Functional mechanisms of specific proteins of pre-catalytic B complex spliceosomes in constitutive and alternative splicing

• Applicants: Professor Dr. Reinhard Lührmann; Professor Dr. Markus C. Wahl
• Subject Area: Biochemistry
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 257515868

For each pre-mRNA splicing event, a spliceosome is assembled de novo by the stepwise recruitment of snRNP and non-snRNP subunits. Spliceosomes of higher eukaryotes contain about 80 factors, which are not present in lower eukaryotes (such as yeast) and which may be required to implement alternative splicing. While initial recognition of the splice sites by the core machinery commits a pre-mRNA to the splicing pathway, the decision on a specific splicing pattern occurs at a later stage. Of particular importance in the latter decision is the conversion of an initial cross-exon to a cross-intron pre-catalytic spliceosome, an aspect of the preferred assembly pathway in higher eukaryotes. At this assembly stage a group of nine proteins are recruited, which are released again in the following spliceosome activation step and which therefore are of prime importance for alternative splicing regulation. Most of these so-called B-specific proteins are not present in yeast. Current data suggest that the B-specific proteins may serve to stably integrate the U4/U6-U5 tri-snRNP or to recruit other stage-specific proteins (the Bact-specific, Prp19 and/or Prp19-related proteins, several of which again lack orthologs in yeast). Interactions involving the B-specific proteins may also function as checkpoints that must be met before spliceosome activation is initiated. B-specific proteins could thereby serve for deciding on a particular splicing pattern and different alternative splicing events may have different requirements for the B-specific proteins. However, presently next to nothing is known about the molecular mechanisms underlying the presumed functions of the B-specific proteins. Here, we therefore suggest a combined biochemical, structural and functional analysis of B-specific proteins. We propose to investigate the protein neighborhoods of B-specific proteins in pre-catalytic spliceosomes, to determine putative sites of interaction with pre-mRNAs, to determine atomic structures of complexes involving B-specific proteins and to engineer protein variants with reduced interaction potential or lacking functional regions. We will assess the effects of depletion/knock-down of B-specific proteins on constitutive and alternative splicing and on the switch from cross-exon to cross-intron complexes. We will then use recombinant wild type and dysfunctional B-specific protein variants in add-back/rescue experiments to determine which properties of these proteins are required for their influences on splicing processes. The expected results will advance our fundamental understanding of the spliceosome and of the principles underlying alternative splicing.

• DFG Programme: Research Grants