Our group is working with the model organism Schizosaccharomyces pombe (fission yeast) to unravel a control mechanism of basic pre-mRNA splicing in eukaryotes. Based on bioinformatic analysis comparing the fission yeast splicing factors with the splicing factors of budding yeast (Sacharomyces cerevisiae) and Homo sapiens we suggested that fission yeast contains a pre-mRNA splicing machinery more closely reflecting the archetype of a spliceosome (Käufer and Potashkin, 2000).
Intensive genetic studies allowed us to establish an epistasis map of many spliceosomal factors and lead to the identification and isolation of Prp4 kinase (Alahari et al. 1993, Gross et al. 1997). Prp4 kinase is one of the 17 fission yeast kinases which are essential for cell growth and is known to be involved in splicing. Multiple genetic interactions have been identified between Prp4 Kinase, Prp1, Prp8, Brr2 and many other spliceosomal proteins (Käufer and Kuhn, 2003, Bottner et al. 2005).
Recently, we showed that Prp4 kinase plays an important role during splice site recognition and the efficient splicing of introns displaying weak exon1/5' splice sites and weak branch sequences. It became clear that there exist two classes of introns: One class needs the Prp4 kinase to be spliced out and therefore these introns are called Prp4-dependent. The other class is called Prp4-independent because these introns do not need the Prp4 kinase activity. Notably, there are only slight differences in the sequence features of the splice sites between these two classes (see figure).
Hypothetical example of RNA-RNA interaction during intron recognition in fission yeast. Consensus sequences of the splice sites of Prp4-independent and -dependent introns are shown.
Nevertheless, analysis of different mutations in exon1/5' splice site and branch sequence showed that increasing the complementarity to the snRNAs U1 and U2 of the spliceosome can change a Prp4-dependent intron into an -independent one and vice versa. Based on this finding it seems that the proper transient interaction between the 5' end of snRNA U1 and the exon1/5' splice site as well as the snRNA U2 and the branch sequence interaction governs the dependence of splicing on Prp4 kinase activity (Eckert et al. 2016).
The kinase is not directly attached to the spliceosome but phosphorylates the spliceosomal protein Prp1 in vivo (Schwelnus et al. 2001) and the SR protein Srp2 in vitro (Lützelberger and Käufer, 2012). How the phosphorylation of these substrates influences the recognition and the efficient splicing of introns with weak exon1/5' splice sites and weak branch sequences remains unclear and is one question for the future.