Many roads to maturity: microRNA biogene

Many roads to maturity: microRNA biogenesis pathways and their regulation(진행중)

슬로싱킹 2024. 11. 23. 08:02

 

winter2009.pdf
0.84MB

 

 

MicroRNAs (miRNAs) are short (20–23-nucleotide), endogenous, single-stranded RNA molecules that regulate gene expression1 . Mature miRNAs and Argonaute (Ago) proteins form the RNA-induced silencing complex (RISC), a ribonucleoprotein complex mediating posttranscriptional gene silencing2–5.

 

Complementary base-pairing of the miRNA guides RISC to target messenger RNAs, which are degraded, destabilized or translationally inhibited by the Ago protein6,7.

 

Proteomic studies have recently uncovered the broad impact of a single miRNA on hundreds of targets8,9. Many cellular pathways are affected by the regulatory function of miRNAs; the most prominent of these pathways control developmental and oncogenic processes10–20.

 

Notably, miRNA processing defects also enhance tumorigenesis21. Although insights into the regulatory function of miRNAs are beginning to emerge, much less is known about the regulation of miRNA expression and activity. Recently, evidence for post-transcriptional control of miRNA activity has been accumulating22–26. In contrast to the linear miRNA processing pathway that was initially thought to be universal for the biogenesis of all mature miRNAs (Fig. 1), multiple discoveries led to the recognition of miRNA-specific differences that open a plethora of regulatory options to express and process individual miRNAs differentially.

 

Here we review the recent progress made in elucidating the complexity of miRNA processing and post-transcriptional regulation. Although we focus predominantly on the mammalian system, related information obtained from other model systems including the fruitfly Drosophila melanogaster, the nematode Caenorhabditis elegans and the plant Arabidopsis thaliana will also be presented where applicable.

 

Early steps: microRNA processing in the nucleus

Transcription of the pri-miRNA. miRNA genes are transcribed by either RNA polymerase II or RNA polymerase III into primary miRNA transcripts (pri-miRNA)27–29. Many pri-miRNAs are polyadenylated and capped — hallmarks of polymerase II transcription.

 

Their transcription is sensitive to treatment with the polymerase II inhibitor α-amanitin, and polymerase II binds to promoter sequences upstream of the miR-23a/ miR-27a/miR-24-2 cluster27,28. In contrast, miRNAs encoded by the largest human miRNA cluster, C19MC, are transcribed by polymerase III29.

 

Both RNA polymerases are regulated differently and recognize specific promoter and terminator elements, facilitating a wide variety of regulatory options. Expression of selected miRNAs is under the control of transcription factors, for example c-Myc or p53 (refs 17, 19), or depends on the methylation of their promoter sequences30–32. In addition, it has been shown that each miRNA located in the same genomic cluster can be transcribed and regulated independently33. However, controls of miRNA transcription steps are not necessarily universal34,35, and regulatory mechanisms at the transcriptional level are beyond the scope of this review.

 

microRNA editing. RNA editing of primary transcripts by ADARs (adenosine deaminases acting on RNA) modifies adenosine (A) into inosine (I). Because the base-pairing properties of inosine are similar to those of guanosine (G), A-to-I editing of miRNA precursors may change their sequence, base-pairing and structural properties and can influence their further processing as well as their target recognition abilities. Several examples of editing-mediated regulation of miRNA processing have been described (see Box 1).

 

 

pri-miRNA cleavage by the Drosha–DGCR8 microprocessor complex. The pri-miRNA is next endonucleolytically cleaved by the nuclear microprocessor complex formed by the RNase III enzyme Drosha (RNASEN) and the DGCR8 (DiGeorge critical region 8) protein (also known as Pasha (Partner of Drosha) in D. melanogaster and C. elegans) 36 (Fig. 2a). DGCR8/ Pasha contains two double-stranded RNA-binding domains and is essential for miRNA processing in all organisms tested37–40. An average human pri-miRNA contains a hairpin stem of 33 base-pairs, a terminal loop and two single-stranded flanking regions upstream and downstream of the hairpin. The double-stranded stem and the unpaired flanking regions are critical for DGCR8 binding and Drosha cleavage, but the loop region or the specific sequences are less important for this step41–43. A single nucleotide polymorphism in a miRNA precursor stem can block Drosha processing44. Nevertheless, many miRNA sequence aberrations observed in human tumours alter the secondary structure without affecting processing, and reveal the structural flexibility of the microprocessor34. The two RNase domains of Drosha cleave the 5´ and 3´ arms of the primiRNA hairpin39, whereas DGCR8 directly and stably interacts with the pri-miRNA and functions as a molecular ruler to determine the precise cleavage site41. Drosha cleaves 11 base pairs away from the single-stranded RNA/double-stranded RNA junction at the base of the hairpin stem. Drosha-mediated cleavage of the pri-miRNA occurs co-transcriptionally and precedes splicing of the protein-encoding or non-coding host RNA that contains the miRNAs. Splicing is not inhibited by Drosha-mediated cleavage, because a continuous intron is not required for splicing45,46.

