GSE264166 shRNA-based miRNA overexpression results in significant off-target effects caused by isomiR production [miRNA-seq PC-3]

Contributors : Diana Maltseva ; Ivan Kirillov ; Anton Zhiyanov ; Roman Suvorov ; Daria Gubani ; Anna Kudriaeva ; Alexey Belogurov ; Alexander TonevitskySeries Type : Non-coding RNA profiling by high throughput sequencingOrganism : Homo sapiensmiRNA 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.

Source: GEO: Gene Expression Omnibus - April 17, 2024 Category: Genetics & Stem Cells Tags: Non-coding RNA profiling by high throughput sequencing Homo sapiens Source Type: research