Trans-splicing in C. Elegans

University Of Colorado, Boulder CO

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Abstract

Operons are an important feature of the C. elegans genome. Their transcripts are polycistronic pre-mRNAs that get processed by 3' end formation and trans-splicing. These two evens occur in close proximity between operon genes. Although 3' end formation is generally accompanied by transcription termination, in operons it is not. We have identified the key sequences and many of the key trans-acting proteins and snRNAs responsible for carrying out these events, and we are determining what roles they play and how. The key sequence required for both the trans-splicing and for preventing transcription termination is the Ur element that occurs ~50 bp downstream of the 3' end cleavage site. We will determine what binds there. We are also studying the roles of the protein components of the snRNP that does this specialized transsplicing event. Functional studies involving mutants and RNAi of these components and biochemical studies in an in vitro splicing extract are proposed. We have discovered that one of these proteins is bound to a novel snRNA, Sm Y, currently the only snRNA whose function is not known. We will study the role of this RNA in trans-splicing and operon pre-mRNA processing. We have identified several proteins likely to play roles in 3' end formation and transcription termination as suppressors of a mutation that inserts a transcription termination site into the first gene of an operon. One of these proteins is an exonuclease, another is an SR protein, and three others all have a domain that indicates they perform their functions by binding directly to RNA polymerase. We will determine what functions these proteins have in 3' end formation. These studies should provide important new insight into how 3' end cleavage can occur without accompanying transcription termination.