SF3B1 phosphorylation is an evolutionarily conserved step in spliceosome activation carried out by the divergent, OTS964-insensitive kinase CRK9 in trypanosomes

Abstract

SF3B1 is a subunit of the heptameric SF3B complex which, as part of the U2 small nuclear ribonucleoprotein, facilitates branch point recognition in pre-mRNA splicing. In addition to this early-stage function, it was recently shown that activation of the spliceosome depends on the phosphorylation of threonine-proline (TP) motifs in SF3B1s N-terminal domain (NTD) by cyclin-dependent kinase 11 (CDK11). This breakthrough result was made possible by the discovery of the CDK11-specific inhibitor OTS964. Trypanosomes are protistan parasites whose proteomes are highly divergent in sequence from those of model organisms, and thus their CDKs were generically named CDC2-related kinases (CRKs). We previously characterized the trimeric CRK9 complex of Trypanosoma brucei and showed that it is essential for spliced leader trans splicing, the predominant splicing mode in the parasite. Although CRK9 and CDK11 deviate from each other substantially, we show that CRK9 activity is required to maintain SF3B1 phosphorylation in vivo, CRK9 directly phosphorylates TP motifs in the SF3B1 NTD in vitro, and the TP motifs themselves are crucial for spliceosome activation, demonstrating evolutionary conservation of this essential splicing step. Contrary to CDK11 and human cells, CRK9 and trypanosomes were rather insensitive to OTS964, indicating potentially exploitable differences in their ATP-binding pockets.