Connecting Spliceosome Complexity with Intron Diversity

Prof. Dr. Florian Heyd

October 01, 2021

Our lab is interested in the regulation and functional consequences of alternative splicing

In higher eukaryotes, in the majority of primary transcripts, the protein coding mRNA sequence is interrupted by one or more non-coding introns. Introns show massive variations in length, structure, sequence composition and the quality of splicing signals. Despite the intronic complexity, it is believed that the same major spliceosome assembles on each of these introns, with less than 1% of all introns being processed by a second (the minor) spliceosome.

The spliceosome is a highly dynamic machinery that assembles on each intron in an ordered manner to catalyze the two transesterification reactions of the splicing process. The spliceosome consists of five preformed RNA-protein particles (snRNPs) and a large number of auxiliary proteins, that often associate with the spliceosome only during a clearly defined stage of the splicing reaction. Despite detailed structural information of stable splicing intermediates the exact localization and function of many auxiliary proteins remains enigmatic.

Here, we plan a systematic analysis of spliceosome-associated proteins to understand their importance and – potentially intron-specific – function in splicing. In a pilot study of siRNA-mediated perturbation of the spliceosomal C* complex we found many C*-specific protein factors regulating a specific subtype of introns with two adjacent alternative 3’- splice sites. We plan to use the same bioinformatics pipeline to find intron-specific effects of other spliceosome associated factors. We will combine publically available RNA sequencing data and create novel datasets for additional components of the spliceosome to reveal protein-specific changes in splice site selection. Ultimately, this will correlate specific intron features with auxiliary splicing factors. For example, only a very small subset of human introns is characterized by a TG dinucleotide at the 3’- splice site but it is not known which auxiliary splicing factors are essential for splicing of this intron subtype. Second, we will perform mutagenesis to further characterize the interplay of the cis-regulatory environment and the trans-acting splicing factors. In a minigene analysis, we will define sequence elements necessary and sufficient for the splicing regulation by a specific factor. In addition, we will also establish complementation assays to define functional domains in the trans-acting factors and we may further address their functionality using structural biology techniques. Finally, by comparing RNA sequencing data from different tissues and development stages, we will correlate expression levels of splicing factors with global changes in certain splicing subtypes to reveal the biological function and regulatory potential of spliceosome-associated factors.

For more information have a look at the website of the RNA Biochemistry group.

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