Gene 544 (2014) 236–240
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
RNA Polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes Andreas R. Gruber ⁎ Computational and Systems Biology, Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland Swiss Institute of Bioinformatics, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland
a r t i c l e
i n f o
Article history: Received 14 March 2014 Received in revised form 25 April 2014 Accepted 28 April 2014 Available online 30 April 2014 Keywords: Promoter elements Noncoding RNA RNA Polymerase III Caenorhabditis elegans
a b s t r a c t RNA Polymerase III is a highly specialized enzyme complex responsible for the transcription of a very distinct set of housekeeping noncoding RNAs including tRNAs, 7SK snRNA, Y RNAs, U6 snRNA, and the RNA components of RNaseP and RNaseMRP. In this work we have utilized the conserved promoter structure of known RNA Polymerase III transcripts consisting of characteristic sequence elements termed proximal sequence elements (PSE) A and B and a TATA-box to uncover a novel RNA Polymerase III-transcribed, noncoding RNA family found to be conserved in Caenorhabditis as well as other clade V nematode species. Homology search in combination with detailed sequence and secondary structure analysis revealed that members of this novel ncRNA family evolve rapidly, and only maintain a potentially functional small stem structure that links the 5′ end to the very 3′ end of the transcript and a small hairpin structure at the 3′ end. This is most likely required for efficient transcription termination. In addition, our study revealed evidence that canonical C/D box snoRNAs are also transcribed from a PSE A–PSE B–TATA-box promoter in Caenorhabditis elegans. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In metazoa, transcription of RNA from its DNA template is orchestrated by three different complexes termed RNA polymerases (Pols) I, II, and III. While RNA Pol II handles both the transcription of mRNAs and noncoding RNAs, RNA Pol I and RNA Pol III have so far only been found to transcribe noncoding RNAs. Many of these latter RNA molecules are considered “housekeeping genes” because of their crucial roles in cellular processes such as splicing and protein synthesis. Recent studies have demonstrated that the repertoire of genes transcribed by RNA Pol III is much larger than previously anticipated (see a recent review and the references therein (White, 2011)), but the bulk of RNA Pol III transcripts is constituted by a small set of highly expressed noncoding, structured RNAs including 5S RNA, tRNAs, Y RNAs, 7SK snRNA, U6 snRNA, and the RNA components of RNaseP and RNaseMRP (Dieci et al., 2007). While transcription of 5S RNA and tRNAs is driven by internal promoter elements, the other RNA Pol III transcripts have been shown to rely on upstream promoter elements. In Caenorhabditis elegans the structure of the external RNA Pol III promoter is well
Abbreviations: ncRNA, noncoding RNA; Pol, Polymerase; PSE, proximal sequence element; PWM, position weight matrix; nt, nucleotide. ⁎ Computational and Systems Biology, Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.gene.2014.04.068 0378-1119/© 2014 Elsevier B.V. All rights reserved.
characterized (Boria et al., 2010; Li et al., 2008) and can be decomposed into the proximal sequence elements (PSE) A and B and the TATA-box. Stretches of four or more T residues (U residues on the RNA level) render the RNA Pol III transcription complex inactive, leading to its release and termination of transcription (Nielsen et al., 2013). Previously, we have used a computational RNA Pol III promoter screen to identify the 7SK snRNA gene in Arthropod species (Gruber et al., 2008a), which conventional strategies had failed to pick up (Gruber et al., 2008b). In this current work, we applied a computational RNA Pol III promoter screen to the genome of C. elegans and uncovered a novel noncoding RNA (ncRNA) family conserved in Caenorhabditis species as well as other clade V nematodes. Analysis of the conserved sequence and structural features suggests that this novel noncoding RNA evolves rapidly, maintaining a few small, potentially functionally important base-paired regions. 2. Materials and methods 2.1. Sequence data and promoter screen In general, genome sequences were retrieved from WormBase (version WS241, www.wormbase.org). The genome sequence of Pristionchus exspectatus was obtained from www.pristionchus.org/ variome (Rödelsperger et al., 2014). Position weight matrices (PWMs) for the PSE A, PSE B, and the TATA-box regions were generated by aligning the sequences of the 100 nucleotides (nt) upstream regions of the
A.R. Gruber / Gene 544 (2014) 236–240
following eleven RNA Pol III-transcribed genes: RNAseP RNA gene (I:13,463,955–13,464,207 (+)), the RNAseMRP RNA gene (II:7,200,233– 7,200,418 (+)), and nine U6 snRNA gene loci (III:5,080,797– 5,080,898 (−), III:9,445,911–9,446,012 (+), III:9,447,758– 9,447,859 (−), III:9,449,499–9,449,545 (+), III:10,989,679– 10,989,780 (+), IV:4,864,634–4,864,735 (−), IV:4,866,811–4,866,912 (+), IV:4,885,548–4,885,649 (−), IV:4,930,954–4,931,055 (+)). fragrep version 2 was then used to screen the C. elegans genome for matches to the inferred promoter elements (Mosig et al., 2006). 2.2. Expression analysis from RNA-seq data Publicly available RNA-seq data from total RNA extracted from N2 mixed-stage embryos (Ikegami and Lieb, 2013) was obtained from NCBI GEO (GSM1048425, www.ncbi.nlm.nih.gov/geo) and mapped to the C. elegans genome using segemehl version 0.1.4-397 (Hoffmann et al., 2009). The read count for sequences that mapped to multiple loci in the genome was weighted by the number of loci the sequences were mapped to. 2.3. Homology search and characterization of conserved structure and sequence elements Standard nucleotide BLAST search was used to screen for homologs in the genomes of nematode species. In order to recover highly diverged homologs, multiple sequence alignments of the identified candidate sequences were generated using muscle (Edgar, 2004) and position weight matrices of conserved sequence elements were constructed. fragrep was then used to screen the genomes of the remaining species for matches to the inferred PWMs. In addition, a covariance model was built from the multiple sequence alignment and cmsearch from the Infernal package (Nawrocki et al., 2009) was used to screen for additional candidate ncRNAs. To obtain a sequence/structure model for the newly identified ncRNA family, sequences detected in C. elegans, Caenorhabditis remanei, and Caenorhabditis japonica were first aligned using LocARNA (Smith et al., 2010). An Infernal sequence/structure model was then built based on the consensus structure prediction obtained with RNAalifold (Bernhart et al., 2008). Sequences of the other detected family members were then added using the program cmalign from the Infernal package. 3. Results In order to detect novel noncoding RNAs in the C. elegans genome that are potentially transcribed by RNA Pol III, we first extracted the 100 nucleotide upstream regions of eleven RNA Pol III-transcribed genes including the RNA genes of RNase P and RNaseMRP, and several U6 snRNA loci. The RNA Pol III promoter structure of these ncRNAs is well characterized in C. elegans (Boria et al., 2010; Li et al., 2008), and we used this knowledge to infer position weight matrices (PWMs) for the PSE A, PSE B, and TATA-box regions (Fig. 1A). Next, a pattern search algorithm that not only evaluates matches to the PWMs but also considers distance constraints between the spacing of the elements was used to screen the C. elegans genome. In addition to the eleven genomic loci that were used to construct the search pattern four new, putative promoter regions could be uncovered (denoted as P1–4, Fig. 1B). In order to identify potential noncoding RNAs that are transcribed from these loci, we screened the 500 nt downstream region of these newly discovered promoter elements for a stretch of four or more consecutive T residues that could serve as a termination signal for the RNA Pol III transcription complex and thus defines the 3′ end of the corresponding transcript. For each of the four genomic loci, potential transcripts ranging in length from 79 to 318 nt could be identified. Intersection with existing gene annotation from WormBase revealed that the transcripts originating from loci P3 and P4 each overlap with C/D box snoRNA genes (WormBase entries W05B2.9 and ZK994.7). It is a well established fact that many snoRNAs are transcribed by RNA Pol III in
