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High-throughput in vitro DROSHA processing on human pri-miRNA variants

ABSTRACT: High-throughput in vitro DROSHA processing on human pri-miRNA variants

PROVIDER: PRJNA595970 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10708144.fastq.gz	Fastqsanger.gz
	SRR10708145.fastq.gz	Fastqsanger.gz
	SRR10708146.fastq.gz	Fastqsanger.gz
	SRR10708147.fastq.gz	Fastqsanger.gz
	SRR10708148.fastq.gz	Fastqsanger.gz

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High-throughput in vitro DROSHA processing on human pri-miRNA variants

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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High-throughput in vitro DROSHA processing on 38,880 pri-miRNA variants

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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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The high-throughput pri-miRNA cleavage assays on 262144 variants

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) 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 investigate the role of a long ncRNA transcribed from an ultraconserved region (T-UCR) in the control of post-transcriptional pri-miRNA processing. The regulation is based on complementarity between the lower stem region in pri-miR-195 transcript and the ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. Mutation of the site in either RNA molecule uncouples regulation in vivo and in vitro. We propose a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. In this work, we characterize a new role for Uc.283+A as a direct interactor and regulator of pri-miRNA-195 maturation at the level of Drosha processing. We combine cellular assays with in vitro biochemical analyses to reveal the first case of RNA-directed downregulation of miRNA biogenesis by a T-UCR In the study presented here, a colorrectal cancer cell line (HCT-116) was transiently transfected with Uc.283+A in order to identify putative miRNA targets for Uc.283+A. Variant 1 represents a SNP variant (8x(T) repeat in the sequence). Variant 2 represents a SNP variant (9x(T) repeat in the sequence).

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