 

microRNA-specific regulation of the microprocessor complex. Drosha-mediated pri-miRNA processing was recently shown to be subject to regulation by miRNA-specific mechanisms. Drosha forms two different complexes, a small microprocessor complex that contains only Drosha and DGCR8 and processes many pri-miRNAs, and a larger complex that contains RNA helicases, double-stranded RNA binding proteins, heterogeneous nuclear ribonucleoproteins and Ewing’s sarcoma proteins38. The RNA helicases p72 and p68 are part of the large Drosha complex and might act as specificity factors for the processing of a subset of pri-miRNAs (Fig. 2b). Expression levels of several miRNAs are reduced in homozygous p68−/− or p72−/− knockout mice, whereas other miRNAs remain unaffected47. Drosha-mediated cleavage can also be regulated for individual miRNAs: the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds specifically to pri-miR-18a and facilitates its processing. Loss of hnRNP A1 diminishes the abundance of mature miR-18a (Fig. 2c), but hnRNP A1 does not have any impact on other miRNAs that are located in the same miR-17 genomic cluster, demonstrating the extraordinary specificity of miR-18a biogenesis48. hnRNP A1 binds to the conserved loop of the pri-miR-18a and changes the hairpin conformation to create a more favourable cleavage site for Drosha49. About 14% of the human pri-miRNA loops are conserved between different species and could provide anchor points for similar regulatory mechanisms. Transforming growth factor-β (TGF-β) and bone morphogenetic factors (BMPs) induce the maturation of miR-21 by regulating the microprocessor activity. TGF-β and BMP bring about the recruitment of ligand-specific signal transducers (the SMAD proteins) to the primiR-21 transcript in complex with the RNA helicase DDX5 (p68). As a consequence, Drosha-mediated processing of pri-miR-21 is strongly enhanced and the abundance of mature miR-21 increases, ultimately resulting in a contractile phenotype in vascular smooth muscle cells (Fig. 2d)50.

 

Mirtrons: splicing replaces Drosha cleavage. Surprisingly, Droshamediated processing of pri-miRNAs into pre-miRNAs is not obligatory. Intron-derived miRNAs are released from their host transcripts after splicing (Fig. 2e). If the intron resulting from the action of the splicing machinery and the lariat debranching enzyme has the appropriate size to form a hairpin resembling a pre-miRNA, it bypasses Drosha cleavage and is further processed in the cytoplasm by Dicer51,52. These miRNAs, called mirtrons, have been discovered in several species including mammals, D. melanogaster and C. elegans51–53.

 

Lin-28 regulates let-7 processing and precursor stability. Lin-28 is a stem-cell-specific regulator of let-7 processing that uses multiple mechanisms54–58. Lin-28 was found to be necessary and sufficient to block microprocessor-mediated cleavage of the pri-miRNA (Fig. 3a)54. Mature let-7g increases during embryonic stem cell differentiation but the pri-miRNA levels remain constant, indicating post-transcriptional regulation of maturation. Recombinant Lin-28 blocks pri-miRNA processing, and knockdown of Lin-28 facilitates the expression of mature let-7 (ref. 54). The miRNA binding site of the Drosha competitor Lin-28 maps to conserved bases in the terminal loop of pri-let-7 (refs 56, 57). Intriguingly, although the loop region is considered dispensable for microprocessor action, many miRNAs have evolutionarily conserved loops potentially containing regulatory information49.

 

Post-transcriptional self-regulation of the microprocessor complex. The miRNA processing factors are also regulated post-transcriptionally or post-translationally. For example, the two components of the microprocessor complex regulate each other. DGCR8 stabilizes Drosha through an interaction between its conserved carboxy-terminal domain with the middle domain of Drosha (Fig. 4a)59. In turn, Drosha cleaves two hairpin structures in the 5´ untranslated region and the coding sequence of the Dgcr8 mRNA60. The Dgcr8 mRNA is then degraded, resulting in a negative feedback loop reducing Dgcr8 expression when sufficient microprocessor activity is available (Fig. 4b). The discovery that Drosha can directly cleave hairpin structures in mRNAs also points to the possibility that the two Drosha complexes in the cell regulate mRNAs independently of miRNAs.

 

Exportin-5–Ran-GTP mediate the export of the pre-miRNA. After nuclear processing, the pre-miRNA is exported into the cytoplasm by Exportin-5 (XPO5) in complex with Ran-GTP61. Knockdown of Exportin-5 leads to a decreased abundance of mature miRNAs but not to a nuclear accumulation of the pre-miRNA, indicating that Exportin-5 also protects pre-miRNAs against nuclear digestion61–63. Exportin-5 recognizes the pre-miRNA independently of its sequence or the loop structure. A defined length of the double-stranded stem and the 3´ overhangs are important for successful binding to Exportin-5, ensuring the export of only correctly processed pre-miRNAs63–65.