Minor intron splicing revisited: identification of new minor intron-containing genes and tissue-dependent retention and alternative splicing of minor introns

Abstract

Background Mutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA (microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1)) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues. Results We employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns. Conclusion Here we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at https://midb.pnb.uconn.edu . Electronic supplementary material The online version of this article (10.1186/s12864-019-6046-x) contains supplementary material, which is available to authorized users.

Minor spliceosome disruption causes limb growth defects without altering patterning

“…Since minor intron retention can introduce a premature stop codon and result in nuclear degradation or nonsense mediated decay (NMD), we analyzed the effect of minor intron retention on the open reading frame (ORF) of these 152 MIGs 3,9 . Indeed, we found that minor intron retention almost invariably resulted in the introduction of a premature stop codon (150/152; 98.6%) (Supp.…”

“…2c; Supp. Data File 1) 3,9 . Some of these MIGs are crucial for cell cycle regulation, including Cdc45 , Mau2 , and Spc24 , which suggested that the limb defects observed in our mutant mice stem from cell cycle dysregulation (Fig.…”

“…The minor spliceosome, which consists of five small nuclear RNAs (snRNAs) U11, U12, U4atac, U5, and U6atac and associated proteins, splices <0.5% of introns, termed minor introns, found in <2% of genes, termed minor intron-containing genes (MIGs) 1–3 . In metazoans, the minor spliceosome, MIGs, and the position of minor introns within MIGs are all highly conserved 4–6 .…”

The minor and major spliceosome interact to regulate alternative splicing around minor introns

“…The canonical spliceosome, also called the major spliceosome, splices major introns, whereas the minor spliceosome splices a small subset of introns called minor introns, that have divergent consensus sequences (Turunen et al, 2013). These minor introns are found in genes that are predominately made up of major introns, and thus the expression of these minor intron-containing genes (MIGs) requires the coordinated action of both the major and the minor spliceosome (Olthof et al, 2019). The splicing reactions executed by these two spliceosomes are analogous, as are the components.…”

“…In order to specifically study retention and AS, we have previously developed a customized bioinformatics pipeline that was used to identify several tissue-specific AS events around minor introns (Olthof et al, 2019). Here, we leveraged this pipeline to understand the effect of minor spliceosome inhibition on minor intron retention and AS.…”