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C. elegans (Dieci et al., 2007; Ikegami and Lieb, 2013), but so far only promoter elements that resemble A box and B box motifs of the internal tRNA promoter have been identified to drive the expression of canonical snoRNAs. Next, our efforts focused on the characterization of the transcripts originating from loci P1 and P2. WormBase entries C38C6.7 and C38C6.8, which are listed as probable noncoding RNAs of unknown function, partially cover regions of the inferred transcripts (see red bars in Fig. 1C). More evidence that the two inferred transcripts are indeed noncoding RNAs was provided by screening with the Coding Potential Calculator (http://cpc.cbi.pku.edu.cn) that clearly classified both transcripts as “noncoding” (both CP scores b −1.5) (Kong et al., 2007). Compared to other ncRNA genes transcribed by RNA Pol III in C. elegans like U6 snRNA with a length of 102 nt or even RNaseP with a length of 252 nt, the length of 300 nt of the two inferred transcripts is outstanding. Intersection with data obtained from a recent experimental noncoding RNA screen in C. elegans (Xiao et al., 2012) confirms the predicted length of the transcript originating from the locus P1. Although Xiao and colleagues noted that the WormBase entry C38C6.7 constituted only a truncated version of the probable noncoding RNA, no attempts were further made to characterize the noncoding RNA in terms of structural domains or promoter region. BLAST search against Rfam (rfam.sanger.ac.uk), a database of known noncoding RNAs, and the nucleotide database at NCBI did not reveal any hit to known noncoding RNAs. Sequence analysis of the transcripts derived from loci P1 and P2 revealed that they share more than 98% sequence identity. This strongly suggests that the two ncRNAs are paralogs and constitute a novel RNA family. Next, we used publicly available RNA-seq data prepared from total RNA from C. elegans embryos (Ikegami and Lieb, 2013) to demonstrate that the two inferred noncoding RNAs are indeed expressed. Fig. 1C depicts the RNA-seq read per base coverage for the RNA genes of RNaseP and RNaseMRP, and the two inferred noncoding RNAs. This data provides clear evidence that the two proposed ncRNAs are abundantly expressed, at levels comparable to those of the RNAseP and RNAseMRP RNA genes. In an effort to further characterize and infer potential functional domains of this novel ncRNA class, we conducted homology search in other nematode genomes. While homologs in closely related species could easily be uncovered by nucleotide BLAST searches, this strategy failed on distantly related species. A multiple sequence alignment of family members identified thus far (CE1, CE2, CN1, CN2 — see Table 1) was generated and PWMs for highly conserved sequence elements were constructed. Additionally, we used the multiple sequence alignment and consensus structure prediction (Bernhart et al., 2008) to build a sequence/structure model for a refined search. The pattern matching program fragrep (Mosig et al., 2006) and cmsearch from the Infernal toolbox (Nawrocki et al., 2009) were then used to screen the remaining genomes for matches. Newly identified sequences were added to the multiple sequence alignment and the process was repeated. In this manner, we could identify members of this novel ncRNA class in several Caenorhabditis species as well as other clade V species such as Pristionchus pacificus (Table 1). No hits were recovered for nematode species outside of the clade V branch. Except for Caenorhabditis species 5 and Haemonchus contortus, multiple genomic loci of this new ncRNA family could be detected in each species. Members of this new ncRNA family vary considerably in length, ranging from 282 nt in P. pacificus to 461 nt in Caenorhabditis briggsae (Table 1) and show only weak conservation at the sequence level in pairwise sequence comparisons (Fig. 1D). Such high divergence at the nucleotide level makes it particularly difficult to construct accurate multiple sequence alignments and to identify conserved structural features. We therefore first constructed a multiple sequence alignment with a limited set of sequences using an alignment algorithm that considers both sequence identity and RNA secondary structure conservation (Smith et al., 2010) and then used the Infernal toolbox to align the remaining sequences (Nawrocki et al., 2009). The inferred consensus secondary structure model is depicted in Fig. 1E. In general, only a few
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Fig. 1. Results of the computational RNA Pol III promoter screen and subsequent homology search. (A) Sequence logos of the promoter structure of known RNA Pol III transcripts used to scan the C. elegans genome. (B) Nucleotide sequences of the four newly discovered RNA Pol III promoter loci in the C. elegans genome. See (A) for a comparison with established RNA Pol III promoter loci. (C) Per base coverage of RNA-seq reads obtained from total RNA of C. elegans embryos for two known RNA Pol III transcripts (RNaseP, RNaseMRP) and the two newly identified ncRNAs originating from genomic loci P1 and P2. The black line at the bottom of each panel represents the inferred full-length transcript in 5′ to 3′ direction. The red line represents gene annotation from WormBase. (D) Matrix representation of percent sequence identity obtained from pairwise alignments generated with muscle. For explanation of labels see Table 1. Copies of the ncRNA in the same species are highly identical, while inter-species comparison shows only moderate values. (E) Visualization of the derived consensus secondary structure model from selected ncRNA family members in Caenorhabditis species (excluding CJ1, CJ2, CA1, and CA2). The gray box represents the model inferred including all family members. R2R was used to generate the secondary structure visualizations (Weinberg and Breaker, 2011). (F) Nucleotide sequences of the inferred promoter elements of the identified novel ncRNAs. Nucleotide sequences upstream of ncRNA loci C5 and CJ2 could not be retrieved from the corresponding contig in sufficient length. For H. contortus, P. pacificus and P. exspectatus the corresponding regions from other RNA Pol III-transcribed ncRNAs are included for comparison (HCU6: scaffold8533:10,176–10,269 (+), PPP: Ppa_Contig1:55,159–55,420 (−), PEP: scaffold718:49,799–50,050 (−), PPU6: Ppa_Contig83:488,148–488,240 (−), PEU6: scaffold92:102,617–102,709 (+)). The ‘~’ character denotes a region of 13–14 nt.
