RESEARCH ARTICLE

Cbc2p, Upf3p and eIF4G are components of the DRN (Degradation of mRNA in the Nucleus) in Saccharomyces cerevisiae Satarupa Das, Upasana Saha & Biswadip Das Department of Life Science and Biotechnology, Jadavpur University, Kolkata, India

Correspondence: Satarupa Das, Department of Life Science and Biotechnology, Jadavpur University, 188 Raja S.C. Mullick Road, Kolkata – 700 032, West Bengal, India. Tel.: 91 9748324755; fax: 91 33 2414 6414; e-mail: [email protected] Received 24 January 2014; revised 2 July 2014; accepted 3 July 2014. Final version published online 28 July 2014. DOI: 10.1111/1567-1364.12180 Editor: Ian Dawes Keywords mRNA degradation; cap-binding protein; DRN; RRP6; eIF4G; UPF3.

Abstract Messenger RNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system designated DRN (Degradation of mRNA in the Nucleus) that is dependent on the nuclear mRNA cap-binding protein, Cbc1p, as well as nuclear exosome component Rrp6p, a 30 to 50 exoribonuclease. DRN has been shown to act on RNAs preferentially retained in the nucleus, such as: (1) global mRNAs in export defective nup116-D mutant strains at the restrictive temperature; (2) a certain class of normal mRNAs called special mRNAs (e.g. IMP3 and YLR194c mRNAs); and (3) mutant mRNAs for example, lys2187 and cyc1-512. In this study, we further identify three novel components of DRN (Cbc2p, Upf3p and Tif4631p) by employing a genetic screen and by considering proteins/factors that interact with Cbc1p. Participation of these components in DRN was confirmed by demonstrating that null alleles of these genes resulted in stabilization of the rapid decay of global mRNAs in the export defective nup116-D strain and of representative special mRNAs. Depletion of Tif4632p, an isoform of Tif4631p, also exhibited a partial impairment of DRN function and is therefore also considered to play a functional role in DRN. These findings clearly establish that CBC2, UPF3, and TIF4631/32 gene products participate in DRN function.

YEAST RESEARCH

Introduction The rate of synthesis and degradation of mRNAs are major determinants in the regulation of gene expression. All normal eukaryotic mRNAs undergo degradation via a default pathway primarily in the cytoplasm following translation. The default decay is initiated by deadenylation followed by decapping and exonucleolytic degradation of the transcript body, either from a 50 ?30 direction (Tucker & Parker, 2000; Mitchell & Tollervey, 2001; Coller & Parker, 2004; Parker & Song, 2004; Parker, 2012; Das & Das, 2013) or from a 30 ?50 direction (Anderson & Parker, 1998; Chen et al., 2001; Wang & Kiledjian, 2001; Mukherjee et al., 2002; Parker, 2012; Das & Das, 2013). In addition, individual mRNAs undergo sequencespecific endonucleolytic cleavage by siRNAs or miRNAs, which in turn trigger further degradation (Dodson & Shapiro, 2002; Kim, 2005). Specialized surveillance pathways also operate, both in the nucleus and cytoplasm, to recognize and selectively degrade various types of aberrant and defective mRNAs, ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

thus contributing to the quality control of gene expression (Maquat & Carmichael, 2001). Three such surveillance pathways, nonsense-mediated decay (NMD), nonstop decay (NSD) and the no-go decay (NGD) operate in the cytoplasm (Parker, 2012; Das & Das, 2013). NMD is a widely studied and well-understood cytoplasmic mRNA surveillance mechanism and selectively degrades messenger RNAs harboring an in frame premature termination codon (PTC) in a translation-dependent manner (Muhlrad & Parker, 1994; Jacobson & Peltz, 1996; Mitchell & Tollervey, 2003; Maquat, 2004; Parker, 2012). Interestingly, a substantial number of wild-type mRNAs are also degraded by NMD (Lelivelt & Culbertson, 1999; He et al., 2003; Guan et al., 2006; Metzstein & Krasnow, 2006). NSD specifically acts on defective transcripts lacking a natural nonsense codon, which arises due to internal polyadenylation event within an ORF (Frischmeyer et al., 2002). Such transcripts are degraded by Ski2p/Ski3p/Ski7p and cytoplasmic exosome in a 30 ?50 direction after recruitment to translating ribosomes at the extreme 30 -end of the poly(A) tail (Dodson & Shapiro, FEMS Yeast Res 14 (2014) 922–932

