Browse
Submit Data
Databases
API
Help

Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo

ABSTRACT: Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies. Various shRNAs were expressed in Cell and processed by the RNase III enzyme Dicer. The profiles of the siRNA products were generated by deep sequencing with or without the Ago2-IP.

ORGANISM(S): Homo sapiens

SUBMITTER: shuo gu

PROVIDER: E-GEOD-41292 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Json Xml

Shared Molecules

Only show the datasets with similarity scores above: 0.5

Threshold

0.5

Similar Datasets

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo

Project description:Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

2012-12-25 | GSE41292 | GEO

Determinants of selectivity in the dicing mechanism III

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.

2024-07-13 | GSE272135 | GEO

Determinants of selectivity in the dicing mechanism

2024-06-01 | GSE252434 | GEO

Determinants of selectivity in the dicing mechanism

2024-06-01 | GSE252433 | 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. Examination of small RNA profiles from Dicer-null mouse embryonic stem cells transfected with either wild-type or 5' pocket mutant Dice expression plasmids.

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

The effect of 22-bulge of pre-miRNAs on DICER cleavage in human cells.

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 | GSE192613 | GEO

The effect of 22-bulge of pre-miRNAs on DICER cleavage in human cells

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

Dicer Partner Proteins Tune the Length of Mature miRNAs in Flies and Mammals

Project description:Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. loquacious (loqs) encodes three Dicer partner proteins, Loqs-PA, Loqs-PB, and, Loqs-PD, generated by alternative splicing. To understand the function of each Loqs isoform, we constructed loqs isoform-specific rescue flies. Loqs-PD promotes siRNA production in vivo by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: Loqs-PB is required to produce a specific subset of miRNAs. Surprisingly, Loqs-PB tunes the product size cleaved by Dicer-1 from pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The mouse and human Dicer-binding partner TRBP, a homolog of Loqs-PB, similarly tunes the site of pre-miR-132 cleavage by mammalian Dicer. Thus, Dicer-binding partner proteins can change the choice of cleavage site by Dicer, producing miRNAs with different target specificities than those that would be made by Dicer alone. Examination of Dicer-binding proteins on small RNA profiles of female fly heads, fly ovaries, mouse embryonic fibroblasts, and mouse tail fibroblasts.

2012-12-07 | E-GEOD-37443 | biostudies-arrayexpress

Endogenous shRNAs, siRNAs, and Other Microprocessor-independent, Dicer-dependent Small RNAs in Mouse ES Cells

Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes. Small RNAs were sequenced from wt, dgcr8(-), and dicer(-) mouse ES cells and the frequencies of small RNA types compared between the three. This record includes Illumina-platform-generated datasets from all three samples and 454-platform-generated datasets from wt and dgcr8(-) samples. [raw data files are unavailable]

2008-09-09 | E-GEOD-12521 | biostudies-arrayexpress

The effect of 22-bulge of shRNAs on DICER cleavage in human cells

Project description:The effect of 22-bulge of shRNAs on DICER cleavage in human cells

| PRJNA757441 | ENA