MOXEKymPHÄS EHomma, 2011, moM 45, № 2, c. 225-230
TEHOMHKA. ^^^^^^^^^^^^^^ TPAHCKPHOTOMHKA
UDC 577.218
GENOMIC ANALYSIS OF SILKWORM microRNA PROMOTERS AND CLUSTERS
© 2011 Yong Huang1, 2, Xing Jia Shen1, 2*, Quan Zou3, Jin Shan Huang1, 2, Shun Ming Tang1, 2
Jiang Su University of Science and Technology, Zhenjiang City, 212018, Jiangsu Province, China
2The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang City, 212018, Jiangsu Province, China 3School of Information Science and Technology of Xiamen University, Xiamen City, 361005, Fujian Province, China
Received February 08, 2010 Accepted for publication April 21, 2010
MicroRNAs (miRNAs) are endogenous single-stranded RNAs of 18~22 nt in length, which can regulate the complementary mRNAs at the post-transcriptional level by cleavage or repression of translation of the target mRNAs. Studies have shown that the majority of animal miRNAs are transcribed from independent transcription units, and some are transcribed together with their host genes. However, the nature of the primary transcript of intergenic miRNAs remains unknown. Silkworm (Bombyx mori) miRNAs are representative of those of the Lepidoptera insects and many of them are conserved in Caenorhabditis elegans and other animal species. To date, little is known about the transcriptional regulation of silkworm miRNA genes. We performed the genomic analysis on the silkworm miRNA transcripts around the promoter region including the transcription start site (TSS) and the TATA-box, and on the organization of the miRNA cluster. In 73 pre-miRNAs from the silkworm 131 promoters were detected via a bioinformatics approach. Among them the portion of non-conserved promoters is greater than that of the conserved ones. The genomic organization of pre-miRNAs of the silkworm was globally analyzed and it was determined that 11 of them were organized into five clusters. Sequence alignment showed that paralogs existed for some of the miRNAs in the cluster. These results may increase the understanding of the specific sequences upstream of the pre-miRNAs and of the functional implications of miRNA clusters in the silkworm.
Keywords: microRNA, Bombyx mori, promoter, miRNA cluster.
MicroRNAs (miRNAs), encoded by specific genes, are endogenous single-strand RNAs ranging form 19—25 nt in length existing in various organisms from viruses to plants and mammals [1—4]. They play key roles in the regulation of translation or degradation of target mRNAs by inducing post-transcriptional silence through base pairing to the complementary target sites. The precursor miRNAs are transcribed from the genome as pri-miRNA precursors and cleaved by the Drosha endonuclease in the nucleus to form approximately 70 nt pre-miRNAs with a characteristic hairpin structure. Then the pre-miRNAs are transported to the cytoplasm by Exportin-5, and the loop region of the hairpin is removed by the Dicer endonuclease. One strand of the matured double-stranded miRNA is incorporated into a large protein complex, the RNA-induced silencing complex (RISC), in order to guide the RISC to complementary mRNA targets. The RISC either inhibits translation elongation or triggers mRNA degradation, depending upon the degree ofcomplementarity ofthe miRNA with its target [5, 6]. MiRNAs are involved in numerous cellular processes including development, differentiation, proliferation, apoptosis and response to stress. Dysregulation of miRNA expression also contributes to disease pathology.
The biogenesis of miRNA is complicated and several lines of evidence have shown that most miRNAs are en-
* E-mail: shenxj63@yahoo.com.cn
coded by their own genes located in the intergenic regions or the antisense strands ofannotated genes [7—9]. The in-tergenic miRNA genes are believed to be transcribed independently to form a new gene family. However the intronic miRNAs and the ones interspersed in the human genome with mobile Alu elements are transcribed with their host genes [5, 10]. Studies showed that more than half of all known mammalian miRNAs are located in the introns of either protein-coding or noncoding transcriptional units (TUs), and about 10% ofmiRNAs are encoded by exons of long nonprotein-coding transcripts, also known as mRNA-like noncoding RNAs [10, 11]. Unlike plant miRNAs, miRNAs in animals are primarily encoded in intronic regions [12], suggesting that transcription of animal pri-miRNA may differ from that ofplants [13, 14]. Many pieces of evidence have indirectly suggested that miRNA genes are class-II genes which are transcribed by RNA polymerase II, suggesting that transcription of miRNA may be regulated by a similar mechanism as was established for protein-coding genes and miRNAs may contain promoter elements similar to those of protein-coding genes [15]. It is known that the class-II promoters consist of two parts: the core promoter and the upstream element. The core promoter contains at least two elements: a TATA box beginning at the approximate position of -30 and an initiator centered on the transcription start site (TSS). In Arabidopsis 63 miRNA TSSs were identified
via 5'-RACE and most of them contain a TATA-box in the core promoter region [16]. However, there are exceptions to this rule, for example, promoters of miR-23a, -27a, -24—2 lack the common promoter elements required for transcription initiation such as the TATA-box and the initiator element, and a large portion of pri-miRNAs does not contain a 5'-cap or a poly (A) tail [14]. Such TATA-less promoters are often found in housekeeping genes [15]. It is therefore necessary to study the upstream sequences including promoters, transcription start sites or specific elements, to understand the location and length of pri-miRNAs, expression patterns of miRNAs, miRNA-mediated regulatory pathways and their network.