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2002; Frischmeyer et al., 2002; van Hoof et al., 2002). The NGD pathway degrades mRNAs harboring a nonphysiological secondary structure that causes a strong pause in translational elongation (Doma & Parker, 2006). In addition to the above cytoplasmic decay pathways, aberrant messages undergo quality control and selective degradation in the nucleus primarily by two mechanisms (Moore, 2002; Vasudevan & Peltz, 2003). These include nuclear RNA exosome-mediated decay (Imataka & Sonenberg, 1997; Bousquet-Antonelli et al., 2000; Burkard & Butler, 2000; Torchet et al., 2002) and DRN (Degradation of mRNA in the Nucleus) (Das et al., 2000, 2003, 2006; Kuai et al., 2005). The RNA exosome is a large multiprotein complex of eleven 30 ?50 exoribonucleases and few associated nuclear and cytoplasmic cofactors that acts on a multitude of RNA substrates (Mitchell et al., 1997; Allmang et al., 1999; Butler, 2002; Parker, 2012; Das & Das, 2013). It degrades precursor noncoding RNAs (pre-rRNAs, pre-snoRNAs and pre-tRNAs) (Gudipati et al., 2012; Schneider et al., 2012), as well as aberrant pre-mRNAs such as those resulting from inefficient splicing (Bousquet-Antonelli et al., 2000) or 30 -end processing (Imataka & Sonenberg, 1997; Burkard & Butler, 2000; Torchet et al., 2002). The nuclear exosome also targets normal mRNAs that fail to achieve an export competent mRNP conformation in THO mutant strains (Libri et al., 2002; Rougemaille et al., 2007; Assenholt et al., 2008). The DRN pathway is dependent on Cbc1p, large subunit of the nuclear cap-binding complex (CBC), and on Rrp6p, a 30 ?50 exoribonuclease and a component of nuclear exosome. DRN acts on several abnormal and normal mRNA substrates that are exported slowly from nucleus (Das et al., 2000, 2003). These substrates include (1) global mRNAs retained in the nucleus of export defective nup116-D strains (Das et al., 2003); (2) a small subset of normal mRNAs referred to as special mRNAs, such as IMP3 and YLR194c that inherently are exported slowly (Kuai et al., 2005); and (3) mutant mRNAs lys2187 and cyc1-512 (Das et al., 2000, 2006). The half-life of ACT1 mRNA, representing a typical mRNA, is decreased nearly 40% in a nuclear export defective nup116-D strain, whereas upon deletion of either CBC1 or RRP6 gene in the nup116-D mutant, its stability was restored (Das et al., 2003). More recently, Kuai et al. (2005) identified hundreds of nonaberrant mRNAs (referred to as special mRNAs) that degrade rapidly in a normal strain which is stabilized in either cbc1-D and rrp6-D strains. They further demonstrated that a representative special mRNA, SKS1 are retained preferentially in the nucleus (Kuai et al., 2005). Mutationally altered mRNAs such as lys2187 and cyc1-512 mRNAs are also retained in the nucleus and subjected to Cbc1p-dependent decay (Das et al., 2000, 2006). Based on these collective observations, Das FEMS Yeast Res 14 (2014) 922–932

et al. (2000, 2003, 2006) and Kuai et al. (2005) proposed that all mRNAs may potentially be subjected to DRN, suggesting that kinetic competition exists between nuclear decay by DRN and nuclear export. A kinetic competition is supported by the observation that nuclear retention of global messages in several conditionally lethal export mutant strains (e.g. nup116-D and rat7-1) leads to massive destabilization. When DRN was knocked out by deleting either the CBC1 or RRP6 gene, their stabilization was restored (Das et al., 2000, 2003; Kuai et al., 2005). Delineating the underlying mechanism of this nuclear mRNA decay pathway is therefore crucial to the understanding of the mechanistic details of cellular mRNA quality control. To uncover molecular mechanism of DRN, we used a genetic screen to isolate extragenic suppressors of cyc1512 and lys2-187 alleles and identify UPF3 and CBC2 as two novel components of DRN. Additionally, we asked if Tif4631p (eIF4GI) plays a role in DRN as its mammalian homolog physically associates with CBP80 in the nucleus of Hela cells (McKendrick et al., 2001). Our results indicate that deletion of CBC2, UPF3, TIF4631, or TIF4632 impairs DRN function thus establishing Cbc2p, Upf3p, Tif4631p, and Tif4632p as new components of DRN.

Materials and methods Nomenclature, strains, media, and yeast genetics

Standard genetic nomenclature is used to designate wildtype alleles (e.g. CYC1, LYS2, NUP116, TIF4631), recessive mutant alleles (e.g. cyc1-512, lys2-187, nup116-D, tif4631-1) and disruptants or deletions (e.g. cbc1::URA3, cbc1-D). An example of denoting a protein encoded by a gene is as follows: Tif4631p encoded by TIF4631. CBC denotes the Cap Binding Complex composed of Cbc1p and Cbc2p. The genotypes of Saccharomyces cerevisiae strains used in this study are listed in Supporting Information, Table S1. Standard YPD, YPG, SC-Leu (leucine omission), and other omission media were used for testing and for growth of yeast (Sherman, 1991). Yeast genetic analysis was carried out by standard procedures (Sherman, 1991). Extraction and purification of total RNA

