We investigate the mechanism and regulation of minor splicing, and the associated diseases.
An essential and conserved machinery
Minor spliceosome is an essential nuclear machinery that is required for co-transcriptional removal of highly conserved minor intron sequences. Mammalian genomes contain ~700-800 genes , which each contain typically a single minor intron, also called as U12-type intron. This represents approximately 0.35% of all introns in mammals. In contrast, major spliceosome catalyses the removal of the majority (>99.5% ) of mammalian introns. The number of U12-type introns in Drosophila is 19, while in plants (Arabidopsis) the number is ~250.
U12-type introns are enriched in a subset of cellular pathways. The gene set containing U12-type introns codes for various essential cellular functions such as gene expression pathway (transcription, mRNA processing and translation), cytoskeleton organization, voltage-gated ion channels, MAPK signaling pathway (including nearly all cellular MAP kinases) and tumor suppressor functions.
The main regulatory function for U12-type introns is their slow or inefficient splicing. This is thought to limit the expression of genes containing U12-type introns. Consequently, the cellular levels of unspliced U12-type introns are higher than the levels of U2-type introns.
Regulation via unique snRNA and protein components
Minor spliceosome has unique snRNA and protein components. Both spliceosomes are composed of small nuclear RNAs (snRNAs) that associate with protein components to make up small nuclear ribonucleoproteins. Four of the five snRNPs are unique to the minor spliceosome. These are U11, U12, U4atac and U6atac snRNPs. U5 snRNP is shared with the major spliceosome. Additionally, minor spliceosome contains 7 unique protein components not found from the major spliceosome. They are all associated with the U11/U12 di-snRNP.
Unique snRNA and protein components regulate the minor spliceosome. We have identified two proteins involved in the regulation of minor spliceosome activity. The levels of U11-48K protein (SNRNP48 locus) are regulated through a negative feedback loop that leads to formation of stop-codon containing 48K mRNA that is subsequently degraded through the NMD pathway. The levels of U11/U12-65K protein (RNPC3 locus) are regulated at the level of nucleo-cytoplasmic export of the 65K mRNA and also through termination of transcription. This process is also regulated during neuronal differentiation. In addition, Dreyfuss lab has shown that U6atac snRNA levels are regulated by the p38-MAPK pathway.
Our current focus is to understand the mechanism and significance of U11-48K and U11/U12-65K regulatory pathways.
Mutations in the minor spliceosome
Six human diseases have been directly linked to the minor spliceosome. Of these, congenital mutations in U4atac snRNA lead to Microcephalic osteodysplastic primordial dwarﬁsm/Taybi-Linder Syndrome (MOPD1/TALS), Roifman syndrome (RFMN), and Lowry-Wood Syndrome (LWS). In U12 snRNP, congenital mutations in U12 snRNA lead to Early-Onset Cerebellar Ataxia (EOCA), and mutations in U11/U12-65K protein to Isolated growth hormone deficiency type I (IGHD). Additionally, somatic mutations in ZRSR2 protein have been associated with Myelodysplastic syndrome (MDS).
Mutations lead to defects in the splicing of U12-type introns. Disease-causing mutations targeting minor spliceosome components cause a partial loss-of-function phenotype, with a subset of U12-type introns failing to splice properly. Consequently, elevated levels of unspliced U12-type introns can be detected in the patient cells. Additionally, a subset of mRNAs show an activation of cryptic U2-type splice sites near the U12-type introns. These splicing defects can lead to formation of altered or defective mRNAs that are either destabilized by the NMD pathway, retained in the nucleus, or lead to formation of truncated or altered proteins.
Our research on minor spliceosome diseases is focused on molecular mechanisms of the disease-causing mutations and gene-expression consequences of the splicing defects.
Novel bioinformatics tools for intron retention
Increased retetion of U12-type introns. A main characteristic of U12-type introns is that they show a ~2-fold higher intron retention values compared to the nearby U2-type introns in the same gene in the cellular steady-state RNA pool. We have developed IntEREst, i.e. an R/Bioconductor tool for accurate intron retention quantification and comparison. Using IntEREst we have shown that the increased retention is a general feature of U12-type introns. Furthermore, we have shown that disease-causing mutations in the minor spliceosome components lead to further increase in the retention of U12-type introns.