Structural basis for pre-miRNA-31 biogenesis
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE230167 | GEO | 2023/04/24
REPOSITORIES: GEO
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Project description:Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). Over 1,800 miRNA loci are annotated in humans, but it remains largely unknown if and at which sites the pri-miRNAs are cleaved by DROSHA. Here we performed in vitro processing on a full set of human pri-miRNAs (miRBase v21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs based on DROSHA-dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing as well as unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
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Project description:The cellular abundance of mature microRNAs (miRNAs) is dictated by the efficiency of nuclear processing of primary miRNA transcripts (pri-miRNAs) into pre-miRNA intermediates. The Microprocessor complex of Drosha and DGCR8 carries this out, but it has been unclear what controls Microprocessor's differential processing of various pri-miRNAs. Here, we show that Drosophila DGCR8 (Pasha) directly associates with the C terminal domain of the RNA polymerase II elongation complex when it is phosphorylated by the Cdk9 kinase (pTEFb). When association is blocked by loss of Cdk9 activity, a global change in pri-miRNA processing is detected. Processing of pri-miRNAs with a UGU sequence motif in their apical junction domain increases, while processing of pri-miRNAs lacking this motif decreases. Therefore, phosphorylation of RNA polymerase II recruits Microprocessor for co-transcriptional processing of non-UGU pri-miRNAs that would otherwise be poorly processed. In contrast, UGU-positive pri-miRNAs are robustly processed by Microprocessor independent of RNA polymerase association.
2017-09-15 | GSE103234 | GEO
Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs that repress gene expression. In plants, the RNase III enzyme Dicer-like (DCL1) processes primary miRNAs (pri-miRNAs) into miRNAs. Here, we show that SMALL1 (SMA1), a homolog of the DEAD-box pre-mRNA splicing factor Prp28, plays essential roles in miRNA biogenesis in Arabidopsis. A hypomorphic sma1-1 mutation causes growth defects and reduces miRNA accumulation correlated with increased target transcript levels. SMA1 interacts with the DCL1 complex and positively influences pri-miRNA processing. Moreover, SMA1 binds the promoter region of genes encoding pri-miRNAs (MIRs) and is required for MIR transcription. Furthermore, SMA1 also enhances the abundance of the DCL1 protein levels through promoting the splicing of the DCL1 pre-mRNAs. Collectively, our data provide new insights into the function of SMA1/Prp28 in regulating miRNA abundance in plants.
2018-06-09 | GSE114073 | GEO
Project description:DEAD-box RNA-binding proteins (RBPs) play a significant role in RNA metabolism to achieve cellular homeostasis, including miRNA biogenesis and transcription. Hypoxia induces stemness cell-like characteristics in cancer cells and promotes malignant progression. Despite the fact that hypoxia can induce the changes in protein and RNA modification, thereby regulating downstream gene expressions, how modifications at different molecular layers interplay with each other are poorly understood. Here we show that hypoxia induces HectH9-mediated K63-linked polyubiquitination of the DEAD-box protein DDX17 as well as reduces N6-methyladenosine (m6A) marks in pri-miRNAs. While m6A potentiates DDX17 binding to pri-miRNAs, decreased m6A modifications of pri-miRNAs and increased polyubiquitination of DDX17 under hypoxia lead to decreased DDX17 binding to pri-miRNAs binding. These events enhance the association of DDX17 with the ubiquitin receptor p300 and lead to a decrease in miRNA biogenesis, especially for miRNAs regulating stemness and stemness-related genes. In addition, polyubiquitinated DDX17 together with p300 upregulates H3K56Ac levels on the stemness and stemness-related genes, resulting in enhancement of tumor initiating ability. Post-transcriptionally, decreased miRNA production, including those targeting stemness genes or stemness-related genes, also facilitates tumor initiation. Together, hypoxia triggers DDX17 poly-ubiquitination, which orchestrates dual mechanisms to increase tumor initiating ability and promote tumor progression.
