pre-piRNA biogenesis mimics the pathway of miRNA

Biochemical Systematics and Ecology 43 (2012) 200–204

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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco

pre-piRNA biogenesis mimics the pathway of miRNA Meenakshisundaram Kandhavelu a, *, Jeyalakshumi Kandhavelu b a b

A.I. Virtanen Institute for Molecular Science, University of Kuopio, 70211, Finland School of Veterinary Sciences, University of Camerino, 62024, Italy

a r t i c l e i n f o Article history: Received 1 September 2011 Accepted 18 March 2012 Available online 22 April 2012 Keywords: RNA system biology microRNA piRNA pre-piRNA snoRNA Biogenesis

1. Introduction Non-coding RNAs (ncRNAs) have an important role in several cellular processes such as replication (Christov et al., 2006), transcription (Ren, 2010), and transcriptional regulation (Goodrich and Kugel, 2006), including repression (Eddy, 2001), translation, protein degradation and translocation (Storz, 2002). It has been reported that, even after the completion of genome sequencing project, both the number and diversity of ncRNA genes remain largely unknown (Eddy, 2001). Recent studies show that ncRNAs are far more abundant and important than was initially thought. They have been identified by different methods, such as cloning and sequencing (Eddy, 2001; Mardis, 2008), identification of conserved coding exons by comparative genome analysis, and computational gene prediction (Eddy, 2002). But the information regarding ncRNAs has been restricted to biochemically abundant species and anecdotal discoveries. Lately, several different systematic screens have identified a surprisingly large number of ncRNA families, such as snoRNA (Lestrade and Weber, 2006), snRNA (Hernandez, 2001), rasiRNA, miRNA, piRNA, etc. In the past few years, miRNA research has rapidly increased. Giving importance to these ncRNAs will enable researchers to find the previously unknown regulatory pathways in the human genome (Kandhavelu et al., 2009). miRNA genes are transcribed by RNA polymerase II (or III) to generate primary transcripts (pri-miRNAs) (Lee et al., 2004). The initiation step is mediated by RNA III enzyme Drosha–DGCR8 complex (Microprocessor complex), located mainly in the nucleus (Han et al., 2004). The Dicer cleaves off the loop of the miRNA hairpin, thereby generating a short dsRNA of about 20–25 nucleotides (nt) in length (Bohnsack et al., 2004; Grishok et al., 2001; Hutvagner et al., 2001). The structure of this precursor miRNA (Pre-miRNA) might serve as a signature motif that is recognized by the nuclear export factor exportin-5 (Yi et al., 2003). This export factor and its cofactor Ran (the GTP-bound form) form a transport complex with the Pre-miRNA, which is often called a miRNPs (Bohnsack et al., 2004; Lund et al., 2004). The cytoplasmic RNase III dicer involved in the secondary dicing

* Corresponding author. Tel.: þ358 449662364; fax: þ39 0737 404002. E-mail address: meenakshisundaram.kandhavelu@tut.fi (M. Kandhavelu). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2012.03.012

