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SiRNA Seed Region Is Divided into Two Functionally Different Domains in RNA Interference in Response to 2'-OMe Modifications.

ABSTRACT: In RNA interference (RNAi), small interfering RNA (siRNA) functions to suppress the expression of its target mRNA with perfect sequence complementarity. In a mechanism different from above, siRNA also suppresses unintended mRNAs with partial sequence complementarities, mainly to the siRNA seed region (nucleotides 2-8). This mechanism is largely utilized by microRNAs (miRNAs) and results in siRNA-mediated off-target effects. Thus, the siRNA seed region is considered to be involved in both RNAi and off-target effects. In this study, we revealed that the impact of 2'-O-methyl (2'-OMe) modification is different according to the nucleotide positions. The 2'-OMe modifications of nucleotides 2-5 inhibited off-target effects without affecting on-target RNAi activities. In contrast, 2'-OMe modifications of nucleotides 6-8 increased both RNAi and off-target activities. The computational simulation revealed that the structural change induced by 2'-OMe modifications interrupts base pairing between siRNA and target/off-target mRNAs at nucleotides 2-5 but enhances at nucleotides 6-8. Thus, our results suggest that siRNA seed region consists of two functionally different domains in response to 2'-OMe modifications: nucleotides 2-5 are essential for avoiding off-target effects, and nucleotides 6-8 are involved in the enhancement of both RNAi and off-target activities.

SUBMITTER: Kobayashi Y

PROVIDER: S-EPMC8771963 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype-phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.

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SiRNA Seed Region Is Divided into Two Functionally Different Domains in RNA Interference in Response to 2'-OMe Modifications.

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