Total RNA was isolated from appropriate strains of S. cerevisiae as described by Das et al. (2000, 2003). Appropriately grown yeast cells were washed with washing buffer after harvesting at 4 °C, followed by extraction of total cellular RNA by vortexing in the presence of phenol– chloroform–IAA (25 : 24 : 1) for three times. Following ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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extraction, RNA was purified and recovered by precipitation with RNAase free ethanol and further quantified by UV absorption spectrophotometry. Analysis of the steady state levels and stability of specific mRNAs

The stabilities of various mRNAs were determined by inhibition of transcription with the transcription inhibitor thiolutin, as described by Das et al. (2000, 2003). Briefly, yeast strains were grown at 30 °C till mid-logarithmic phase when thiolutin was added to the growing culture at 4 lg mL1 final concentration followed by withdrawal of 25 mL of culture at various times after thiolutin addition. Steady state levels of specific mRNAs were determined by harvesting yeast cells at 30 °C. Message levels were determined by either real-time PCR (described below) or by Northern blot analysis as described (Das et al., 2000, 2003), except that probe was prepared using the psoralenbiotin labeling kit (Applied Biosystems) and mRNA levels quantified using a phosphorimager. Signals associated with specific message were normalized against SCR1 signals. The decay rates and half-lives of specific mRNAs were estimated with the regression analysis program (Origin Pro 8, OriginLab Corporation, MA) using a single exponential decay formula y = 100 ebx, assuming mRNA decay follows a first order kinetics. RT-PCR, DNase treatment, and cDNA preparation

For reverse transcription, total RNA was treated with DNase I (Fermentas Inc., Pittsburgh, PA). RNA purity was assessed by capillary electrophoresis (Agilent 2100 Bioanalyzer; Agilent Technologies, Inc., Santa Clara, CA) to determine RNA integrity numbers (range 8.7–9.5). DNase I-treated RNA (0.5 lg) was reverse transcribed with random primers using Superscript III enzyme (Invitrogen, Carlsbad, CA) for 1 h at 50 °C. The resultant cDNA was purified with the QIAquick PCR purification system (Qiagen). RNA and cDNA were quantified using the Quant-iT RNA assay kit and the Quant-iT dsDNA HS assay kit, respectively, with the Qubit fluorimeter (Invitrogen). Real-time PCR

One nanogram of quantified cDNA was used to quantify the levels of mRNAs by Q-PCR using target specific primers and standard Cyber Green Technology with Power Cyber Green PCR Master Mix (Applied Biosystem). Q-PCR assays were run in triplicate and conducted using an Applied Biosystems StepOneTM real-time PCR ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

system. For each target, purified DNA templates were prepared by PCR, followed by gel purification and serial dilutions to generate standard curves from 102 to 109 copy number/reaction vs. threshold cycle (Ct), determined by the StepOneTM software version 2.2.2 (Applied Biosystems). Standard curves were used to determine the amplification efficiencies (E = 10(1/n), where n = slope of the standard curve) in Cyber Green assays as well as copy numbers of templates in experimental samples.

Results Identification of upf3 and cbc2 as suppressors of cyc1-512 and lys2-187 mutations

The rationale for our genetic screening approach was based on previous findings that null alleles of CBC1 and RRP6 (cbc1-D and rrp6-D), suppress both, cyc1-512 (Das et al., 2000) and lys2-187 (Das et al., 2006) mutant alleles. Both mutations resulted in formation of aberrant transcripts retained preferentially in the nucleus and degraded by DRN in a Cbc1p-/Rrp6p-dependent manner (Das et al., 2000, 2006). Additionally, cyc1-512 and lys2-187 mutations were suppressed by nonsense suppressors for example, upf1, upf2, and upf3 (components of NMD pathway). In contrast, cbc1-D or rrp6-D mutations could not suppress nonsense mutations such as leu2-1 (UAA), his4-166 (UGA), or met8-1 (UAG) (Das et al., 2000, 2006). Therefore, suppressors that concomitantly act on cyc1-512 and lys2-187, but do not suppress nonsense mutations might identify novel components of the DRN pathway. The screen was initiated by mutagenizing the yeast strain yBD-20 (MATa cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1) by UV and plating on lysine-omission medium (SC-Lys) to score the suppressors of lys2-187 (Lys2+). Suppression of the cyc1-512 mutation was assessed by higher levels of cytochrome c in the Lys2+ suppressors. A total of 1314 Lys2+ suppressor mutants were obtained and further tested for their ability to grow on SL medium, which requires increased cytochrome c levels. A total of 401 mutants grew better on SL medium, and their cytochrome c levels were determined by low temperature (196 °C) spectroscopic examination. Of these, 74 candidates contained higher levels of cytochrome c than the parental strain, yBD-20 (Table 1). Genetic complementation tests were then carried out with these mutants to assist in identification of suppressors acting on cyc1-512 and lys2-187. Each of them was first individually crossed to yBD-27 (MATa cyc1-512 lys2187 leu2-1 ura3-52 his 4-166 trp1-289). The observed low levels of cytochrome c and Lys2p in each of these resulting diploid strains established that these suppressors were FEMS Yeast Res 14 (2014) 922–932

