The high-throughput DICER cleavage assays on 23296 shRNA variants
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA757440 | ENA |
REPOSITORIES: ENA
Dataset's files
| 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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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.
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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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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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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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Project description:The experiments were conducted to examine the effects of 22-bulge of pre-miRNAs on DICER cleavage in human cells.
2022-02-21 | GSE183552 | GEO
Project description:The experiments were conducted to examine the effects of 22-bulge of pre-miRNAs on DICER cleavage in human cells.
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Project description:RNA silencing relies on specific and efficient processing of dsRNA by DICER which produces microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, our current knowledge of DICER’s specificity is restricted to the secondary structures of its substrates: a dsRNA than 22 bp with a 2-nt 3′ overhang. We recently found evidence pointing to additional sequence-dependent determinant(s) beyond these features. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with over a million pre-miRNA variants. Our analyses revealed a highly conserved cis-acting element, termed the “GYM” motif (paired G, paired pyrimidine, and mismatched C or A) at the cleavage site, which strongly promotes processing at a specific position. We find that the C-terminal double-stranded RNA binding domain (dsRBD) of DICER recognizes the GYM motif. Mutation of the dsRBD alters pre-miRNA cleavage sites and impairs processing in a motif-dependent fashion, which in turn affects the miRNA repertoire in cells. Consistently, integrating the GYM motif into short hairpin RNA (shRNA) or Dicer substrate siRNA (DsiRNA) potentiates RNA interference.
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