A.R. Gruber / Gene 544 (2014) 236–240 Table 1 Novel ncRNA loci identified in clade V nematodes. ID
Species
Location
Length (nt)
CE1 CE2 CB1 CB2 C5 CR1 CR2 CR3 CN1 CN2 CN3 C111 C112 C113 CJ1 CJ2 CA1 CA2 HC1 PP1 PP2 PE1 PE2
C. elegans C. elegans C. briggsae C. briggsae C. species 5 C. remanei C. remanei C. remanei C. brenneri C. brenneri C. brenneri C. species 11 C. species 11 C. species 11 C. japonica C. japonica C. angaria C. angaria H. contortus P. pacificus P. pacificus P.exspectatus P.exspectatus
II:14,641,314–14,641,615 (−) II:14,635,795–14,636,093 (−) II:4,369,112–4,369,571 (−) II:4,441,127–4,441,587 (+) Csp5_scaffold_07862:64–514 (+) Crem_Contig39:353,992–354,419 (−) Crem_Contig39:382,190–382,619 (−) Crem_Contig1:3,172,995–3,173,312 (+) Cbre_Contig340:62,653–62,967 (−) Cbre_Contig60:322,822–323,138 (−) Cbre_Contig239:159,626–159,894 (−) Scaffold630:13,081,031–13,081,390 (−) Scaffold630:13,144,364–13,144,723 (+) Scaffold629:15,452,009–15,452,280 (+) Cjap.Contig17560:8974–9267 (−) Cjap.Contig2137:1059–1354 (−) Cang_2012_03_13_00152:27,009–27,273 (+) Cang_2012_03_13_00367:75,363–75,608 (+) Scaffold6670:4151–4352 (+) Ppa_Contig87:141,150–141,431 (−) Ppa_Contig85:295,111–295,393 (−) Scaffold65:142,038–142,311 (−) Scaffold159:16,733–17,014 (+)
302 299 460 461 451 428 430 318 315 317 269 360 360 272 294 296 265 246 202 282 283 274 282
sequence and structural features are conserved even when comparing a selected set of genes from Caenorhabditis species only. In particular, the very 5′ end of the ncRNAs forms a conserved stem with a region in close proximity to the 3′ end. In the loop formed by this stem, we could further identify a small conserved stem and sequence stretches. In close proximity to the terminator sequence we found a small hairpin structure that might serve as a structural motif to ensure efficient transcription termination (Nielsen et al., 2013). When the analysis was extended to all uncovered ncRNA family members, only the 5′-to-3′ stem structure and the small stem structure likely to be required for efficient transcription termination remain as conserved features (Fig. 1E gray box). Moreover, the small termination stem is only conserved at the structural level and not at the sequence level. Finally, we were interested in whether the expression of the newly identified ncRNAs is also driven by RNA Pol III promoter elements in species other than C. elegans. Fig. 1F depicts the inferred PSE A, PSE B, and TATA-box regions for the ncRNA family members where an upstream promoter region could be inferred. For H. contortus, P. pacificus, and P. exspectatus, the corresponding regions from other RNA Pol III transcripts in the same species are included for comparison. Each of the ncRNA family members shows clear characteristics of a conserved PSE B–PSE A–TATA-box promoter. This high conservation of the promoter elements and the conserved stretch of T residues at the 3′ end of the ncRNA genes suggests that the newly discovered ncRNAs are bona fide RNA Pol III transcripts in all species investigated. 4. Discussion In this work we used a computational screen for RNA Pol III promoter elements in C. elegans to uncover a novel ncRNA family conserved in Caenorhabditis, as well as other clade V nematodes. We initially started with a stringent set of promoter regions of known RNA Pol III-transcribed genes, and identified four new, high-confidence RNA Pol III promoter regions. It has to be noted, that inclusion of the newly identified promoter regions as well as promoter regions of yet other RNA Pol III transcripts such as Y RNAs or tRNA-SeC genes would help to uncover an even greater number of potential RNA Pol III-transcribed loci. However, with the addition of more diverged promoter elements the search becomes less sensitive and experimental evidence in the form of a RNA Pol III ChiP-seq (see e. g. Ikegami and Lieb (2013) for a publicly available data set) would be needed to validate the huge number of predicted loci
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that would surely emerge. In this work, two of the four newly discovered RNA Pol III promoter loci seem to be responsible for the transcription of two C/D box snoRNA genes, while the two other loci P1 and P2 for the transcription of members of the newly characterized ncRNA family. Even though parts of the two noncoding RNA genes originating from loci P1 and P2 in the C. elegans genome were previously labeled as potential ncRNAs (C. elegans Sequencing Consortium, 1998), this work defines the full-length transcript boundaries and showed clear evidence that members of this ncRNA family are bona fide RNA Pol III transcripts. In C. elegans embryos, expression levels of the two novel ncRNA genes originating from loci P1 and P2 are comparable to those