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Current Perspectives in Intronic Micro Rnas (Mirnas)

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Current Perspectives in Intronic Micro Rnas (Mirnas) Journal of Biomedical Science (2006) 13:5–15 5 DOI 10.1007/s11373-005-9036-8 Current perspectives in intronic micro RNAs (miRNAs) Shao-Yao Ying & Shi-Lung Lin Department of Cell & Neurobiology, Keck School of Medicine, BMT-403, University of Southern California, 1333 San Pablo Street, Los Angeles, CA, 90033, USA Received 27 May 2005; accepted 14 September 2005 Ó 2005 National Science Council, Taipei Key words: fine-tuning of gene function, functional/structural genomics, gene expression, genetic regula- tion, intronic microRNA, miRNA biogenesis, miRNA, post-translational modification, regulatory gene Summary MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a post-transcriptional gene silencing mechanism. Intronic miRNAs, derived from introns by RNA splicing and Dicer processing, can interfere with intracellular mRNAs to silence that gene expression. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of type II RNA polymerases (Pol-II) and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in Caenorhabditis elegans, mouse and human cells; however, neither their function nor application has been reported. To this day, the computer searching program for miRNA seldom include the intronic portion of protein-coding RNAs. The functional significance of artificially generated intronic miRNAs has been successfully ascertained in several biological systems such as zebrafishes, chicken embryos and adult mice, indicating the evolutionary pres- ervation of this gene regulation system in vivo. Multiple miRNAs can be generated from the same cluster of introns; however, non-homologous miRNAs may have different targets and functions while homologous miRNA may be derived from different intronic clusters. Taken together, the model of intronic miRNA- mediated transgenic animals provides a tool to investigate the mechanism of miRNA-associated diseases in vivo and will shed light on miRNA-related therapies. Introduction whereas introns are thought to be junk genes. We know now that this perception needs to be The intronic microRNA (miRNA) is a form of gene modified. that regulates the function of other genes. It The Human Genome Project has completed the probably plays a critical role as gene regulators in process of determining, sequencing, and mapping fine-tuning many biological manifestations. The more than 36,000 genes and about 3 billion gene is conventionally defined as the specific region nucleotides bases of the human genome. What of a DNA molecule that codes for the synthesis of a dose the vast majority of the nucleotides do in our particular protein. This specific region consists of body? The purpose of this article is to provide an exons and introns; exons code the protein sequences up-to-date summary of new knowledge that is directly resulted from the studying of the intronic *To whom correspondence should be addressed. Fax: +323- miRNA as an example to illustrate the role of the 442-3466; E-mail: [email protected] so-called ‘‘junk genes.’’ 6 The intronic miRNA is defined as small RNAs, types of small RNA molecules usually interfere derived from introns, processed by splicing and with expression of genes intracellularly based on pre-miRNA degradations and targets the intracel- their completely or partially complementary of the lular mRNA for silencing of that gene expression. small RNAs to the target gene, respectively. The intron occupies the largest proportion of non- Basically, siRNAs are double-stranded RNAs that coding sequences in the protein-coding DNA of a degrade target gene transcripts based on nearly genome. The transcription of the genomic protein- perfect complementarity between these two RNA coding DNA generates precursor messenger RNA molecules [7, 8]. miRNAs, unlike the stringent (pre-mRNA). complementarity of siRNAs to their RNA targets, The pre-mRNA consists four major parts: are single-stranded that pair with target RNAs, the 5¢-untranslational region (UTR), the protein- which have partial complementarity to the miR- coding exon, the non-coding intron and the NAs [9, 10]. Many native miRNAs have been 3¢-UTR region. Both the 5¢- and 3¢-UTR regions located in hairpin-like RNA precursors in eukary- can be broadly defined as a kind of intron otes, including yeast (Schizosaccharomyces pom- extension, however, the processing of these two be), plant (Arabidopsis spp.), nematode (C. regions during mRNA translation is different from elegans), fly (Drosophila melanogaster), mouse, the intron located between two protein-coding and human. These native miRNAs are usually exons, termed the in-frame intron. The in-frame involved in defense against viral infections and intron can be as big as several 10 k-nucleotides regulation of gene expression during development and was thought to be a huge genetic waste in gene [11–21]. In contrast, native siRNAs are mainly