Regulation of microRNA biogenesis Ha, Minju; Kim, V Narry
Nature reviews. Molecular cell biology,
08/2014, Letnik:
15, Številka:
8
Journal Article
Recenzirano
Odprti dostop
MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and ...pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Global investigation of the 3′ extremity of mRNA (3′-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences ...and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50–100 nt in HeLa and NIH 3T3 cells. Poly(A) length correlates with mRNA half-life, but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (<25 nt), while the G tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing.
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•We develop TAIL-seq that reveals the transcriptome-wide landscape of RNA 3′ ends•Poly(A) length correlates with mRNA half-life, but not with translation efficiency•We found widespread uridylation and guanylation at the 3′ ends of poly(A) tail•The U tails are generally found on short poly(A) tails (<25 nt)
Eukaryotic mRNAs have poly(A) tails that can affect mRNA stability and translational activity. Chang et al. describe a method, called TAIL-seq, that allows high-resolution, transcriptome-wide investigation of 3′ end sequences. They use this approach to profile 3′ ends and to identify several different modifications at the ends of poly(A) tails.
Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and ...cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.
SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic. Although the SARS-CoV-2 genome was reported recently, its transcriptomic architecture is unknown. Utilizing two complementary ...sequencing techniques, we present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous discontinuous transcription events. In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif, AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the modification and the 3′ tail. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.
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•We provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome•The transcriptome is complex owing to numerous discontinuous transcription events•In addition to 10 canonical RNAs, SARS-CoV-2 produces RNAs encoding unknown ORFs•We discover at least 41 potential RNA modification sites with an AAGAA motif
The SARS-CoV-2 transcriptome and epitranscriptome reveal a complex array of canonical and non-canonical viral transcripts with RNA modifications.
Abstract
MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to ...investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification of miRNAs has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here, we quantify miRNAs and their variants (known as isomiRs) by an improved sRNA-seq protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which minimize the ligation bias during library preparation. Measurement using AQ-seq allows us to correct the previously misannotated 5′ end usage and strand preference in public databases. Importantly, the analysis of 5′ terminal heterogeneity reveals widespread alternative processing events which have been underestimated. We also identify highly uridylated miRNAs originating from the 3p strands, indicating regulations mediated by terminal uridylyl transferases at the pre-miRNA stage. Taken together, our study reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).
RNA tailing, or the addition of non-templated nucleotides to the 3' end of RNA, is the most frequent and conserved type of RNA modification. The addition of tails and their composition reflect RNA ...maturation stages and have important roles in determining the fate of the modified RNAs. Apart from canonical poly(A) polymerases, which add poly(A) tails to mRNAs in a transcription-coupled manner, a family of terminal nucleotidyltransferases (TENTs), including terminal uridylyltransferases (TUTs), modify RNAs post-transcriptionally to control RNA stability and activity. The human genome encodes 11 different TENTs with distinct substrate specificity, intracellular localization and tissue distribution. In this Review, we discuss recent advances in our understanding of non-canonical RNA tails, with a focus on the functions of human TENTs, which include uridylation, mixed tailing and post-transcriptional polyadenylation of mRNAs, microRNAs and other types of non-coding RNA.
Although more than 100 types of RNA modification have been described thus far, most of them were thought to be rare in mRNAs and in regulatory noncoding RNAs. Recent developments have unveiled that ...at least some of the modifications are considerably abundant and widely conserved. This Minireview summarizes the molecular machineries and biological functions of methylation (N6-methyladenosine, m(6)A) and uridylation (U-tail).
Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in ...non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell-cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle.
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•TAIL-seq reveals dynamic changes in poly(A) tail length during somatic cell cycle•Oligo-dT capture can lead to a bias in quantification of deadenylated mRNAs•Poly(A) tail length correlates with translation rate only to a threshold of ∼20 nt•Genes with the TOP element escape translational suppression in M phase
By combining transcriptome, 3′-terminome, and translatome data, Park et al. unveil dynamic regulation of poly(A) tail length and translation during somatic cell cycle. They discover the impact of poly(A) tail on translation within a limited range (<∼20 nt) and find the selective escape of TOP genes from mitotic translational suppression.
MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main ...determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed “formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq),” which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.
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•We develop fCLIP-seq, which identifies the substrates of DROSHA and their cleavage sites•Primary microRNA processing sites are mapped on a genomic scale•fCLIP-seq reveals widespread end modification and alternative processing of microRNA•Dozens of non-miRNA targets are discovered, suggesting noncanonical functions of DROSHA
Kim et al. develop “fCLIP-seq” and use it to identify the targets of DROSHA, a microRNA-processing endonuclease. They map hundreds of primary microRNA cleavage sites and find evidence for microRNA end modification events and alternative DROSHA processing. They also discover many non-microRNA targets, suggesting non-canonical functions of DROSHA.
MicroRNA (miRNA) maturation is initiated by Microprocessor composed of RNase III DROSHA and its cofactor DGCR8, whose fidelity is critical for generation of functional miRNAs. To understand how ...Microprocessor recognizes pri-miRNAs, we here reconstitute human Microprocessor with purified recombinant proteins. We find that Microprocessor is an ∼364 kDa heterotrimeric complex of one DROSHA and two DGCR8 molecules. Together with a 23-amino acid peptide from DGCR8, DROSHA constitutes a minimal functional core. DROSHA serves as a “ruler” by measuring 11 bp from the basal ssRNA-dsRNA junction. DGCR8 interacts with the stem and apical elements through its dsRNA-binding domains and RNA-binding heme domain, respectively, allowing efficient and accurate processing. DROSHA and DGCR8, respectively, recognize the basal UG and apical UGU motifs, which ensure proper orientation of the complex. These findings clarify controversies over the action mechanism of DROSHA and allow us to build a general model for pri-miRNA processing.
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•Microprocessor is a trimeric complex with one DROSHA and two DGCR8•Functional core of Microprocessor contains DROSHA and the C-terminal tail of DGCR8•DROSHA serves as a ruler by recognizing the basal elements•DGCR8 interacts with the apical elements to ensure fidelity of processing
Functional reconstruction of human Microprocessor defines its molecular stoichiometry and the specific role of each component in substrate recognition and orientation, revealing a comprehensive processing mechanism of Microprocessor.
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