Regulation and functional consequences of alternative 3’ splice site selection

Prof. Dr. Florian Heyd

October 07, 2022

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

In higher eukaryotes, most pre-mRNAs undergo alternative splicing, generating multiple mRNAs and proteins from a single gene. Alternative splicing is dynamically regulated, often in a tissue- or development specific manner. Exon inclusion or intron retention programs are regulated by RNA binding proteins that can activate or repress splicing, and their fundamental biological relevance has been demonstrate in diverse biological systems. In contrast, much less is known for alternative 5′ or 3′ splice site selection, which affects the length of an exon by altering its 5′ or 3′ end. Alternative 3’ splice site (A3’ss) selection – especially for adjacent A3’ss – occurs during the second step of splicing, when the 5’ splice site and the branch point are already defined. A3’ss therefore requires a different mode of splicing regulation. 

In a recent study, we have identified several metazoan specific splicing factors, regulating adjacent A3’ss in human cells (unpublished data). These factors are first recruited to the spliceosome at the C* stage, the complex catalyzing the second step in the splicing reaction, revealing a paradigm for splicing regulation after the first step. Here, we plan to further investigate mechanistic principles and to reveal biological functions of adjacent A3’ss selection. Combining a bioinformatics analysis of C* factor specific A3’ss with mutagenesis of reporter minigenes will allow us to decipher the cis-regulatory code that controls A3’ss selection – for example in a tissue or development specific manner. In addition, we will use our Cryo-EM map of the C* complex to correlate the positioning of C* factors relative to potential cis-regulatory elements to reveal regulatory principles at atomic resolution. Finally, comparing A3’ss changes upon C* factor knockdowns with publically available RNA sequencing data of different tissues, developmental stages or disease conditions, will guide us towards understanding biological functions of regulated A3’ss selection. Interestingly, we find that the majority of C* factors control a concerted switch in A3’ss selection – i.e. global use of either the distal or proximal acceptor site. Such a concerted switch in A3’ss selection has been observed in a cell density dependent manner, making C* factors prime candidates for regulated A3’ss selection in this context. We will therefore investigate signaling pathways controlling C* factor levels or activity, which will ultimately help us to understand how these regulated changes in A3’ss selection control cellular functionally in different conditions. Altogether, we will combine RNA-Seq and structural biology with RNA biochemistry, cell culture and in vivo experiments to understand the mechanism and functional impact of alternative 3’ splice site selection.  

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

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