of the housekeeping ncRNAs RNaseP and RNaseMRP as demonstrated on publicly available RNA-seq data (Ikegami and Lieb, 2013). Despite this high level of expression, it is the unusual length of more than 300 nt for a RNA Pol III-transcribed noncoding RNA and its rapid evolution that most likely allowed it to slip through previous experimental as well as computational ncRNA screens. Indeed, the rate at which members of this ncRNA family evolve is yet another demonstration why the computational detection of ncRNAs is a particularly difficult problem (Menzel et al., 2009). The most conserved feature is a short stem structure with the 5′ part being separated more than 200 nt from the 3′ part of the stem. Since this long range interaction of base-pairs is the only truly conserved feature both at the sequence and the structural levels, it renders a case like this almost impossible to be picked up by computational ncRNA gene finders that rely on the prediction of local, conserved structural features (Gruber et al., 2010; Pedersen et al., 2006). Structural features that also show a high degree of conservation at the nucleotide level are often binding regions for protein complexes, as it is the case for many small ncRNAs such as the 7SK snRNA or Y RNAs (Boria et al., 2010; Gruber et al., 2008a). These protein–RNA complexes are not only needed to ultimately execute the function of the complex, as for example for the 7SK-protein or snoRNA–protein complexes, but also to protect the RNA component from degradation. The fact that the set of RNA Pol III-transcribed genes consists of many housekeeping genes with essential roles in the metabolism of cells suggests that this novel ncRNA family might also be in control of important cellular pathways. Identification of the potential protein binding partners would be a first step to functionally characterize this ncRNA family. Conflict of interest The authors declare no conflict of interest. Acknowledgments I would like to thank Peter F. Stalder for his support on the pre-study of this work and Alexander Kanitz and Aaron Grandy for comments on the manuscript. The work was supported by the Swiss National Science Foundation (grant no. 31003A-143977 to Walter Keller). References Bernhart, S.H., Hofacker, I.L., Will, S., Gruber, A.R., Stadler, P.F., 2008. RNAalifold: improved consensus structure prediction for RNA alignments. BMC Bioinformatics 9, 474. Boria, I., Gruber, A.R., Tanzer, A., Bernhart, S.H., Lorenz, R., Mueller, M.M., Hofacker, I.L., Stadler, P.F., 2010. Nematode sbRNAs: homologs of vertebrate Y RNAs. Journal of Molecular Evolution 70, 346–358. C. elegans Sequencing Consortium, 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018. Dieci, G., Fiorino, G., Castelnuovo, M., Teichmann, M., Pagano, A., 2007. The expanding RNA polymerase III transcriptome. Trends in Genetics 23, 614–622. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 1792–1797. Gruber, A.R., Kilgus, C., Mosig, A., Hofacker, I.L., Hennig, W., Stadler, P.F., 2008a. Arthropod 7SK RNA. Molecular Biology and Evolution 25, 1923–1930. Gruber, A.R., Koper-Emde, D., Marz, M., Tafer, H., Bernhart, S., Obernosterer, G., Mosig, A., Hofacker, I.L., Stadler, P.F., Benecke, B.-J., 2008b. Invertebrate 7SK snRNAs. Journal of Molecular Evolution 66, 107–115. Gruber, A.R., Findeiß, S., Washietl, S., Hofacker, I.L., Stadler, P.F., 2010. RNAz 2.0: improved noncoding RNA detection. Pac. Symp. Biocomput, pp. 69–79.
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Hoffmann, S., Otto, C., Kurtz, S., Sharma, C.M., Khaitovich, P., Vogel, J., Stadler, P.F., Hackermüller, J., 2009. Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS Computational Biology 5, e1000502. Ikegami, K., Lieb, J.D., 2013. Integral nuclear pore proteins bind to Pol III-transcribed genes and are required for Pol III transcript processing in C. elegans. Molecular Cell 51, 840–849. Kong, L., Zhang, Y., Ye, Z.-Q., Liu, X.-Q., Zhao, S.-Q., Wei, L., Gao, G., 2007. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Research 35, W345–W349. Li, T., He, H., Wang, Y., Zheng, H., Skogerbo, G., Chen, R., 2008. In vivo analysis of Caenorhabditis elegans noncoding RNA promoter motifs. BMC Molecular Biology 9, 71. Menzel, P., Gorodkin, J., Stadler, P.F., 2009. The tedious task of finding homologous noncoding RNA genes. RNA 15, 2075–2082. Mosig, A., Sameith, K., Stadler, P., 2006. Fragrep: an efficient search tool for fragmented patterns in genomic sequences. Genomics, Proteomics & Bioinformatics 4, 56–60. Nawrocki, E.P., Kolbe, D.L., Eddy, S.R., 2009. Infernal 1.0: inference of RNA alignments. Bioinformatics 25, 1335–1337. Nielsen, S., Yuzenkova, Y., Zenkin, N., 2013. Mechanism of eukaryotic RNA polymerase III transcription termination. Science 340, 1577–1580.
