Identifying the binding aites of CFIM-1 in C. elegans
3’untranslated regions (3’UTRs) are important noncoding elements at the ends of mRNA transcripts containing various sequence elements targeted by gene regulatory factors. 3’UTR lengths are defined by a process known as RNA cleavage and polyadenylation, which is performed by a large, multi-subunit complex known as the RNA cleavage and polyadenylation complex (CPC).
The CPC recognizes and binds to various sequence elements within 3’UTRs, including bipartite elements identified by a CPC subcomplex known as cleavage factor 1 (CF1), which acts as an enhancer of the cleavage reaction.
Dysregulation of CF1 leads to altered gene expression, which in turn contributes to many diseases, including cancers and neurological disorders.
Despite its significance, CF1 is poorly characterized. Our project focuses on identifying the RNA binding site of CFIM-1, a member of CF1, in the model organism C. elegans. Since 3’UTR biology is highly conserved among metazoans, our results can later be used to gain insights into 3’UTR processing in humans.
We are using two convergent strategies: immunoprecipitation and sequencing of RNA bound to tagged CFIM-1, and direct RNA editing on 3’UTRs by generating a transgenic worm expressing a chimeric protein consisting of the RNA base editor APOBEC-1 fused to CFIM-1. This second approach enables precise editing of CFIM-1-bound RNAs that can be identified via RNA sequencing.
Currently, we are constructing transgenes for these experiments, which will advance our understanding of CF1’s role in RNA cleavage and polyadenylation and how it contributes to disease when it becomes dysregulated.