2018-03-09 | PXD006059 | JPOST Repository
Project description:microRNAs (miRNAs) play important roles in the regulation of gene expression. In Arabidopsis, mature miRNAs are processed from primary miRNA transcripts (pri-miRNAs) by nuclear HYL1/SE/DCL1 complexes that form Dicing bodies (D-bodies). Here we report that a RNA-binding protein MOS2 bound to pri-miRNAs and was required for their efficient processing. MOS2 did not interact with HYL1, SE, and DCL1 and was not localized in D-bodies. Interestingly, in the absence of MOS2, the recruitment of pri-miRNAs by HYL1 was greatly reduced and the localization of HYL1 in D-bodies was compromised. These data suggest that MOS2 facilitates pri-miRNA processing through facilitating the recruitment of pri-miRNAs by the Dicing complexes.
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Project description:MiRNAs are key regulators of gene expression. They are processed from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by DNA-dependent polymerase II (Pol II). MiRNA levels need to be precisely controlled to maintain various biological processes. In this manuscript, we report a discovery that STV1, a conserved ribosomal protein, acts in miRNA biogenesis in Arabidopsis. A portion of STV1 localizes at the nucleus and binds pri-miRNAs. Using pri-miR172b as a reporter, we show that STV1 binds the stem-loop with a short 5’ flanking sequencing within pri-miRNAs. Lack of STV1 in stv1-1 reduced the association of pri-miRNAs with their processing complex. These data suggest that STV1 promotes miRNA biogenesis through facilitating the recruitment of pri-miRNAs to their processing complex. Furthermore, we show that STV1 indirectly influence the occupancy of Pol II at the promoters of miRNA coding genes (MIR) and MIR promoter activities. Based on these results, we propose that STV1 refines the accumulation of miRNAs through its combined effects on pri-miRNA processing and transcription. This study uncovers an extra-ribsomal function of STV1.
2017-03-06 | GSE91013 | GEO
Project description:Hairpin-containing pre-miRNAs are precursors of microRNAs (miRNAs) that play important roles in cellular processes and various human diseases. Pre-miRNAs are produced from longer primary transcripts (pri-miRNAs). The falsity in cellular expression and sequences of pre-miRNAs might cause cellular defects or human diseases. However, the current pre-miRNA quantification methods by qPCR cannot discriminate between pre-miRNAs and their parental pri-miRNAs. In addition, the ligation of the sequencing adapter to 5’-end of pre-miRNAs is inefficient, therefore, pre-miRNA sequencing is highly impractical. Here, we developed a method, called the intramolecular ligation method (iLIME) for pre-miRNA quantification and sequencing. This method utilized T4 RNA ligase 1 to convert hairpin-pre-miRNAs into circularized RNAs that do not naturally exist in cells. The resulting circuliarized RNAs allow us to design unique primers to quantify pre-miRNAs by qPCR specicially, and thus this qPCR can distinguish pre-miRNA from pri-miRNAs. In addition, the iLIME also allows us to sequence pre-miRNAs using next-generation sequencing. The iLIME method offers a simple and effective way to quantify and sequence pre-miRNAs. This will be useful in investigating pre-miRNAs for addressing research questions for medical applications. The iLIME can be potentially extended to other hairpin-containing RNAs, such as tRNAs and snRNAs.
2021-10-11 | GSE185524 | GEO
Project description:MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem-loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base-pairing throughout the remainder of the stem, and a narrow stem-length preference of 35±1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs, in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence, yet processed more efficiently than natural pri-miRNAs. Three major experiments are included in the submitted data. 1) Pools of synthetic DNA containing barcodes and pri-miRNA variants were sequenced to infer the barcode-pri-miRNA linkages, and the pri-miRNA variants were assayed for their cleavage efficiency ("selection"), which can be quantified by comparing their representations in the input and after cleavage. 2) Artificial miRNAs produced in HEK293T cells were sequenced. 3) Transcriptomes of artificial miRNA-expressed cells were sequenced to quantify mRNA changes.
2015-09-24 | E-GEOD-67937 | biostudies-arrayexpress
Project description:Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). Over 1,800 miRNA loci are annotated in humans, but it remains largely unknown if and at which sites the pri-miRNAs are cleaved by DROSHA. Here we performed in vitro processing on a full set of human pri-miRNAs (miRBase v21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs based on DROSHA-dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing as well as unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
2021-07-27 | GSE174222 | GEO
Project description:Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). Over 1,800 miRNA loci are annotated in humans, but it remains largely unknown if and at which sites the pri-miRNAs are cleaved by DROSHA. Here we performed in vitro processing on a full set of human pri-miRNAs (miRBase v21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs based on DROSHA-dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing as well as unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
2021-07-27 | GSE174143 | GEO