Pedersen, J.S., Bejerano, G., Siepel, A., Rosenbloom, K., Lindblad-Toh, K., Lander, E.S., Kent, J., Miller, W., Haussler, D., 2006. Identification and classification of conserved RNA secondary structures in the human genome. PLoS Computational Biology 2, e33. Rödelsperger, C., Neher, R.A., Weller, A.M., Eberhardt, G., Witte, H., Mayer, W.E., Dieterich, C., Sommer, R.J., 2014. Characterization of genetic diversity in the nematode Pristionchus pacificus from population-scale resequencing data. Genetics 196, 1153–1165. Smith, C., Heyne, S., Richter, A.S., Will, S., Backofen, R., 2010. Freiburg RNA Tools: a web server integrating INTARNA, EXPARNA and LOCARNA. Nucleic Acids Research 38, W373–W377. Weinberg, Z., Breaker, R.R., 2011. R2R—software to speed the depiction of aesthetic consensus RNA secondary structures. BMC Bioinformatics 12, 3. White, R.J., 2011. Transcription by RNA polymerase III: more complex than we thought. Nature Reviews. Genetics 12, 459–463. Xiao, T., Wang, Y., Luo, H., Liu, L., Wei, G., Chen, Xiaowei, Sun, Y., Chen, Xiaomin, Skogerbø, G., Chen, R., 2012. A differential sequencing-based analysis of the C. elegans noncoding transcriptome. RNA 18, 626–639.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
RNA Polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes Andreas R. Gruber ⁎ Computational and Systems Biology, Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland Swiss Institute of Bioinformatics, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland
a r t i c l e
i n f o
Article history: Received 14 March 2014 Received in revised form 25 April 2014 Accepted 28 April 2014 Available online 30 April 2014 Keywords: Promoter elements Noncoding RNA RNA Polymerase III Caenorhabditis elegans
a b s t r a c t RNA Polymerase III is a highly specialized enzyme complex responsible for the transcription of a very distinct set of housekeeping noncoding RNAs including tRNAs, 7SK snRNA, Y RNAs, U6 snRNA, and the RNA components of RNaseP and RNaseMRP. In this work we have utilized the conserved promoter structure of known RNA Polymerase III transcripts consisting of characteristic sequence elements termed proximal sequence elements (PSE) A and B and a TATA-box to uncover a novel RNA Polymerase III-transcribed, noncoding RNA family found to be conserved in Caenorhabditis as well as other clade V nematode species. Homology search in combination with detailed sequence and secondary structure analysis revealed that members of this novel ncRNA family evolve rapidly, and only maintain a potentially functional small stem structure that links the 5′ end to the very 3′ end of the transcript and a small hairpin structure at the 3′ end. This is most likely required for efficient transcription termination. In addition, our study revealed evidence that canonical C/D box snoRNAs are also transcribed from a PSE A–PSE B–TATA-box promoter in Caenorhabditis elegans. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In metazoa, transcription of RNA from its DNA template is orchestrated by three different complexes termed RNA polymerases (Pols) I, II, and III. While RNA Pol II handles both the transcription of mRNAs and noncoding RNAs, RNA Pol I and RNA Pol III have so far only been found to transcribe noncoding RNAs. Many of these latter RNA molecules are considered “housekeeping genes” because of their crucial roles in cellular processes such as splicing and protein synthesis. Recent studies have demonstrated that the repertoire of genes transcribed by RNA Pol III is much larger than previously anticipated (see a recent review and the references therein (White, 2011)), but the bulk of RNA Pol III transcripts is constituted by a small set of highly expressed noncoding, structured RNAs including 5S RNA, tRNAs, Y RNAs, 7SK snRNA, U6 snRNA, and the RNA components of RNaseP and RNaseMRP (Dieci et al., 2007). While transcription of 5S RNA and tRNAs is driven by internal promoter elements, the other RNA Pol III transcripts have been shown to rely on upstream promoter elements. In Caenorhabditis elegans the structure of the external RNA Pol III promoter is well
Abbreviations: ncRNA, noncoding RNA; Pol, Polymerase; PSE, proximal sequence element; PWM, position weight matrix; nt, nucleotide. ⁎ Computational and Systems Biology, Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland. E-mail address: [email protected].
http://dx.doi.org/10.1016/j.gene.2014.04.068 0378-1119/© 2014 Elsevier B.V. All rights reserved.
characterized (Boria et al., 2010; Li et al., 2008) and can be decomposed into the proximal sequence elements (PSE) A and B and the TATA-box. Stretches of four or more T residues (U residues on the RNA level) render the RNA Pol III transcription complex inactive, leading to its release and termination of transcription (Nielsen et al., 2013). Previously, we have used a computational RNA Pol III promoter screen to identify the 7SK snRNA gene in Arthropod species (Gruber et al., 2008a), which conventional strategies had failed to pick up (Gruber et al., 2008b). In this current work, we applied a computational RNA Pol III promoter screen to the genome of C. elegans and uncovered a novel noncoding RNA (ncRNA) family conserved in Caenorhabditis species as well as other clade V nematodes. Analysis of the conserved sequence and structural features suggests that this novel noncoding RNA evolves rapidly, maintaining a few small, potentially functionally important base-paired regions. 2. Materials and methods 2.1. Sequence data and promoter screen In general, genome sequences were retrieved from WormBase (version WS241, www.wormbase.org). The genome sequence of Pristionchus exspectatus was obtained from www.pristionchus.org/ variome (Rödelsperger et al., 2014). Position weight matrices (PWMs) for the PSE A, PSE B, and the TATA-box regions were generated by aligning the sequences of the 100 nucleotides (nt) upstream regions of the
A.R. Gruber / Gene 544 (2014) 236–240
following eleven RNA Pol III-transcribed genes: RNAseP RNA gene (I:13,463,955–13,464,207 (+)), the RNAseMRP RNA gene (II:7,200,233– 7,200,418 (+)), and nine U6 snRNA gene loci (III:5,080,797– 5,080,898 (−), III:9,445,911–9,446,012 (+), III:9,447,758– 9,447,859 (−), III:9,449,499–9,449,545 (+), III:10,989,679– 10,989,780 (+), IV:4,864,634–4,864,735 (−), IV:4,866,811–4,866,912 (+), IV:4,885,548–4,885,649 (−), IV:4,930,954–4,931,055 (+)). fragrep version 2 was then used to screen the C. elegans genome for matches to the inferred promoter elements (Mosig et al., 2006). 2.2. Expression analysis from RNA-seq data Publicly available RNA-seq data from total RNA extracted from N2 mixed-stage embryos (Ikegami and Lieb, 2013) was obtained from NCBI GEO (GSM1048425, www.ncbi.nlm.nih.gov/geo) and mapped to the C. elegans genome using segemehl version 0.1.4-397 (Hoffmann et al., 2009). The read count for sequences that mapped to multiple loci in the genome was weighted by the number of loci the sequences were mapped to. 2.3. Homology search and characterization of conserved structure and sequence elements Standard nucleotide BLAST search was used to screen for homologs in the genomes of nematode species. In order to recover highly diverged homologs, multiple sequence alignments of the identified candidate sequences were generated using muscle (Edgar, 2004) and position weight matrices of conserved sequence elements were constructed. fragrep was then used to screen the genomes of the remaining species for matches to the inferred PWMs. In addition, a covariance model was built from the multiple sequence alignment and cmsearch from the Infernal package (Nawrocki et al., 2009) was used to screen for additional candidate ncRNAs. To obtain a sequence/structure model for the newly identified ncRNA family, sequences detected in C. elegans, Caenorhabditis remanei, and Caenorhabditis japonica were first aligned using LocARNA (Smith et al., 2010). An Infernal sequence/structure model was then built based on the consensus structure prediction obtained with RNAalifold (Bernhart et al., 2008). Sequences of the other detected family members were then added using the program cmalign from the Infernal package. 