Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sequence determinants of dsRNA processing by DICER [Rescue Experiment]

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE202533 | GEO | 2022/12/16

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Sequence determinants of dsRNA processing by DICER [Massively Parallel Assay]

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.

2022-12-16 | GSE202530 | GEO

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

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.

2023-11-30 | GSE247114 | GEO

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

2023-11-30 | GSE247113 | 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.

2023-09-06 | GSE226874 | GEO

Structure of the human DICER-pre-miRNA complex in a dicing state I

Project description:Dicer plays a key role in small RNA biogenesis by processing double-stranded RNAs (dsRNAs). Human DICER (hDICER) is specialized in processing of small hairpins such as pre-microRNAs (pre-miRNAs) with a limited activity towards long dsRNAs, unlike its homologs in lower eukaryotes and plants which cleave long dsRNAs. While the mechanism of long dsRNA cleavage has been well documented, our understanding of pre-miRNA processing is limited due to lack of the structure of hDICER in a catalytic state. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state, uncovering the structural basis for pre-miRNA processing.

2022-11-23 | GSE203062 | GEO

Structure of the human DICER-pre-miRNA complex in a dicing state II

2022-11-23 | GSE215866 | GEO

The effects of the 5' pocket motif of Dicer on miRNA biogenesis

Project description:A hallmark of RNA silencing is a class of ~22 nt RNAs which are processed from dsRNA precursor by Dicer. Accurate processing by Dicer is critical for the functionality of microRNAs (miRNAs). According to the current model, Dicer measures the length by anchoring the 3' overhang of the dsRNA terminus. Here we find that human Dicer binds to the 5' end of RNA and utilizes the 5' end as an additional reference point for cleavage site selection (5' counting rule). We further identify a novel motif (5'-pocket) in Dicer, which recognizes the 5' end of RNA. By analyzing miRNA population from 5'-pocket mutant Dicer expressing Dicer-null mES, we provide that the 5' pocket is significant for Dicer processing in vivo.

2011-07-13 | GSE27903 | GEO

The effects of the 5' pocket motif of Dicer on miRNA biogenesis

2011-07-12 | E-GEOD-27903 | biostudies-arrayexpress

Determinants of selectivity in the dicing mechanism

Project description:The RNase III enzyme, DICER, is instrumental in the production of small RNAs, including miRNAs and siRNAs, by cleaving their precursors, such as pre-miRNAs, shRNAs, and long duplex RNAs. Utilizing High-throughput (HT) cleavage assays, our study delves into the cleavage activity of DICER. We challenge the widely accepted 2-nt loop counting rule in the previous study, revealing a divergent mechanism, the bipartite base pairing rule. This rule directs DICER's cleavage sites via the RNase III domain. Moreover, we demystify the recognition mechanism of the previously identified YCR motif. Building on this understanding, a secondary YCR motif that also influences DICER's cleavage sites has been discovered. We also address a long-debated issue concerning DICER's cleavage sites on long stem RNAs, such as pre-siRNAs or long shRNAs/pre-miRNAs. Our study shows that the dsRBD plays a crucial role in determining the cleavage sites of DICER in long-stem RNAs. In sum, our research provides a comprehensive understanding of several fundamental DICER mechanisms, challenging the long-standing model of the loop counting rule. This newfound knowledge reshapes our understanding of DICER's mechanisms, providing a robust foundation for future studies investigating the vast number of DICER mutations linked to various diseases.

2024-06-01 | GSE252434 | GEO

Determinants of selectivity in the dicing mechanism

2024-06-01 | GSE252433 | GEO