The effect of mismatches and wobble base pairs in the upper stem of pri-miRNAs on DROSHA cleavage using small RNA sequencing
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ABSTRACT:
PROVIDER: PRJNA595962 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10704454.fastq.gz | Fastqsanger.gz | |||
| SRR10704455.fastq.gz | Fastqsanger.gz | |||
| SRR10704456.fastq.gz | Fastqsanger.gz | |||
| SRR10704457.fastq.gz | Fastqsanger.gz |
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Project description:The experiments were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage and thereby miRNA biogenesis.
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Project description:The in vitro high-throughput human pri-miRNA processing assays were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage.
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Project description:MicroRNA (miRNA) biogenesis begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the processing of pri-miR-9-1 sequence variants, which encode the same mature miRNA but differ in the surrounding scaffold. We show that, in addition to previously known features, the overall structural flexibility of pri-miRNA impacts Drosha cleavage fidelity. Internal loops and nearby G·U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5’ isomiR production. Here, we report the the miRNA-seq data of HEK293T cell lines transfected with different pri-miR-9 constructs.
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Project description:We report the ability of the Drosha null/conditional-null mouse model to enable the identification of pri-miRNA transcripts. The conditional-null allele of Drosha phenocopies the null allele both in mESC and in mice, upon conversion to the null state with Cre.
2015-02-09 | GSE55735 | GEO
Project description:Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRES). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.
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Project description: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 gate-keeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated on a genomic scale, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here we establish a protocol termed ‘formaldehyde crosslinking and immunoprecipitation followed by sequencing (fCLIP-seq)’ that allows identification of DROSHA cleavage sites at single nucleotide resolution. fCLIP identifies numerous cleavage sites that do not match the ends of annotated mature miRNAs, particularly at the 3′ termini, suggesting widespread end modifications during miRNA maturation such as trimming and tailing. fCLIP also finds many pri-miRNAs undergoing alternative processing, yielding multiple miRNA isoforms. Moreover, we discover dozens of novel substrates that are bound and cleaved by DROSHA. These substrates are processed less efficiently than canonical pri-miRNAs, and produce only minute amounts of small RNAs. Depletion of DROSHA results in an increase of the hairpin-containing transcripts. Thus, the hairpins may serve mainly as cis-elements for DROSHA-mediated gene regulation rather than as miRNA genes. fCLIP-seq not only accurately maps the cleavage sites of DROSHA and suggests noncanonical functions of DROSHA, but also could be a general tool for investigating interactions between dsRNA binding proteins and structured RNAs.
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Project description: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 gate-keeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated on a genomic scale, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here we establish a protocol termed ‘formaldehyde crosslinking and immunoprecipitation followed by sequencing (fCLIP-seq)’ that allows identification of DROSHA cleavage sites at single nucleotide resolution. fCLIP identifies numerous cleavage sites that do not match the ends of annotated mature miRNAs, particularly at the 3′ termini, suggesting widespread end modifications during miRNA maturation such as trimming and tailing. fCLIP also finds many pri-miRNAs undergoing alternative processing, yielding multiple miRNA isoforms. Moreover, we discover dozens of novel substrates that are bound and cleaved by DROSHA. These substrates are processed less efficiently than canonical pri-miRNAs, and produce only minute amounts of small RNAs. Depletion of DROSHA results in an increase of the hairpin-containing transcripts. Thus, the hairpins may serve mainly as cis-elements for DROSHA-mediated gene regulation rather than as miRNA genes. fCLIP-seq not only accurately maps the cleavage sites of DROSHA and suggests noncanonical functions of DROSHA, but also could be a general tool for investigating interactions between dsRNA binding proteins and structured RNAs.
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Project description:MicroRNAs (miRNAs) are small RNAs that regulate gene expression. miRNAs are produced from primary miRNAs (pri-miRNAs), the cleavage of which is catalyzed by the Microprocessor complex. Microprocessor therefore plays a key role in determining the efficiency and precision of miRNA production, and thus the function of the final miRNA product. In this study, we utilized high-throughput sequencing-integrated enzymology with purified Microprocessor proteins and randomized pri-miRNA sequences to investigate the catalytic mechanism of Microprocessor. We identified multiple mismatches and wobble base pairs in the upper stem of pri-miRNAs, which determine the efficiency and accuracy of pri-miRNA processing. The existence of these RNA elements helps to explain the alternative cleavage mechanism of Microprocessor, which occurs for some human pri-miRNAs. We also showed that these RNA elements are targets of RNA-editing or single nucleotide polymorphisms (SNPs) for regulating miRNA biogenesis. These findings considerably improve our understanding of pri-miRNA processing mechanisms, and provide a foundation for interpreting differential miRNA expression by several mechanisms, such as RNA modifications and SNPs.
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Project description:MicroRNA (miRNA) biogenesis begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the processing of three pri-miR-9 paralogs (pri-miR-9-1, pri-miR-9-2 and pri-miR-9-3), which encode the same miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its kinked tertiary structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Here, we report the the miRNA-seq data of HEK293T and HeLa cell lines transfected with different pri-miR-9 constructs.
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