B to mark the inner centromere. GFP-Aurora-B signal in second panel has been exaggerated in Photoshop to highlight spindle-localized Aurora-B. Scale bar is 5μm. D. Aurora-B or control IgG IPs from extracts containing replicated sperm nuclear DNA. Fcr1
Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 17
Author Manuscript
and Xl19006 RNAs were detected by RT-PCR. E. Interaction of full-length, untagged recombinant Aurora-B with fcr1 sense,antisense, α-satellite sense, antisense, and MCS from pCR2.1 RNAs examined by EMSA. F. Binding curves, Kds, and Hill coefficient for AuroraB binding to each RNA calculated from three independent experiments. Also see Supplemental Figure 3 for additional EMSA gels and additional quantification.
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 18
Author Manuscript Author Manuscript
Figure 3. Transcription regulates Aurora-B localization to the inner centromere
Author Manuscript
A–C. Localization of the antigens indicated on the left to the inner centromere in control and triptolide-treated extracts. D. Quantification of the normalized signal for the indicated antigens at the centromere region in triptolide-treated extracts. N=3 extracts for all antigens except endogenous Aurora-B where n=4. E. Enrichment of pAurora-B at the inner centromere in control and triptolide treated extracts. * indicates a p-value less than 0.05, *** indicates a p-value less than 0.005, all other p-values are not significant. P-values from D calculated using a single-sample t-test. P-value for E calculated using a Wilcox signed rank test. F–G. Images of H2A pT120 and H3 pT3 in control and triptolide-treated extracts. Scale bar is 5μm.
Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 19
Author Manuscript Author Manuscript Author Manuscript Figure 4. Fcr1 antisense RNA regulates Aurora-B localization to the inner centromere
Author Manuscript
A. Localization of Bub1 and endogenous Aurora-B in extracts treated with the indicated antisense LNA-gapmer. B. Fraction of fcr1 antisense FISH positive nuclei (normalized to GAPDH control) for each indicated antisense LNA-gapmer (n=at least 100 nuclei from 3 independent extracts). C. Quantification of centromere intensity of Bub1 and Aurora-B (normalized to GAPDH-treated extracts)(n=3 extracts). * indicates a p-value less than 0.05 by single sample t-test. D. Distribution of normalized Aurora-B centromere intensity in extracts treated with the indicated antisense LNA-gapmers. E. Boxplot and points of the normalized centromere intensity of Aurora-B for each indicated antisense LNA-gapmer. At least 100 centromeres were measured per extract from three independent extracts. All data points are combined in this plot. F. Localization of Bub1 and phosphorylated Aurora-B in Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 20
Author Manuscript
extracts treated with antisense LNA-gapmers and triptolide. G. Quantitation of normalized signal for Bub1 and pAurora-B at the inner centromere region in gapmer-treated extracts. 50–200 centromere regions were quantified for each condition from three independent extracts. Error bars are standard deviation. H. Boxplot of the enrichment of Aurora-B at the inner centromere region in gapmer and triptolide-treated extracts. For this plot all enrichment values from three replicate experiments are combined into a single plot. *** indicates a p-value less that 0.005 by Wilcox Rank Sum test. Scale bar is 5μm.
Author Manuscript Author Manuscript Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
Blower
Page 21
Author Manuscript Author Manuscript
Figure 5. Transcription regulates kinetochore microtubule attachment
Author Manuscript
A. Intact spindles were prepared from control or triptolide-treated extracts containing GFPAurora-B and stained for Bub1 to mark the positions of the kinetochores. Single optical sections are shown for both spindles. The majority of control kinetochores exhibit clear bipolar attachment to the spindle (arrow), while ~half of kinetochores in triptolide-treated extracts exhibit monopolar attachment to the kinetochore. Insets are magnified 3X in Photoshop. B. Quantification of kinetochore attachment in control and triptolide-treated extracts. 100–200 kinetochores were scored in each of two extracts. P-value was calculated using a Wilcox signed rank test. C. Normalized position of each kinetochore was calculated and control and triptolide-treated extracts are plotted as a density histogram. Scale bar is 5μm.
Author Manuscript Cell Rep. Author manuscript; available in PMC 2016 May 31.
