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

ABSTRACT: The experiments were conducted to examine the effects of 22-bulge on DICER cleavage in human cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182700 | GEO | 2022/02/21

REPOSITORIES: GEO

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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.

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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.

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

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

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

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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.

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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.

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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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