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ShRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE241303 | GEO | 2023/08/21

REPOSITORIES: GEO

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shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq]

Project description:miRNA overexpression strategy based on RNA Polymerase (Pol) III mediated expression of short hairpin RNA (shRNA) coding particular miRNA is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that extra \texttt{U} residues (up to five) added Pol III at the 3$'$-end of the transcribed shRNA associated with a shift in the Dicer cleavage position of the shRNA. This results in the undesirable formation of 5$'$-end miRNA isoforms (5$'$-isomiRs), which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. The predominant expression of 5$'$-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5$'$-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved.

2023-08-22 | GSE241301 | GEO

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [mRNA-seq]

2023-08-22 | GSE241300 | GEO

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq PC-3]

2024-04-17 | GSE264166 | GEO

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [DU-145 cells]

2023-11-17 | GSE247987 | GEO

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production

Project description:shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production

| PRJNA1007655 | ENA

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq]

Project description:shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq]

| PRJNA1007668 | ENA

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [mRNA-seq]

Project description:shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [mRNA-seq]

| PRJNA1007663 | ENA

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq PC-3]

Project description:shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq PC-3]

| PRJNA1101081 | ENA

shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [DU-145 cells]

Project description:shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [DU-145 cells]

| PRJNA1041356 | ENA

Synthetic Pre-miRNA-Based shRNA as Potent RNAi Triggers.

Project description:RNA interference (RNAi) is a powerful tool for studying gene function owing to the ease with which it can selectively silence genes of interest, and it has also attracted attention because of its potential for therapeutic applications. Chemically synthesized small interfering RNAs (siRNAs) and DNA vector-based short hairpin RNAs (shRNAs) are now widely used as RNAi triggers. In contrast to expressed shRNAs, the use of synthetic shRNAs is limited. Here we designed shRNAs modeled on a precursor microRNA (pre-miRNA) and evaluated their biological activity. We demonstrated that chemically synthetic pre-miRNA-based shRNAs have more potent RNAi activity than their corresponding siRNAs and found that their antisense strands are more efficiently incorporated into the RNA-induced silencing complex. Although greater off-target effects and interferon responses were induced by shRNAs than by their corresponding siRNAs, these effects could be overcome by simply using a lower concentration or by optimizing and chemically modifying shRNAs similar to synthetic siRNAs. These are challenges for the future.

| S-EPMC3139136 | biostudies-literature

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