3. Results In order to detect novel noncoding RNAs in the C. elegans genome that are potentially transcribed by RNA Pol III, we first extracted the 100 nucleotide upstream regions of eleven RNA Pol III-transcribed genes including the RNA genes of RNase P and RNaseMRP, and several U6 snRNA loci. The RNA Pol III promoter structure of these ncRNAs is well characterized in C. elegans (Boria et al., 2010; Li et al., 2008), and we used this knowledge to infer position weight matrices (PWMs) for the PSE A, PSE B, and TATA-box regions (Fig. 1A). Next, a pattern search algorithm that not only evaluates matches to the PWMs but also considers distance constraints between the spacing of the elements was used to screen the C. elegans genome. In addition to the eleven genomic loci that were used to construct the search pattern four new, putative promoter regions could be uncovered (denoted as P1–4, Fig. 1B). In order to identify potential noncoding RNAs that are transcribed from these loci, we screened the 500 nt downstream region of these newly discovered promoter elements for a stretch of four or more consecutive T residues that could serve as a termination signal for the RNA Pol III transcription complex and thus defines the 3′ end of the corresponding transcript. For each of the four genomic loci, potential transcripts ranging in length from 79 to 318 nt could be identified. Intersection with existing gene annotation from WormBase revealed that the transcripts originating from loci P3 and P4 each overlap with C/D box snoRNA genes (WormBase entries W05B2.9 and ZK994.7). It is a well established fact that many snoRNAs are transcribed by RNA Pol III in
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C. elegans (Dieci et al., 2007; Ikegami and Lieb, 2013), but so far only promoter elements that resemble A box and B box motifs of the internal tRNA promoter have been identified to drive the expression of canonical snoRNAs. Next, our efforts focused on the characterization of the transcripts originating from loci P1 and P2. WormBase entries C38C6.7 and C38C6.8, which are listed as probable noncoding RNAs of unknown function, partially cover regions of the inferred transcripts (see red bars in Fig. 1C). More evidence that the two inferred transcripts are indeed noncoding RNAs was provided by screening with the Coding Potential Calculator (http://cpc.cbi.pku.edu.cn) that clearly classified both transcripts as “noncoding” (both CP scores b −1.5) (Kong et al., 2007). Compared to other ncRNA genes transcribed by RNA Pol III in C. elegans like U6 snRNA with a length of 102 nt or even RNaseP with a length of 252 nt, the length of 300 nt of the two inferred transcripts is outstanding. Intersection with data obtained from a recent experimental noncoding RNA screen in C. elegans (Xiao et al., 2012) confirms the predicted length of the transcript originating from the locus P1. Although Xiao and colleagues noted that the WormBase entry C38C6.7 constituted only a truncated version of the probable noncoding RNA, no attempts were further made to characterize the noncoding RNA in terms of structural domains or promoter region. BLAST search against Rfam (rfam.sanger.ac.uk), a database of known noncoding RNAs, and the nucleotide database at NCBI did not reveal any hit to known noncoding RNAs. Sequence analysis of the transcripts derived from loci P1 and P2 revealed that they share more than 98% sequence identity. This strongly suggests that the two ncRNAs are paralogs and constitute a novel RNA family. Next, we used publicly available RNA-seq data prepared from total RNA from C. elegans embryos (Ikegami and Lieb, 2013) to demonstrate that the two inferred noncoding RNAs are indeed expressed. Fig. 1C depicts the RNA-seq read per base coverage for the RNA genes of RNaseP and RNaseMRP, and the two inferred noncoding RNAs. This data provides clear evidence that the two proposed ncRNAs are abundantly expressed, at levels comparable to those of the RNAseP and RNAseMRP RNA genes. In an effort to further characterize and infer potential functional domains of this novel ncRNA class, we conducted homology search in other nematode genomes. While homologs in closely related species could easily be uncovered by nucleotide BLAST searches, this strategy failed on distantly related species. A multiple sequence alignment of family members identified thus far (CE1, CE2, CN1, CN2 — see Table 1) was generated and PWMs for highly conserved sequence elements were constructed. Additionally, we used the multiple sequence alignment and consensus structure prediction (Bernhart et al., 2008) to build a sequence/structure model for a refined search. The pattern matching program fragrep (Mosig et al., 2006) and cmsearch from the Infernal toolbox (Nawrocki et al., 2009) were then used to screen the remaining genomes for matches. Newly identified sequences were added to the multiple sequence alignment and the process was repeated. In this manner, we could identify members of this novel ncRNA class in several Caenorhabditis species as well as other clade V species such as Pristionchus pacificus (Table 1). No hits were recovered for nematode species outside of the clade V branch. Except for Caenorhabditis species 5 and Haemonchus contortus, multiple genomic loci of this new ncRNA family could be detected in each species. Members of this new ncRNA family vary considerably in length, ranging from 282 nt in P. pacificus to 461 nt in Caenorhabditis briggsae (Table 1) and show only weak conservation at the sequence level in pairwise sequence comparisons (Fig. 1D). Such high divergence at the nucleotide level makes it particularly difficult to construct accurate multiple sequence alignments and to identify conserved structural features. We therefore first constructed a multiple sequence alignment with a limited set of sequences using an alignment algorithm that considers both sequence identity and RNA secondary structure conservation (Smith et al., 2010) and then used the Infernal toolbox to align the remaining sequences (Nawrocki et al., 2009). The inferred consensus secondary structure model is depicted in Fig. 1E. In general, only a few
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Fig. 1. Results of the computational RNA Pol III promoter screen and subsequent homology search. (A) Sequence logos of the promoter structure of known RNA Pol III transcripts used to scan the C. elegans genome. (B) Nucleotide sequences of the four newly discovered RNA Pol III promoter loci in the C. elegans genome. See (A) for a comparison with established RNA Pol III promoter loci. (C) Per base coverage of RNA-seq reads obtained from total RNA of C. elegans embryos for two known RNA Pol III transcripts (RNaseP, RNaseMRP) and the two newly identified ncRNAs originating from genomic loci P1 and P2. The black line at the bottom of each panel represents the inferred full-length transcript in 5′ to 3′ direction. The red line represents gene annotation from WormBase. (D) Matrix representation of percent sequence identity obtained from pairwise alignments generated with muscle. For explanation of labels see Table 1. Copies of the ncRNA in the same species are highly identical, while inter-species comparison shows only moderate values. (E) Visualization of the derived consensus secondary structure model from selected ncRNA family members in Caenorhabditis species (excluding CJ1, CJ2, CA1, and CA2). The gray box represents the model inferred including all family members. R2R was used to generate the secondary structure visualizations (Weinberg and Breaker, 2011). (F) Nucleotide sequences of the inferred promoter elements of the identified novel ncRNAs. Nucleotide sequences upstream of ncRNA loci C5 and CJ2 could not be retrieved from the corresponding contig in sufficient length. For H. contortus, P. pacificus and P. exspectatus the corresponding regions from other RNA Pol III-transcribed ncRNAs are included for comparison (HCU6: scaffold8533:10,176–10,269 (+), PPP: Ppa_Contig1:55,159–55,420 (−), PEP: scaffold718:49,799–50,050 (−), PPU6: Ppa_Contig83:488,148–488,240 (−), PEU6: scaffold92:102,617–102,709 (+)). The ‘~’ character denotes a region of 13–14 nt.