However, up to date we have limited knowledge on the transcription of miRNA genes, which is the first and important step of miRNA biogenesis. Understanding the mechanism of miRNA gene expression is fundamentally important. One of the main goals for miRNA research is to elucidate how pri-miRNA genes are transcribed and how is miRNA involved in the complicated gene regulatory network [17]. Recently, five transcription factor (TF) binding motifs have been identified relating to protein-coding gene promoter sequences: AtMYC2, ARF, SORLREP3, LFY and TATA-box [18]. Identification of promoters of intergenic miRNA genes in Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana and Oryza sativa revealed that most known miRNA genes in these four species contain the same type of promoters as the protein-coding genes [19]. As is known, the promoter of a gene is a crucial control region for transcription initiation [20, 21].
Silkworm (Bombyx mori) has been domesticated for over 5000 years and is well-known for its industrial importance in sericulture. In addition it is a model system for the Lepidoptera insects with a completed genome sequence, which is in great favor for functional genomic research. At present, there are 91 silkworm microRNAs in the miRBase. Although millions ofsmall RNAs from the silkworm were identified via high-throughput sequencing [22] and the number of non-conserved miRNAs identified in the silkworm is growing and is greater than in Drosophila and other animal species [23, 24], little is known about the promoters of miRNAs in the silkworm. The method of full-length cDNA sequence alignment may allow to identify some promoters in the silkworm, but most of the cDNA sequence clones do not extend to the transcriptional start site (TSS) [25—27]. Full understanding of miRNA transcription requires a complete description of the location and extent of pri-miRNAs, including transcription start sites and promoters [13, 18, 19, 28].
We are interested in the known miRNA genes that contain their own promoters, and are focused on (1) the identification of specific sequences or motifs adjacent to pre-miRNAs, which relate to the expression of miRNAs, and (2) the pattern of miRNA clusters associated with the upstream specific sequences ofpre-miRNA in the silkworm.
EXPERIMENTAL
Upstream sequences of silkworm miRNAs. The silkworm (B. mori) pre-miRNA sequences were obtained from the miRBase database (miRBase Sequence Database, http://microrna.sanger.ac.uk; released 14.0 September 2009). The silkworm genome sequences were downloaded from the SilkDB (http://silkworm.genom-ics.org.cn/) and the Silkworm Genome Research Program (http://sgp.dna.affrc.go.jp/index.html). As there were no reports on silkworm miRNAs in introns, intergenic miRNAs of the silkworm were divided into two groups: non-conserved miRNAs and conserved miRNAs.
The upstream sequences of pre-miRNAs in the intergenic regions were organized according to the method described previously [19, 29]. Briefly, ifa pre-miRNA and its upstream gene were in the same direction and the distance between them was more than 2400 bp, the 2000 bp sequence upstream of the pre-miRNA was retrieved by Apollo (a genome annotation tool) on basing on the GFF file ('Gene-Finding Format' or 'General Feature Format') of silkworm miRNAs from the genome sequences [30]. Meanwhile, two hundred random sequences 2000 bp in length were automatically generated by a computer as a control.
Prediction of specific sequences upstream of silkworm miRNA genes. Sequences of the TSS and the TATA-box were predicted using an online web approach (ht-tp://www-bimas.cit.nih.gov/molbio/proscan/) and the Eponine method [31].
Clustering of silkworm miRNA genes. For analysis of miRNA clustering, both upstream and downstream sequences with pairwise distance less than 10 kb were considered as clustered miRNAs. When the clustered miRNAs were organized, the 5'-end sequences of the first miRNAs in the upstream regions were fetched following the same rule mentioned above
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