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Table 1. Number of mutants obtained in the screen with yBD-20 Total Lys+ mutants examined Lys+ mutants with increased growth on lactate medium Lys+ mutants with increased cytochrome c levels* Approximately 45% normal level Approximately 30% normal level Approximately 25% normal level Approximately 20% normal level Lys+ mutants with increased cytochrome c containing suppressors cbc1 suppressors cbc2 suppressors upf1 suppressors upf2 suppressors upf3 suppressors Novel suppressors Novel suppressors not showing mendelian segregation Novel suppressors showing mendelian segregation Intragenic suppressors Extragenic suppressors, sut4-1

1314 401 74 25 33 14 2 74 2 2 4 3 49 15 11 4 3 1

*The original yBD-20 strain has approximately 5–10% the normal level of cytochrome c.

recessive. They were then crossed individually to derivatives of yBD-27 containing one of the following deletions: cbc1-D, cbc2-D, upf1-D, upf2-D, and upf3-D (Table 1 and Table S1) to examine if these suppressors contain any of the above five mutations. The rationale of inclusion of a cbc2-D strain was based on our assumption that CBC2 could be a component of DRN along with CBC1 as both Cbc1p and Cbc2p function together in a nuclear complex (Izaurralde et al., 1994). The other three deletions of nonsense suppressors, upf1-D, upf2-D, and upf3-D, were included in the complementation tests because we expected these three suppressors to be picked up in the screen. The higher cytochrome c levels in some of the diploids resulting from these crosses, indicative of the lack of complementation, allowed assignment of 59 of the 74 suppressors to the CBC1, CBC2, UPF1, UPF2, and UPF3 loci, as summarized in Table 1. Of these 59 suppressors, Cbc1p was previously shown to function in DRN, whereas Upf1p was not (Das et al., 2000, 2003). Therefore, the functional role of the three suppressors, cbc2, upf2, and upf3, were further evaluated by specific mRNA decay assay (see below). Eleven of the remaining fifteen mutants did not show a 1 : 1 spore segregation pattern when their corresponding diploids (made with yBD 27) were sporulated, indicating the mutant phenotype arose due to mutations in multiple genes in a single cell, presumably involved in the same DRN pathway (Table 1). None of these mutants were analyzed further. The four mutants showed Mendelian segregation patterns upon tetrad analyses. Three of them constituted intragenic suppressors of cyc1-512 (revertants FEMS Yeast Res 14 (2014) 922–932

in CYC1 gene itself) and were therefore not analyzed further. The last one in this category, sut4-1, did not diminish DRN function when disrupted (data not shown), thus ruling out the possibility that it could be a component of DRN. In summary, the screen yielded potential and testable candidates, CBC2, UPF2, and UPF3. Defects in Cbc2p, Tif4631p, and Upf3p suppress the rapid degradation of global mRNAs retained in the nucleus of the nup116-D strain

Cbc1p interacts with a large repertoire of proteins involved in mRNA processing (splicing and 30 -end formation), mRNA export and in translation (Fortes et al., 2000; Oeffinger et al., 2007; Collins et al., 2007). In HeLa cells, homologs of translation initiation factor Tif4631p and Cbc1p reportedly associate within the nucleus (McKendrick et al., 2001). Moreover, Tif4631p is considered as the hub of a multi-component switch controlling gene expression at the level of translation initiation. Nevertheless, the physiological significance of nuclear shuttling of Tif4631p and its interaction with Cbp1p is unclear. McKendrick et al. (2001) suggested Tif4631p may be a component of a nuclear mRNA decay pathway. We therefore determined whether Tif4631p and three other proteins identified in our genetic screen (Upf2p, Upf3p and Cbc2p) participate in DRN. The half-lives of three representative global mRNAs (CYC1, CYH2, and ACT1) were determined in nup116-D strains carrying one additional deletion (cbc1-D, upf1-D, upf2-D, upf3-D, cbc2-D, or tif4631-D) to verify whether these deletions stabilize the rapid decay of any of these mRNAs. As shown in Fig. 1, the decay rates of each of the three mRNAs were rapid in nup116-D, nup116-Dupf1-D, and nup116-Dupf2-D strains (Fig. 1 and summarized in Table 2). Interestingly, when each of the three genes was deleted in the nup116-D genetic background the half-lives of all three mRNAs increased 2.5- to 3-fold (Fig. 1 and Table 2). Deletion of each of the three gene products, Cbc2p, Upf3p, and Tif4631p, is able to stabilize the rapid decay of global mRNAs trapped in the nucleus. This suggests that Cbc2p, Upf3p, and Tif4631p play a role in DRN. Cbc2p, Tif4631p, and Upf3p are involved in the degradation of special mRNAs