M. Kandhavelu, J. Kandhavelu / Biochemical Systematics and Ecology 43 (2012) 200–204

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process to form the miRNA duplexes, i.e. one strand of the duplex is integrated into an active RNA-induced silencing complex (RISC), can be formed by thousands of nucleotides and contains multiple pre-miRNAs (Kim, 2005). It has also been reported that, on the separated duplex, one strand is selected as the mature miRNA and another strand is degraded (Kim, 2005). In some cases, however, pre-miRNAs are contained in introns of protein-coding genes and are excised by splicing. In the RNA interference (RNAi) or siRNA pathway, long dsRNA is processed by Dicer as well (Bernstein et al., 2001). The mature siRNA is incorporated into the RISC. Ender et al. experimentally showed that a class of small RNAs in human cells originate from snoRNAs, by identifying several human snoRNAs with miRNA-like processing signatures (Ender et al., 2008). The report also confirms that snoRNAs function like miRNAs, and require Dicer activity but are independent of Drosha/DGCR8 (Ender et al., 2008). Small ncRNA biogenesis has almost been understood clearly, but piRNAs remain the only poorly understood ncRNA family. Comparative genomics studies have revealed that Dicer might be involved in the piRNA biogenesis pathway, but have raised a further question about dicer dependent piRNAs and dicer independent piRNAs (Kandhavelu et al., 2009). ncRNA research leads to several questions: How many genes contain ncRNAs? How many ncRNAs are encoded by a genome? How important are they? What functions does a cell delegate to RNA instead of protein, and why? Given the absence of a diagnostic open reading frame, how these genes can be identified? How can all the functions of ncRNAs be elucidated? How to identify its biogenesis pathway clearly? In regard to the above queries, it appears likely that ncRNAs are a critical component of gene regulation in complex organisms. Its maturation process and stepwise process are well characterized, yet we lack many of the details (Kim, 2005). In the miRNA biogenesis pathway, Drosha, DGCR8, and Dicer play an important role in pre-miRNA processing. In addition, a number of human miRNAs have been found in association with eIF2C2, Gemin3 and Gemin4 to form an RNP complex (Mourelatos et al., 2002). The function of this complex and each component protein remains to be understood clearly. Notably the expression profiles of clustered miRNAs are also highly similar, raising the possibility that transcription of these miRNAs is controlled by common regulatory sequences. It could be possible that the process is regulated by the presence of a single promoter for all transcribed clustered miRNAs, especially when the transcript is polycistronic (Seifinejad et al., 2010). In-vitro and in-vivo studies have so far clarified the miRNA maturation step processing with two possible modes of action: (a) processing of the parturient transcript, such as pri-miRNA, to the long precursor, and (b) processing of the long precursor to the short mature miRNA (Lee et al., 2003, 2002). Alternatively, it is also possible that the long precursor itself is the parturient transcript. It has been shown that some miRNAs have a self-promoter to directly produce the mature miRNA (Inui et al., 2010). The understanding of ncRNA requires new approaches in functional genomics. To address the above questions, new systematic gene discovery approaches need to be developed that are specifically aimed at ncRNAs. It is also believed that other families are still to be discovered. Comparative genomic analysis constitutes a powerful approach for the systematic understanding of the genome. Studies of the biogenesis pathway of ncRNA, its transport and its expression are likely to reveal important aspects of functional human genomics. In this study, we explored a class of ncRNAs by deep sequence comparison in which several human piRNAs show miRNA-like processing signatures. We found that sets of pri-ncRNAs are processed as a pre-miRNA to produce both miRNA and piRNA. Finally, we have identified cellular targets that are regulated by these classes of small RNAs through computational prediction. 2. Methodology 2.1. miRNA/piRNA and snoRNA/miRNA cluster prediction To search for miRNAs, miRBase was used (http://microrna.sanger.ac.uk/sequences/). The mature miRNAs of all species were downloaded from the miRNA registry. From that, mature miRNAs of Homo sapiens were retrieved. This is an online database consisting of miRNA gene sequences and predicted target sites. Version 13.0 of miRBase contains 703 human miRNA gene sequences that were used for the deep sequence comparison analysis (GriffithsJones, 2004; Griffiths-Jones et al., 2006, 2008) (Supplementary file 1). Mature miRNA gene sequences were analysed using BLAST with piRNA database (http://pirnabank.ibab.ac.in/index.shtml) to identify the miRNA/piRNA clusters in the same locus of the gene. The secondary structures were constructed using Mfold for the illustration of loop formation and base pair complementation. To elucidate its evolutionary conservation, multiple sequence alignment was performed by genome BLAST (http://www.ncbi.nlm.nih.gov). 3. Results and discussion 3.1. Unique primary precursor for miRNAs and piRNAs In this study, we explore a class of ncRNAs by deep sequence comparison in which several human piRNAs show miRNAlike processing signatures (Fig. 1a). As described in Methodology, we find that the sets of pri-ncRNA are processed as a precursor miRNA (pre-miRNA) to produce miRNA and also pre-piRNA to produce piRNA (Fig. 1b). Interestingly, pri-ncRNAs play an important role in the biogenesis of piRNAs and are also interlinked with the miRNA biogenesis pathway. This characteristic pattern also shows evolutionary signatures shown by its precise selective significance and synteny. Our initial approach to address these issues was to search for transcripts that are unique for both miRNA and piRNA (Bernstein et al., 2001). These long transcripts are also called primary transcripts and may be involved in the production of

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Fig. 1. Stepwise processing of several miRNA/piRNA clusters in human: (a) miRNA and piRNA originating from human miRNA/piRNA clusters that have been found in deep sequence comparison. This Table shows six mature miRNA/piRNA clusters. The complete strand is the piRNA; the miRNA is shown in red. Supportive gene sequence IDs are also given. (b) Secondary structure analysis of the miRNA/piRNA clusters, represented by schematic diagrams in which miRNAs and piRNAs are highlighted in red and green, respectively. (c) The graphical representation of nucleotide (nt) distribution in miRNA/piRNA. It shows that length of the precursors f GC % and nt length f (1/AT) %. nt lengths are 110, 82, 80, 71, 70, 53, corresponding to hsa-miR-182, 106b, 7a-1, 18a, 1974, 1978. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

