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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.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE41292 | GEO | 2012/12/25

SECONDARY ACCESSION(S): PRJNA176460

REPOSITORIES: GEO

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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. 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.

2012-12-25 | E-GEOD-41292 | biostudies-arrayexpress

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

Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects.

Project description:RNA interference (RNAi) is an evolutionarily conserved phenomenon of post-transcriptional gene silencing mediated by small interfering RNAs (siRNAs) generated from short hairpin RNAs (shRNAs) by Dicer cleavage. Here, we report that siRNA precursors can be divided into two categories with different processing mechanisms and silencing activities that are dependent on stem loop length. We designed an alternative siRNA precursor for triggering RNAi named single-stranded Argonaute2 (Ago2)-processed interfering RNA (saiRNA). saiRNA is composed of a 16-18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleavage ribozyme derived from hepatitis delta virus (HDV) to the 3’ end of the saiRNA dramatically improved its silencing activity. Unlike classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during its processing not only eliminated the passenger strand but also avoided the association of mature siRNA with non-nucleolytic Ago proteins, thereby further reducing the risk of off-target effects. Additionally, saiRNA exhibited less competition with the biogenesis of endogenous miRNAs. Therefore, HDV ribozyme-enhanced saiRNA provides a reliable tool for RNAi applications.

2015-06-01 | GSE68631 | GEO

A sliding-bulge structure at the Dicer processing site of pre-miRNA regulates alternative Dicer processing to generate 5’-isomiRs (miR-203)

Project description:5’-isomiRs expand the repertoire of miRNA targets. However, how they are generated is not well understood. Here, we showed that, for some miRNAs in mammalian cells, 5’-isomiRs are generated by alternative Dicer processing by using miRNA offset RNAs (moRs) to determine Drosha cleavage sites in cells. In addition, we showed that in miR-203, alternative Dicer processing is regulated by a conserved sliding-bulge structure at the Dicer processing site, which allows the pre-miRNA molecule to fold into two different structures that are processed differently by Dicer. So far no RNA motif that slides to change conformation and alter a protein–RNA interaction has been reported. Thus, our study revealed a novel RNA motif that regulates 5’-isomiR generation in some miRNAs. It might also contribute to regulating protein–RNA interactions in other biological processes, since it takes only one point mutation to generate the sliding bulge, and there are a large number of different RNAs in the cell.

2016-09-01 | GSE80772 | GEO

A sliding-bulge structure at the Dicer processing site of pre-miRNA regulates alternative Dicer processing to generate 5’-isomiRs (pre-miR-203)

2016-09-01 | GSE80771 | 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

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

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.

2023-11-30 | GSE247113 | GEO

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