The half-lives of three representative special mRNAs, SKS1, IMP3, and YLR194c, were determined in deletion mutants, cbc1-D, cbc2-D, rrp6-D, upf1-D, upf2-D, upf3-D or tif4631-D, as well as in the parent strain. Results shown in Fig. 2 and summarized in Table 2 clearly demonstrate that decay rates of all three special mRNAs are impaired in cbc1-D, cbc2-D, rrp6-D, upf3-D, and tif4631-D mutant strains with the concomitant increase in their half-lives ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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nup116-Δ nup116-Δ cbc1-Δ nup116-Δ rrp6-Δ nup116-Δ upf1-Δ nup116-Δ upf2-Δ nup116-Δ upf3-Δ nup116-Δ tif4631-Δ (b)

57

28

52

52

29

56

16

12

21

20

15

22

47

25

49

38

34

55

CYH2

100

30

40

50

Time after addition of thiolutin (Min)

40 50

30

20

0

10

ACT1

100

50

10

10

20

50

20

50

10

t1/2

12

50

0

SCR1

30 40

20

10

t1/2

0

50

40

30

ACT1

16

CYC1

100

Percent mRNA remaining

0

t1/2

10

50

40

20 30

0

10

Time (min)

20

CYH2

CYC1

(a)

10

0

10

20

30

40

50

Time after addition of thiolutin (Min)

0

10

20

30

40

50

Time after addition of thiolutin (Min)

Fig. 1. (a) Northern blot analysis reveals increased degradation of CYC1, CYH2, and ACT1 mRNAs that are retained in the nucleus in the export deficient nup116-D strain. Also, degradation is suppressed by cbc1-D, rrp6-D, upf3-D, and tif4631-D, but not by upf1-D or upf2-D. An isogenic series consisting of nup116-D (yBD-43), nup116-D cbc1-D (yBD-44), nup116-D rrp6-D (yBD-45), nup116-D tif4631-D (yBD-50), nup116-D upf3-D (yBD-47), nup116-D upf1-D (yBD-46), and nup116-D upf2-D (yBD-51) strains were grown at 25 °C to mid-logarithmic phase. Subsequently, each culture was transferred to the restrictive temperature of 37 °C, incubated for 1 h and transcription inhibited by addition of 4 lg mL1 thiolutin. Cells were harvested at various times after thiolutin addition. mRNA half-lives (min) at 37 °C were normalized against SCR1 RNA and are presented beside each panel, as well as in Table 2. Values are the averages of three separate experiments (see b), not from the representative blot shown. (b) Decay rates of CYC1, CYH2, and ACT1 mRNA in thiolutin-treated cells of the following strains: yBD-43: nup116-D (■), yBD-44: nup116-D cbc1-D (●), yBD-45: nup116-D rrp6-D (▲), yBD-50: nup116-D tif4631-D (▼), yBD-47: nup116-D upf3-D (◄), yBD-46: nup116-D upf1D (►), yBD-51: nup116-D upf2-D (♦). Rates were determined from signals obtained from Northern blot analysis at the times indicated after addition of 4 lg mL1 thiolutin. Signals were normalized to SCR1 RNA, and normalized signals (mean values  SD from three independent experiments) presented as the percentage of remaining RNA (with respect to normalized signals at 0 min) vs. time of incubation in the presence of thiolutin.

by about three- to five-fold. On the other hand, half-lives were comparable to wild-type strain in the upf1-D and upf2-D strains. These results are consistent with those obtained in the previous experiment in demonstrating ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

that decay of mRNAs retained in the nucleus is dependent on Cbc1p, Cbc2p, Rrp6p, Upf3p, and Tif4631p, thus further supporting CBC2, UPF3, and TIF4631 gene products as novel components of DRN. FEMS Yeast Res 14 (2014) 922–932

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Table 2. Half-life (min) of various mRNAs in different nup116-D and NUP116 strains* nup116-D Pertinent genotype

CYC1

– cbc1-D cbc2-D rrp6-D upf1-D upf2-D upf3-D tif4631-D

16.3 57.3 – 52.0 16.3 19.7 47.3 38.0

CYH2

 1.5  2.5     

7.0 1.5 1.5 5.9 4.0

12.3 27.6 – 29.3 11.7 15.0 24.7 34.3

ACT1

 3.2  5.7     

2.3 1.5 4.4 3.1 4.9

19.7 51.7 – 56.0 21.3 22.0 48.7 55.0

SKS1

 2.1  8.5     

2.0 2.3 4.4 6.0 6.6

9.8 26.3 27.3 27.7 6.0 8.5 28.4 23.0

IMP3        

2.3 4.0 4.2 4.2 1.4 0.7 4.2 4.6

3.7 11.7 15.0 16.3 3.5 5.5 15.0 16.3

YLR194C        

1.2 2.1 3.0 4.0 0.7 0.7 1.7 3.8

3.7 22.7 23.7 27.7 5.5 6.0 28.7 26.0

       

