Dataset Information

Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.

ABSTRACT: Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effector protein AGO2 at sites harboring partially complementary sequences to the siRNA remains unknown. Here, we report a strategy to establish a comprehensive picture of siRNA cleaved and noncleaved off-targets by performing SpyCLIP using wild-type and catalytically inactive AGO2 mutants in parallel. Additionally, we investigated naturally occurring cleavage events mediated by endogenous miRNAs using the same strategy. Our results demonstrated that AGO2 SpyCLIP is a powerful method to identify both the cleaved and noncleaved targets of siRNAs, providing valuable information for improving siRNA design rules.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC7666362 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.

Zhang Yao Y Teng Yilan Y Xiao Wangwen W Xu Beiying B Zhao Ya Y Li Weihua W Wu Ligang L

Molecular therapy. Nucleic acids 20201014

Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effect ...[more]

PMID: 33251041

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Project description:BACKGROUND: The discovery of RNA interference phenomenon (RNAi) and understanding of its mechanisms has revolutionized our views on many molecular processes in the living cell. Among the other, RNAi is involved in silencing of transposable elements and in inhibition of virus infection in various eukaryotic organisms. Recent experimental studies demonstrate few cases of viral replication suppression via complementary interactions between the mammalian small RNAs and viral transcripts. PRESENTATION OF THE HYPOTHESIS: It was found that >50% of the human genome is transcribed in different cell types and that these transcripts are mainly not associated with known protein coding genes, but represent non-coding RNAs of unknown functions. We propose a hypothesis that mammalian DNAs encode thousands RNA motifs that may serve for antiviral protection. We also presume that the evolutional success of some groups of genomic repeats and, in particular, of transposable elements (TEs) may be due to their ability to provide antiviral RNA motifs to the host organism. Intense genomic repeat propagation into the genome would inevitably cause bidirectional transcription of these sequences, and the resulting double-stranded RNAs may be recognized and processed by the RNA interference enzymatic machinery. Provided that these processed target motifs may be complementary to viral transcripts, fixation of the repeats into the host genome may be of a considerable benefit to the host. It fits with our bioinformatical data revealing thousands of 21-28 bp long motifs identical between human DNA and human-pathogenic adenoviral and herpesviral genomes. Many of these motifs are transcribed in human cells, and the transcribed part grows proportionally to their lengths. Many such motifs are included in human TEs. For example, one 23 nt-long motif that is a part of human abundant Alu retrotransposon, shares sequence identity with eight human adenoviral genomes. TESTING THE HYPOTHESIS: This hypothesis could be tested on various mammalian species and viruses infecting mammalian cells. IMPLICATIONS OF THE HYPOTHESIS: This hypothesis proposes that mammalian organisms may use their own genomes as sources of thousands of putative interfering RNA motifs that can be recruited to repress intracellular pathogens like proliferating viruses. REVIEWERS: This article was reviewed by Eugene V. Koonin, Valerian V. Dolja and Yuri V. Shpakovski.

Dataset Information

Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.

Publications

Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.

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