A.R. Gruber / Gene 544 (2014) 236–240 Table 1 Novel ncRNA loci identified in clade V nematodes. ID
Species
Location
Length (nt)
CE1 CE2 CB1 CB2 C5 CR1 CR2 CR3 CN1 CN2 CN3 C111 C112 C113 CJ1 CJ2 CA1 CA2 HC1 PP1 PP2 PE1 PE2
C. elegans C. elegans C. briggsae C. briggsae C. species 5 C. remanei C. remanei C. remanei C. brenneri C. brenneri C. brenneri C. species 11 C. species 11 C. species 11 C. japonica C. japonica C. angaria C. angaria H. contortus P. pacificus P. pacificus P.exspectatus P.exspectatus
II:14,641,314–14,641,615 (−) II:14,635,795–14,636,093 (−) II:4,369,112–4,369,571 (−) II:4,441,127–4,441,587 (+) Csp5_scaffold_07862:64–514 (+) Crem_Contig39:353,992–354,419 (−) Crem_Contig39:382,190–382,619 (−) Crem_Contig1:3,172,995–3,173,312 (+) Cbre_Contig340:62,653–62,967 (−) Cbre_Contig60:322,822–323,138 (−) Cbre_Contig239:159,626–159,894 (−) Scaffold630:13,081,031–13,081,390 (−) Scaffold630:13,144,364–13,144,723 (+) Scaffold629:15,452,009–15,452,280 (+) Cjap.Contig17560:8974–9267 (−) Cjap.Contig2137:1059–1354 (−) Cang_2012_03_13_00152:27,009–27,273 (+) Cang_2012_03_13_00367:75,363–75,608 (+) Scaffold6670:4151–4352 (+) Ppa_Contig87:141,150–141,431 (−) Ppa_Contig85:295,111–295,393 (−) Scaffold65:142,038–142,311 (−) Scaffold159:16,733–17,014 (+)
302 299 460 461 451 428 430 318 315 317 269 360 360 272 294 296 265 246 202 282 283 274 282
sequence and structural features are conserved even when comparing a selected set of genes from Caenorhabditis species only. In particular, the very 5′ end of the ncRNAs forms a conserved stem with a region in close proximity to the 3′ end. In the loop formed by this stem, we could further identify a small conserved stem and sequence stretches. In close proximity to the terminator sequence we found a small hairpin structure that might serve as a structural motif to ensure efficient transcription termination (Nielsen et al., 2013). When the analysis was extended to all uncovered ncRNA family members, only the 5′-to-3′ stem structure and the small stem structure likely to be required for efficient transcription termination remain as conserved features (Fig. 1E gray box). Moreover, the small termination stem is only conserved at the structural level and not at the sequence level. Finally, we were interested in whether the expression of the newly identified ncRNAs is also driven by RNA Pol III promoter elements in species other than C. elegans. Fig. 1F depicts the inferred PSE A, PSE B, and TATA-box regions for the ncRNA family members where an upstream promoter region could be inferred. For H. contortus, P. pacificus, and P. exspectatus, the corresponding regions from other RNA Pol III transcripts in the same species are included for comparison. Each of the ncRNA family members shows clear characteristics of a conserved PSE B–PSE A–TATA-box promoter. This high conservation of the promoter elements and the conserved stretch of T residues at the 3′ end of the ncRNA genes suggests that the newly discovered ncRNAs are bona fide RNA Pol III transcripts in all species investigated. 4. Discussion In this work we used a computational screen for RNA Pol III promoter elements in C. elegans to uncover a novel ncRNA family conserved in Caenorhabditis, as well as other clade V nematodes. We initially started with a stringent set of promoter regions of known RNA Pol III-transcribed genes, and identified four new, high-confidence RNA Pol III promoter regions. It has to be noted, that inclusion of the newly identified promoter regions as well as promoter regions of yet other RNA Pol III transcripts such as Y RNAs or tRNA-SeC genes would help to uncover an even greater number of potential RNA Pol III-transcribed loci. However, with the addition of more diverged promoter elements the search becomes less sensitive and experimental evidence in the form of a RNA Pol III ChiP-seq (see e. g. Ikegami and Lieb (2013) for a publicly available data set) would be needed to validate the huge number of predicted loci
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that would surely emerge. In this work, two of the four newly discovered RNA Pol III promoter loci seem to be responsible for the transcription of two C/D box snoRNA genes, while the two other loci P1 and P2 for the transcription of members of the newly characterized ncRNA family. Even though parts of the two noncoding RNA genes originating from loci P1 and P2 in the C. elegans genome were previously labeled as potential ncRNAs (C. elegans Sequencing Consortium, 1998), this work defines the full-length transcript boundaries and showed clear evidence that members of this ncRNA family are bona fide RNA Pol III transcripts. In C. elegans embryos, expression levels of the two novel ncRNA genes originating from loci P1 and P2 are comparable to those of the housekeeping ncRNAs RNaseP and RNaseMRP as demonstrated on publicly available RNA-seq data (Ikegami and Lieb, 2013). Despite this high level of expression, it is the unusual length of more than 300 nt for a RNA Pol III-transcribed noncoding RNA and its rapid evolution that most likely allowed it to slip through previous experimental as well as computational ncRNA screens. Indeed, the rate at which members of this ncRNA family evolve is yet another demonstration why the computational detection of ncRNAs is a particularly difficult problem (Menzel et al., 2009). The most conserved feature is a short