transcripts. Recently, the discovery of the intron- demonstrated in plants and low-level animals derived microRNA (intronic miRNA) completely (worms and flies) and their prevalence in mammals changed this perception. remains to be established [10]. Due to the fact that The intronic miRNA is a new class of small miRNAs are widely detected in eukaryotes, these single-stranded regulatory RNAs and is different small RNAs have recently been used to design from previously identified miRNAs. First, the reagents against cancers and viral infections as a intronic miRNA is derived from the processing novel gene therapy [1, 22]. Indeed, gene silencing of pre-mRNA introns. Second, it requires unique mechanisms involving miRNAs have been pro- Pol-II and RNA splicing components for its posed to be an intracellular defense system as a biogenesis [1–3]. Third, about 1030% of the means to eliminate undesired transgenes and spliced intron are exported out of the nucleus and foreign RNAs, including viral infections and have a moderate half-life in the cytoplasm [4]. The retrotransposon activities [22, 23]. messenger RNA (mRNA) is usually 18–25 oligo- nucleotides capable of either directly degrading its miRNA biogenesis intracellular mRNA target or suppressing the protein translation of its targeted mRNA, depend- The process of miRNA biogenesis in vertebrates is ing on the complementarity between the miRNA presumably involved five steps (Figure 1). First, and its target. In this sense, the intronic miRNA is miRNA is generated as a long primary precursor similar to previously described intergenic miRNAs miRNA (pri-miRNA) which is most likely medi- structurally and functionally. Good examples of ated by RNA polymerases type II (Pol-II) [2, 24]. intronic miRNA have been described in Caenor- Second, the long pri-miRNA is excised by Drosha- habditis elegans, mouse and human genomes [2, 5, like RNase III endonucleases and/or spliceosomal 6] and some of their functions are closely related to components to form the precursor miRNA (pre- RNA interference (RNAi). miRNA). This step is depending on the origin of the pri-miRNA located in an exon or an intron RNAi interference (RNAi) respectively [2, 25]. Third, the pre-miRNA is exported out of the nucleus by Ran-GTP and a RNA interference is a post-transcriptional gene receptor Exportin-5 [26, 27]. In the cytoplasm, silencing mechanism in eukaryotes, which can be Dicer-like nucleases cleave the pre-miRNA to triggered by small RNA molecules such as miRNA form mature miRNA. Lastly, the mature miRNA and small interfering RNA (siRNA). These two is incorporated into a ribonuclear particle (RNP) 7 Figure 1. Comparison of biogenesis and RNAi mechanisms among siRNA, intergenic (exonic) miRNA and intronic miRNA. SiR- NA is likely formed by two perfectly complementary RNAs transcribed from two different promoters (remains to be determined) and further processing into 19–22 bp duplexes by RNase III-familial endonuclease, Dicer. The biogenesis of intergenic miRNA, e.g. lin-4 and let-7, involves a long transcript precursor (pri-miRNA), which is probably generated by a Pol-II or Pol-III RNA pro- moter, while intronic miRNA is transcribed by the Pol-II promoter of its encoded gene and co-expressed in the intron region of the gene transcript (pre-mRNA). After RNA splicing and further processing, the spliced intron may function as a pri-miRNA for intronic miRNA generation. In the nucleus, the pri-miRNA is excised by Drosha-like RNases to form a hairpin-like pre-miRNA template and then exported to cytoplasm for further processing by Dicer* to form mature miRNAs. The asterisk * indicates that the Dicers for siRNA and miRNA pathways are different. All three small regulatory RNAs are finally incorporated into a RNA- induced silencing complex (RISC), which contains either strand of siRNA or the single-strand of miRNA. The effect of miRNA is considered to be more specific and less adverse than that of siRNA because only one strand is involved. On the other hand, siR- NA primarily triggers mRNA degradation, whereas miRNA can induce either mRNA degradation or suppression of protein syn- thesis depending on the sequence complementarity to the target gene transcript. to form the RNA-induced gene silencing complex miRNA is formed as proposed by several labora- (RISC) which executes the RNAi-related gene tories were based on the siRNA model, therefore, silencing effects [28, 29]. Although the in vitro one must distinguish the individual properties and model of siRNA-associated RISC assembly has differences of Dicer and RISC in these two types of been reported, the link between the final miRNA small RNAs processing, particularly for the under- maturation and RISC assembly remains to be standing evolutional and functional relationship determined. The characteristics of Dicer and RISC between these two gene silencing pathways. In in the siRNA and miRNA processing have been addition, the differences between these two gene reported to be distinct [30].
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