two different families of ncRNAs, i.e. miRNAs and piRNAs (Parker and Barford, 2006). These long transcripts are likely to be the nascent ncRNA transcripts that serve as the primary precursors for pre-ncRNAs. From the database, 703 human miRNAs (hsa-miRNAs) were retrieved and analyzed through deep sequence comparison (see Supplementary file 2). All known mature hsa-miRNA sequences were chosen and the conservation of sequences was analysed using BLAST with the known piRNAs so that transcripts covering the whole cluster with the piRNA were deduced. Six miRNA/piRNA clusters were discerned in this study: hsa-let-7a, hsa-miR-18a, hsa-miR-182, hsa-miR-106b, hsa-miR-1974 and hsa-miR-1978, harbouring identical gene sequences with the known piRNA genes (see Supplementary doc, Table 1). These miRNA/piRNA clusters reside in intergenic and sense sequences, making it improbable that the transcripts are unique gene transcripts. The long transcripts’ lengths were confirmed (50 bp and 110 bp), suggesting that the nascent transcripts are monocistronic miRNA/piRNA clusters. It may even be transcribed as single transcriptional unit (Sasaki et al., 2003). Total human genome BLAST showed the presence of these clusters in their corresponding loci (Table 1). Moreover, evolutionary conservation analysis shows that most of these human precursors are highly conserved in Pan troglodytes when compared to other mammals (Table 2). Recent discoveries have revealed by immunoprecipitation and deep sequencing that the hsa-miR-18a, hsa-miR-182, hsa-miR-106b, small RNAs are associated with human Ago1 and Ago2 (Ender et al., 2008; Henras et al., 2004; Munholland and Nazar, 1987). 3.2. Maturation of pri-piRNAs/miRNAs and its evolutionary conservation In order to verify that these long transcripts are indeed precursors of mature miRNAs/piRNAs, we compared all mature miRNA against human piRNAs as a preliminary step. The miRNAs/piRNAs’ identical mature gene sequences were identified and corresponding precursor sequences of miRNAs were retrieved for RNA secondary structure (SS) analysis. For many ncRNA Table 1 Loci and strand position of the six miRNA/piRNA clusters in the human chromosome. Type of cluster

Location in human chromosome

Type of strand

miRNA/piRNA miRNA/piRNA miRNA/piRNA miRNA/piRNA miRNA/piRNA miRNA/piRNA

9:95976060–95980139 13:90799006–90803076 7:99527552–99531633 7:129195459–129199568 5:93928928–93932997 2:149353835–149357887

Plus Plus Negative Negative Negative Negative


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Table 2 Evolutionary conservation of the precursor gene sequences of miRNA/piRNA cluster in chordates. An asterisk represents 100% conservation of precursor gene sequences. Lower identities are not reported. Name of the species

Bos taurus Canis familiaris Equus caballus Homo sapiens Monodelphis domestica Mus musculus Ornithorhynchus anatinus Pan troglodytes Rattus norvegicus Macaca mulatta

miRNA/piRNA cluster let-7a-1

mir-18a

* * * * * * * * * *

* * * * * * * * *

mir-182

mir-106b

mir-1974

mir-1978

*

*

*

*

*

*

molecules, the SS is highly important for their proper function. Any mature sequence development depends upon the complementation of the base pairs in pre-ncRNA. We analyzed all sequences were with Mfold. In Fig. 1b, the precursor sequences of the six ncRNAs in Fig. 1a are shown along with their SSs, with the locations of the mature miRNAs/piRNAs highlighted in red and green, respectively. The maturation process can occur without the 50 - and 30 -end sequences of the primary transcript. Also, most of the mature sequences might have hanging sequences in the 50 and 30 ends. These overhanging sequences are required to produce mature piRNA, according to our verification. Of the six predicted miRNA/piRNA clusters reported here, the piRNA all have overhangs of 3 bp and 10 bp in the 50 and 30 ends. The precursors’ nucleotides show GC richness, nt length f GC %, and nt length f (1/AT) % (Fig. 1c and see Supplementary file 1). Depending upon the complement of these primary transcripts, the RNA-SS could be processed as either miRNA and/or piRNA. Therefore, our results affirm that miRNAs/piRNAs maturation consists of at least two sequential steps: (a) the long primary transcripts are processed into pre-miRNAs, and (b) the pre-miRNAs are then processed into the final mature products (mature miRNAs/ piRNAs). We therefore suggest that the long transcript be designated as ‘pri-miRNAs/piRNAs’ (primary precursor for miRNAs/ piRNAs) (Fig. 2). We inferred the piRNA biogenesis from our findings. It confirms that some of the same machinery involved in miRNA processing could be applied to produce piRNA. In addition, our results suggest that the miRNA maturation steps can be performed in multiple ways which lead to different ncRNA products (Fig. 2). These include neglection and substitution, both of which may not be possible for every miRNA. A detailed functional characterization of all these ncRNAs will provide new insights into the human genome.

Fig. 2. Illustration of the biogenesis machinery of piRNAs and miRNAs proposed here of the primary-miRNAs/piRNAs gene clusters. Pri-miRNA/piRNA’s are produced by pol-II & III, which are processed into pre-miRNA/piRNA. These are recognized and exported by an unidentified export complex, possibly Exportin-5 and other factor(s). Upon export, Dicer and possibly other factors participate in the pre-miRNA/piRNA processing step to produce the mature miRNAs and piRNAs. The question marks indicate unidentified factors.

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