stem structure with the 5′ part being separated more than 200 nt from the 3′ part of the stem. Since this long range interaction of base-pairs is the only truly conserved feature both at the sequence and the structural levels, it renders a case like this almost impossible to be picked up by computational ncRNA gene finders that rely on the prediction of local, conserved structural features (Gruber et al., 2010; Pedersen et al., 2006). Structural features that also show a high degree of conservation at the nucleotide level are often binding regions for protein complexes, as it is the case for many small ncRNAs such as the 7SK snRNA or Y RNAs (Boria et al., 2010; Gruber et al., 2008a). These protein–RNA complexes are not only needed to ultimately execute the function of the complex, as for example for the 7SK-protein or snoRNA–protein complexes, but also to protect the RNA component from degradation. The fact that the set of RNA Pol III-transcribed genes consists of many housekeeping genes with essential roles in the metabolism of cells suggests that this novel ncRNA family might also be in control of important cellular pathways. Identification of the potential protein binding partners would be a first step to functionally characterize this ncRNA family. Conflict of interest The authors declare no conflict of interest. Acknowledgments I would like to thank Peter F. Stalder for his support on the pre-study of this work and Alexander Kanitz and Aaron Grandy for comments on the manuscript. The work was supported by the Swiss National Science Foundation (grant no. 31003A-143977 to Walter Keller). References Bernhart, S.H., Hofacker, I.L., Will, S., Gruber, A.R., Stadler, P.F., 2008. RNAalifold: improved consensus structure prediction for RNA alignments. BMC Bioinformatics 9, 474. Boria, I., Gruber, A.R., Tanzer, A., Bernhart, S.H., Lorenz, R., Mueller, M.M., Hofacker, I.L., Stadler, P.F., 2010. Nematode sbRNAs: homologs of vertebrate Y RNAs. Journal of Molecular Evolution 70, 346–358. C. elegans Sequencing Consortium, 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018. Dieci, G., Fiorino, G., Castelnuovo, M., Teichmann, M., Pagano, A., 2007. The expanding RNA polymerase III transcriptome. Trends in Genetics 23, 614–622. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 1792–1797. Gruber, A.R., Kilgus, C., Mosig, A., Hofacker, I.L., Hennig, W., Stadler, P.F., 2008a. Arthropod 7SK RNA. Molecular Biology and Evolution 25, 1923–1930. Gruber, A.R., Koper-Emde, D., Marz, M., Tafer, H., Bernhart, S., Obernosterer, G., Mosig, A., Hofacker, I.L., Stadler, P.F., Benecke, B.-J., 2008b. Invertebrate 7SK snRNAs. Journal of Molecular Evolution 66, 107–115. Gruber, A.R., Findeiß, S., Washietl, S., Hofacker, I.L., Stadler, P.F., 2010. RNAz 2.0: improved noncoding RNA detection. Pac. Symp. Biocomput, pp. 69–79.
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A.R. Gruber / Gene 544 (2014) 236–240
Hoffmann, S., Otto, C., Kurtz, S., Sharma, C.M., Khaitovich, P., Vogel, J., Stadler, P.F., Hackermüller, J., 2009. Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS Computational Biology 5, e1000502. Ikegami, K., Lieb, J.D., 2013. Integral nuclear pore proteins bind to Pol III-transcribed genes and are required for Pol III transcript processing in C. elegans. Molecular Cell 51, 840–849. Kong, L., Zhang, Y., Ye, Z.-Q., Liu, X.-Q., Zhao, S.-Q., Wei, L., Gao, G., 2007. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Research 35, W345–W349. Li, T., He, H., Wang, Y., Zheng, H., Skogerbo, G., Chen, R., 2008. In vivo analysis of Caenorhabditis elegans noncoding RNA promoter motifs. BMC Molecular Biology 9, 71. Menzel, P., Gorodkin, J., Stadler, P.F., 2009. The tedious task of finding homologous noncoding RNA genes. RNA 15, 2075–2082. Mosig, A., Sameith, K., Stadler, P., 2006. Fragrep: an efficient search tool for fragmented patterns in genomic sequences. Genomics, Proteomics & Bioinformatics 4, 56–60. Nawrocki, E.P., Kolbe, D.L., Eddy, S.R., 2009. Infernal 1.0: inference of RNA alignments. Bioinformatics 25, 1335–1337. Nielsen, S., Yuzenkova, Y., Zenkin, N., 2013. Mechanism of eukaryotic RNA polymerase III transcription termination. Science 340, 1577–1580.
Pedersen, J.S., Bejerano, G., Siepel, A., Rosenbloom, K., Lindblad-Toh, K., Lander, E.S., Kent, J., Miller, W., Haussler, D., 2006. Identification and classification of conserved RNA secondary structures in the human genome. PLoS Computational Biology 2, e33. Rödelsperger, C., Neher, R.A., Weller, A.M., Eberhardt, G., Witte, H., Mayer, W.E., Dieterich, C., Sommer, R.J., 2014. Characterization of genetic diversity in the nematode Pristionchus pacificus from population-scale resequencing data. Genetics 196, 1153–1165. Smith, C., Heyne, S., Richter, A.S., Will, S., Backofen, R., 2010. Freiburg RNA Tools: a web server integrating INTARNA, EXPARNA and LOCARNA. Nucleic Acids Research 38, W373–W377. Weinberg, Z., Breaker, R.R., 2011. R2R—software to speed the depiction of aesthetic consensus RNA secondary structures. BMC Bioinformatics 12, 3. White, R.J., 2011. Transcription by RNA polymerase III: more complex than we thought. Nature Reviews. Genetics 12, 459–463. Xiao, T., Wang, Y., Luo, H., Liu, L., Wei, G., Chen, Xiaowei, Sun, Y., Chen, Xiaomin, Skogerbø, G., Chen, R., 2012. A differential sequencing-based analysis of the C. elegans noncoding transcriptome. RNA 18, 626–639.