0.6 4.0 5.7 3.1 0.7 1.4 4.0 2.7

ACT1 44.7 56.3 54.0 53.7 44.5 41.5 45.3 43.0

       

CYH2 1.5 4.9 7.2 4.5 0.7 2.1 2.5 3.6

29.0 34.0 35.7 34.7 43 36 40.0 42.3

       

7.9 7.9 7.5 10.2 2.8 1.4 2.7 3.2

*The half-life values CYC1, CYH2, and ACT1 mRNAs under nup116-D mutants (shown in the first three columns) were determined at 37 °C temperature and those of SKS1, IMP3, YLR194C, ACT1, and CYH2 in normal strains (shown in the next five columns) were determined at 30 °C.

Both eIF4G isoforms are required for DRN

TIF4632 is considered to be a paralog of TIF4631 which arose from the result of whole genome duplication (Goyer et al., 1993; Tarun et al., 1997; Dominguez et al., 1999). Hence, we tested whether Tif4632p, which has 53% overall identity with Tif4631p, also plays a functional role in DRN. Steady state levels of IMP3, a special mRNA and CYC1, a typical mRNA were compared in a normal, cbc1-D, tif4631-D, and tif4632-D strains. As shown in Fig. 3a, the steady state level of IMP3 increased about 2.5-fold in the tif4632-D strain, comparable to that in the cbc1-D and tif4631-D strains. Steady state level of CYC1, however, did not exhibit significant stabilization in any of the mutant strains, as expected (Fig. 3a). These results clearly indicate both isoforms, Tif4631p and Tif4632p, are components of DRN. The interaction between Cbc1p and Tif4631p is dispensable for DRN function

We further explored whether an association between Cbc1p and Tif4631p is critical for DRN function in the nucleus. It was reported previously that the point mutation DR548/9AA in Tif4631p disrupts interaction between Cbc1p and Tif4641p (Baron-Benhamou et al., 2003). We find the steady state levels of CYC1 and IMP3 mRNAs are comparable in an isogenic series of wild-type and tif4631-DR548/9AA mutant strains (Fig. 3b), suggesting this specific interaction between Cbc1p and Tif4631p is not required for DRN. Stabilization of IMP3 transcripts is similar in yeast strains carrying either or both null alleles of CBC1 and TIF4631 genes

Having identified Tif4631p as a new component of nuclear decay, we further explored whether Tif4631p and FEMS Yeast Res 14 (2014) 922–932

Cbc1p are part of the same degradation machinery. A strain carrying a double null allele, cbc1-D tif4631-D, was used to test if the rapid decay of special mRNA, IMP3 is more efficiently stabilized than in strains carrying each of the null alleles separately. The cbc1-D tif4631-D strain could not support stabilization of IMP3 transcripts any better than strains carrying each of the single null allele (Fig. 3c). The epistatic relationship between CBC1 and TIF4631 genes supports the conclusion that Cbc1p and Tif4631p are involved in the same pathway.

Discussion Regulation of mRNA abundance via selective degradation plays an important role in control of gene expression in eukaryotes. DRN is one such regulatory pathway, which limits the abundance of aberrant messages as well as controls the levels of several special mRNAs by selective degradation in the nucleus. This pathway therefore plays a pivotal role in generating the functional cellular mRNAome. A complete understanding of DRN and its mechanism of action is therefore critical to unveil the complex process of nuclear mRNA surveillance. As part of initial steps in understanding, we identified in the current study new novel components using an improved genetic screening strategy and further considered proteins that directly or indirectly interact with the DRN component, Cbc1p. Our screen yielded two new suppressors, Cbc2p and Upf3p, both of which were confirmed to participate in DRN via mRNA decay assays. In addition, based on putative interaction with Cbc1p, we identify Tif4631p and Tif4632p as additional components of this pathway. The two subunits of eukaryotic CBC, Cbc1p, and Cbc2p (CBP80 and CBP20, respectively, in mammals), bind as a heterodimer to the 7-methylguanosine structure of capped RNAs to influence a number of mRNA processing events (Izaurralde et al., 1994; Colot et al., 1996; Lewis et al., 1996). Both subunits participate in most, if ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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SCR1

CYH2

0 3 5 10 20 30

ACT1

t1/2

0 3 5 10 20 30

t1/2

t1/2

t1/2

10

3

3

44

29

cbc1-Δ

26

11

22

56

34

cbc2-Δ

27

15

23

54

36

rrp6-Δ

28

16

28

53

34

upf1-Δ

6

3

5

44

43

upf2-Δ

8

5

6

41

36

upf3-Δ

28

15

28

45

40

tif4631-Δ

23

16

26

43

42

Percent mRNA remaining

(b)

