The high-throughput cleavage assays on 137 C.elegans pri-miRNAs
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ABSTRACT:
PROVIDER: PRJNA874678 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21291929_1.fastq.gz | Fastqsanger.gz | |||
| SRR21291929_2.fastq.gz | Fastqsanger.gz | |||
| SRR21291930_1.fastq.gz | Fastqsanger.gz | |||
| SRR21291930_2.fastq.gz | Fastqsanger.gz | |||
| SRR21291932_1.fastq.gz | Fastqsanger.gz |
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Project description:Microprocessor (MP), cleaving primary microRNAs (pri-miRNAs) to initiate the biogenesis of thousands of miRNAs, was discovered in animals in 2004. Understanding the molecular mechanism of MP is critical for interpreting the gene-silencing roles of miRNAs in various cellular processes and human diseases. Though the molecular mechanism of human MP (hMP) has been comprehensively investigated for nearly two decades and is well understood, that of Caenorhabditis elegans MP (cMP, cDrosha-Pasha complex) is still unknown. In this study, we revealed a comprehensive molecular mechanism of cMP, distinctively from that of hMP. In the proposed mechanism, cDrosha and Pasha (DGCR8 in humans) measure ~16 bp and ~25 bp of the pri-miRNA stem, respectively, and they are coordinated in determining cleavage sites of cMP in pri-miRNAs. In addition, we also demonstrated the molecular basis for their substrate measurement. Our findings illustrate an unknown molecular mechanism of cMP, clarify differences between the mechanism of hMP and cMP, and provide a foundation for understanding multiple mechanisms regulating miRNA expression acting on cMP.
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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:The high-throughput pri-miRNA cleavage assays on 262144 variants
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Project description:Human Microprocessor cleaves pri-miRNAs to initiate miRNA biogenesis. The accuracy and efficiency of Microprocessor cleavage ensure appropriate miRNA sequence and expression and thus its proper gene regulation. However, Microprocessor cleaves many pri-miRNAs incorrectly, so it requires assistance from its many cofactors. For example, SRSF3 enhances Microprocessor cleavage by interacting with the CNNC motif in pri-miRNAs. However, whether SRSF3 can function with other motifs and/or requires the motifs in a certain secondary structure is unknown. In addition, the function of SRSF7 (a paralog of SRSF3) in miRNA biogenesis still needs to be discovered. Here, we demonstrated that SRSF7 could stimulate Microprocessor cleavage. In addition, by conducting high-throughput pri-miRNA cleavage assays for Microprocessor and SRSF7 or SRSF3, we demonstrated that SRSF7 and SRSF3 function with the CRC and CNNC motifs, adopting certain secondary structures. In addition, SRSF7 and SRSF3 affect the Microprocessor cleavage sites in human cells. Our findings demonstrate the roles of SRSF7 in miRNA biogenesis and provide a comprehensive view of the molecular mechanism of SRSF7 and SRSF3 in enhancing Microprocessor cleavage.
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Project description:The high-throughput human pri-miRNA processing assays
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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 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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Project description:Targeted sequencing at Microprocessor cleavage sites in pri-miRNAs to determine processing efficiency of several pri-miRNAs in vivo in one experiment
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Project description:Targeted sequencing at Microprocessor cleavage sites in pri-miRNAs to determine processing efficiency of several pri-miRNAs in vivo in one experiment
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Project description:MicroRNAs (miRNAs) are short non-coding RNAs that play essential roles in RNA silencing and gene regulation. The human Microprocessor (MP) is the key factor to initiate miRNA biogenesis by cleaving primary microRNAs (pri-miRNAs). However, the Microprocessor alone cannot precisely and efficiently cleave all pri-miRNAs; thus, it requires cofactors to assist its cleavage. SRSF3 interacts with CNNC in pri-miRNAs, enhancing the MP cleavage. However, it is unknown if SRSF3 can function with other non-CNNC motifs and if secondary structure might influce CNNC function. In addition, function of SRSF7, a paralog of SRSF3, in the SR proteins family, in miRNA biogenesis is largely unknown. In this study, by conducting the high-throughput pri-miRNA cleavage assays for the MP with SRSF3 or SRSF7 and randomized pri-miRNAs, we discovered that SRSF7 also stimulate MP clevage. Futhermore, we found that both SRSF3 and SRSF7 can function with some non-CNNC motifs and with CNNC motifs with certain secondary structures. Furthermore, we also demonstrated that SRSF7 and SRSF3 governed the cleavage sites of the Microprocessor in human cells. Our findings disclose the roles SRSF7 in miRNA biogenesis, demonstrate a compresenhive moleucalr mechanism of SFSF3 and SRSF7 in enhancing cleavage of MP and described in more detail the RNA-binding features of SRSF7 and SRSF3.
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