We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3′ and/or 5′ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5′ differences and in support of this we report that a 5′ isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5′ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

Mutations of splice sites, auxiliary splicing elements and the splicing machinery cause a wide range of genetic disease. Here we report that many of the complex effects of splicing mutations can be predicted from background splicing information, with emphasis on BRCA1, BRCA2 and DMD. Background splicing arises from very low level splicing between rarely used background splice sites and from low-level exon skipping between intron splice sites. We show how this information can be downloaded from the Snaptron database of spliced RNA, which we then compared with databases of human splice site mutations. We report that inactivating mutations of intron splice sites typically caused the non-mutated partner splice site to splice to a known background splice site in over 90% of cases and to the strongest background splice site in the large majority of cases. Consequently, background splicing information can usefully predict the effects of splice site mutations, which include cryptic splice activation and single or multiple exon skipping. In addition, de novo splice sites and splice sites involved in pseudoexon formation, recursive splicing and aberrant splicing in cancer show a 90% match to background splice sites, so establishing that the enhancement of background splicing causes a wide range of splicing aberrations. We also discuss how background splicing information can identify cryptic splice sites that might be usefully targeted by antisense oligonucleotides (ASOs) and how it might indicate possible multiple exon skipping side effects of ASOs designed to induce single exon skipping.

We report that low level background splicing by normal genes can be used to predict the likely effect of splicing mutations upon cryptic splice site activation and exon skipping, with emphasis on the DBASS databases, BRCA1, BRCA2 and DMD. In addition we show that background RNA splice sites are also involved in pseudoexon formation, recursive splicing and aberrant splicing in cancer. We discuss how background splicing information might inform splicing therapy.

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