SKS1

100

IMP3

100

0 3 5 10 20 30

YLR194C

0 3 5 10 20 30

t1/2

0 3 5 10 20 30

IMP3

SKS1 0 3 5 10 20 30

(a) Min at 30°C Normal

ACT1

100

50 50

50

10 10

10 1 0

10

20

30

40

50

Time after addition of thiolutin (Min)

0

10

20

30

40

50

Time after addition of thiolutin (Min)

0

10

20

30

40

50

Time after addition of thiolutin (Min)

Fig. 2. (a) Northern blot analysis reveals that mRNAs SKS1, IMP3, and YLR194C are stabilized by cbc1-D, cbc2-D, rrp6-D, upf3-D and tif4631-D, but not by upf1-D or upf2-D. An isogenic series of wild-type strain yBD-20 (Normal) and deletion strains cbc1-D (yBD-21), cbc2-D (yBD-22), rrp6D (yBD-26), tif4631-D (yBD-56), upf3-D (yBD-25), upf1-D (yBD-23), and upf2-D (yBD-24) were grown at 30 °C to an A600 nm of 1.0, treated with 4 lg mL1 of thiolutin and samples removed at various times after treatment as indicated at the top of each panel. mRNAs were detected by Northern blot analysis of total RNA. The blot was sequentially hybridized with probes specific to SKS1, IMP3, YLR194C, ACT1, CYH2, and SCR1. Half-life values (in min) are shown to the right and in Table 2. Signals were normalized to SCR1 RNA. ACT1 CYH2 mRNAs are not affected by DRN, as they are not retained in the nucleus. (b) Decay rates of SKS1, IMP3, and ACT1 mRNA in thiolutin-treated cells in the following strains: yBD-20: Normal (■), yBD-21: cbc1-D (●), yBD-22: cbc2-D (▲), yBD-56: tif4631-D (▼), yBD-25: upf3-D (◄), yBD-26: rrp6-D (►), yBD-24: upf2-D (♦). mRNA decay rates were determined by Northern blot analysis of total RNA extracted from cells treated with 4 lg mL1 of thiolutin at the times indicated. Signals were normalized to SCR1 RNA and normalized signals (mean values  SD from three independent experiments) presented as the percentage of remaining RNA (with respect to normalized signals at 0 min) vs. time of incubation in the presence of thiolutin.

not all, mRNA processing and maturation functions, and in some cases formation of Cbc1p/2p heterodimers is critical, such as in binding of 50 -cap structure (Izaurralde et al., 1994; Colot et al., 1996; Lewis et al., 1996). Our finding of the involvement of both Cbc1p and Cbc2p in DRN therefore raises an issue whether an intact heterodimer is required for a functional DRN pathway even if both subunit contribute in this process. CBC bound at the 50 -cap of newly exported mRNP interacts with cytoplasmic Tif4631p during its exchange ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

for cap-binding protein eIF4E (Fortes et al., 1999, 2000). Tif4631p mediates this exchange (Fortes et al., 1999) and, following exchange of the 50 -cap, can interact with many other proteins (e.g. eIF3, eIF4E, eIF4A, and Pab1p) to recruit 40S ribosomal subunits and promote translation initiation. In both mammals and yeast, Tif4631p has been shown to shuttle between the nucleus and cytoplasm, where in the nucleus, it interacts with the spliceosome machinery and is involved in splicing (Kafasla et al., 2009). McKendrick et al. (2001) found Tif4631p interacts FEMS Yeast Res 14 (2014) 922–932

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CYC1

IMP3

(a)

1.2

Fold stabilized

Fold stabilized

4.0

3.0

2.0

1.0 0.8 0.6 0.4

1.0 0.2 0.0

0.0

IMP3

CYC1

1.0

1.0

0.8

0.8

0.6

0.6

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0.2

0.2

0.0

0.0

CYC1

IMP3

(c) 4.0

Fold stabilized

Fold stabilized

(b)

1.2 1.0

3.0

2.0

0.8 0.6 0.4

1.0 0.2 0.0

0.0

Fig. 3. (a) Both isoforms of eIF4G, Tif4631p, and Tif4632p, function as components of DRN. cDNA was prepared from strains indicated at the bottom of each panel and subjected to either real-time PCR (Bar graph) or end-point PCR analysis (gel panel below the bar graph) using primer sets specific for the special mRNA, IMP3, or the typical mRNA, CYC1. (b) tif4631-DR548/9AA mutation in Tif4631p is dispensable for DRN function. cDNA was prepared from wild-type strain yBD-184 (Normal) and strain yBD-185 (tif4631-DR548/9AA) grown at 30 °C and subjected to either real-time PCR (bar graph) or end-point PCR analysis (gel panel below the bar graph). (c) Yeast strain carrying a cbc1-D tif4631-D deletion stabilize the rapid decay of IMP3 mRNA to an extent similar to strains carrying either a cbc1-D deletion or a tif4631-D deletion, but have no effect on CYC1 mRNA. cDNA was prepared from the strains indicated at the bottom of each panel and subjected to either real-time PCR (bar graph) or end-point PCR analysis (gel panel below the bar graph).

