Characterizing the in vivo functions of cyclin L and its binding partners in pre-mRNA splicing and cell cycle control

Cyclins and cyclin-dependent kinases (CDKs) selectively form a complex that plays a central role in driving cell cycle progression. The functions of both CDKs and cyclins are well conserved across eukaryotes. Notably, the gene families encoding these two types of proteins have undergone significant expansion in multicellular organisms relative to single-celled organisms. However, the in vivo roles of many CDKs and their binding cyclins remain uncharacterized, despite intensive studies of CDKs and their associated cyclins in vitro.

Here we propose to characterize the highly conserved cyclin L (CYL-1), an orthologue of human CYL1, and its binding partners in Caenorhabditis elegans. CYL-1 was pulled out in our genetic screen for genes regulating tissue-specific cell cycle length. The depletion of CYL- 1 decreased the cell cycle length specifically in the daughters of intestine progenitor cell, a phenotype proposed to be associated with transcription. CYL1 forms a complex with CDK11 that is required for RNA splicing of a subset of genes in cultured human cells. Human cyclin L forms a complex with both CDK11 and CDK12, although this association remains controversial. However, our pull-down assay using cyclin L as bait supports the associations of cyclin L with both CDK-11 and CDK-12 in vivo in C. elegans. Importantly, the pull-down assay also identified an association of cyclin L with another highly conserved but uncharacterized protein, Y67D2.7. We hypothesize that the CYL-1, Y67D2.7 and CDK-12 all function in vivo to regulate splicing and cell cycle progression. To test this hypothesis, we will first confirm the interactions between these proteins using independent assays. We will next define the exact roles of these proteins in genome-wide pre-mRNA processing, including alternative use of promoter, exon combination and polyadenylation site, as well as in RNA base modification using direct RNA sequencing technology. We will then investigate the roles of these proteins in the regulation of cell cycle progression. Finally, we will evaluate the extent to which their splicing-related functions are conserved between C. elegans and humans.

The proposed studies will not only establish the precise roles of cyclin L and its binding proteins in pre-mRNA processing and cell cycle regulation in vivo, but will also establish functions for Y67D2.7, a highly conserved yet uncharacterized protein. Given that CDK11 and CDK12 are emerging therapeutic targets for cancer, the characterization of their in vivo functions in splicing and cell cycle control will foster the development of novel therapeutic strategies for cancer intervention.