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The high-throughput DICER cleavage assays on 23296 shRNA variants

ABSTRACT: The high-throughput DICER cleavage assays on 23296 shRNA variants

PROVIDER: PRJNA757440 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR15600188_1.fastq.gz	Fastqsanger.gz
	SRR15600188_2.fastq.gz	Fastqsanger.gz
	SRR15600189_1.fastq.gz	Fastqsanger.gz
	SRR15600189_2.fastq.gz	Fastqsanger.gz
	SRR15600190_1.fastq.gz	Fastqsanger.gz

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The high-throughput DICER cleavage assays on 23296 shRNA variants

Project description:The high-throughput DICER cleavage assays were conducted shRNA variants containing different sequences. We showcase a comprehensive cleavage activity of DICER on different shRNAs containing different secondary structures.

2022-02-21 | GSE182701 | GEO

Two-motif model illuminates DICER's cleavage preferences [DICER]

Project description:DICER is a key regulator of gene expression in animals through its production of miRNAs and siRNAs by processing shRNA. To advance our understanding of this process, we employed high-throughput assays using various shRNA variants and both wild-type and mutant DICER (DICERΔdsRBD). Our analysis revealed that DICER predominantly cleaves shRNAs at two positions, specifically at 21 (DC21) and 22 (DC22) nucleotides from the 5'-end. Our investigation identified two independent motifs, mAHG and YSR, that determine whether DICER cleaves at DC21 or DC22, depending on their location in shRNA/pre-miRNAs. These motifs can work together or independently to determine the cleavage sites of DICER. Furthermore, our findings indicate that dsRBD enhances cleavage at DC21, and mAHG strengthens the interaction between dsRBD and RNA, leading to an even greater enhancement of DC21 cleavage. Conversely, YSR functions independently of dsRBD. Our study proposes a two-motif model that sheds light on the intricate regulatory mechanisms involved in gene expression by elucidating how DICER recognizes its substrate, providing valuable insights into this critical biological process.

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Two-motif model illuminates DICER's cleavage preferences [DICER-mutants]

Project description:In humans, DICER is a key regulator of gene expression in animals through its production of miRNAs and siRNAs by processing miRNA precursors (pre-miRNAs), short-hairpin RNAs (shRNAs), and long double-stranded RNAs (dsRNAs). To advance our understanding of this process, we employed high-throughput assays using various shRNA variants and both wild-type and mutant DICER (DICERΔdsRBD). Our analysis revealed that DICER predominantly cleaves shRNAs at two positions, specifically at 21 (DC21) and 22 (DC22) nucleotides from the 5'-end. Our investigation identified two independent motifs, mWCU and YCR, that determine whether DICER cleaves at DC21 or DC22, depending on their locations in shRNA/pre-miRNAs. These motifs can work together or independently to determine the cleavage sites of DICER. Furthermore, our findings indicate that dsRBD enhances cleavage at DC21, and mWCU strengthens the interaction between dsRBD and RNA, leading to an even greater enhancement of DC21 cleavage. Conversely, YCR functions independently of dsRBD. Our study proposes a two-motif model that sheds light on the intricate regulatory mechanisms involved in gene expression by elucidating how DICER recognizes its substrate, providing valuable insights into this critical biological process.

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Two-motif model illuminates DICER's cleavage preferences [DICER-rescue]

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

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Two-motif model illuminates DICER cleavage preferences

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