with Cbc1p in the nucleus of Hela cells in a manner different from that in the cytoplasm. These authors were unclear as to the physiological significance of Tif4631p nuclear shuttling and its interaction with Cbp1p, although they suggested Tif4631p could itself be a component of nuclear decay. Consistent with this speculation, we demonstrate Tif4631p is a component of DRN and functions in the rapid decay of special mRNAs as well as global mRNAs trapped within the nucleus. Furthermore, Tif4631p has been shown to interact with Cbc1p in S. cerevisiae (Fortes et al., 2000; Gavin et al., 2002; Collins et al., 2007; Oeffinger et al., 2007) which corroborates our findings. Although the mutation (DR548/9AA) in Tif4631p disrupts its interaction with Cbc1p (Baron-Benhamou et al., 2003), we find it has no effect on DRN FEMS Yeast Res 14 (2014) 922–932

function. Furthermore, Baron-Benhamou et al. (2003) found this point mutation has no direct role on cellular growth, translation, and composition of the transcriptome. Collective results therefore support the conclusion that DR548/9AA mutation does not exert any observable phenotype. As mentioned above, eIF4G consists of the two isoforms, Tif4631p and Tif4632p, and both proteins have a common function as evidenced by the lethal double mutant, tif4631-Dtif4632-D (Goyer et al., 1993). Although both isoforms have similar cellular distributions in HeLa cells (McKendrick et al., 2001), a yeast tif4631-D strain displays slow-growth and a cold-sensitive phenotype, whereas the tif4632-D strain has no detectable phenotype (Goyer et al., 1993). Our results indicate both isoforms ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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are functionally active in nuclear mRNA decay, as deletion strains of each isoform displayed stabilization of test RNAs comparable to cbc1-D or rrp6-D deletion. Nonsense-mediated decay (NMD) operates in the cytoplasm in a translation-dependent manner to selectively eliminate mRNAs carrying a PTC. Evidence suggests a surveillance complex is assembled as soon as the translating ribosome stalls, upon encountering a PTC (Mitchell & Tollervey, 2003; Maquat, 2004; Parker, 2012). Interestingly, we find Upf3p, a critical component of the NMD surveillance complex, is a functional component of DRN. Although Upf3p is localized in the cytoplasm in association with polyribosomes (Atkin et al., 1995, 1997), it can enter the nucleus by virtue of its bipartite nuclear localization signal (Shirley et al., 1998). Although mammalian orthologs of yeast Upf3p (i.e. Upf3X and Upf3) were shown to distribute to the nucleus and get deposited onto the spliced transcript in a splicing-dependent manner to form exon–exon junction complex (EJC) (Serin et al., 2001; Maquat, 2004), the significance of nuclear distribution of Upf3p in yeast has remained unclear. Our finding of the involvement of Upf3p in nuclear decay of both aberrant and special mRNAs demonstrates an additional function of for this protein and further explains the physiological significance of its nuclear shuttling in yeast. In summary, we identify additional functional components of DRN, including Cbc2p (a component of nuclear CBC), Upf3p (a component of NMD) and Tif4631p and 32p (both isoforms of eIF4G). Our findings account for previously observed nuclear shuttling of proteins Tif4631p and Tif4632p as well as Upf3p. Moreover, it implied an important link between nuclear quality control of the transcriptome and their cytoplasmic fate of mRNAs in processes such as – translation and/or NMD. Considering the functional coupling between the various steps of mRNA metabolism, these results further define (1) involvement of cytoplasmic factors in nuclear surveillance of mRNA; and (2) functional links between nuclear and cytoplasmic events in an mRNA’s life.

Acknowledgements We gratefully acknowledge the advice of the late Prof. Fred Sherman (University of Rochester, NY USA) in designing the suppressor screening and Prof. David Culp (University of Florida, USA), Prof. Scott Butler (University of Rochester, USA) and Prof. Uma Dasgupta (Jadavpur University, India) for critically reading the manuscript. Assistance of Dr Qinshan Gao (Mount Sinai Medical School, NY, USA) in constructing the cbc2 disruptor plasmid is acknowledged. Thiolutin was a generous gift from Dr Edward Pagani (Pfizer Inc., Groton, CT, USA). We thank Dr Matthias Hentze (EMBL, Heidelberg, Germany) for ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

tif4631-DR548/9AA mutant strains and plasmids. This investigation was supported from research grants from CSIR (Ref. No. 38/1280/11/EMR-II), DST (File No. SR/SO/ BB/0066/2012) and Jadavpur University (to B.D.). S.D. is supported by a Women’s Scientist Scheme from DST (File No. SR/WOS-A/LS-258/2010) and U.S. is a Junior Research Fellow of UGC (Ref. No19-12/2010(i) EU-IV).

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Yeast strains used in this study. Table S2. Plasmids used in this study. Table S3. Oligonucleotides used in this study.

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