Biochemical and drug sensitivity analyses indicate that in metazoans the kinase CDK9 can promote the elongation phase of mRNA transcription, and can phosphorylate RNA Polymerase II (Pol II) on its C-terminal domain (CTD). During the transcription process, the CTD repeat YSPTSPS is first phosphorylated on Ser 5 by the CDK7 kinase, then Ser 2 phosphorylation is observed on elongating polymerases. At most genes, CTD phosphorylation is essential for transcription elongation, and for coupling transcription to mRNA processing steps. CDK9 can phosphorylate the CTD on Ser 2 in vitro , but whether CDK9 is important for Ser 2 phosphorylation in vivo is unknown. The complex of CDK9 with its partner CyclinT is also recruited directly by the human immunodeficiency virus (HIV) protein Tat, which binds to the nascent HIV RNA and is required for production of full-length viral transcripts. It is an important question whether CDK9 is broadly essential for transcription in vivo or has a specialized role. We have addressed this question by studying CDK9/Cyclin T functions in the C. elegans embryo. C. elegans encodes a single
cdk-9 gene, and two cyclin T (cyt) orthologs. Similarly to
ama-1(RNAi) (Pol II) embryos,
cdk-9(RNAi) embryos arrest development at around 100 cells without any evidence of differentiation. Staining with antibodies against Pol II CTD phosphoepitopes indicates that in
cdk-9(RNAi) embryos, CTD Ser 5 phosphorylation is essentially normal, but Ser 2 phosphorylation is undetectable. In
cdk-9(RNAi) embryos we have also not detected expression of any GFP reporters that correspond to early embryonic genes, and have obtained essentially identical results when both cyclin T genes are inhibited. We conclude that CDK9/Cyclin T is essential for mRNA transcription, and may phosphorylate CTD Ser 2 in vivo . Biochemical and some yeast genetic evidence indicates that the protein complex SPT4/SPT5 can inhibit transcription elongation, and that this effect may be counteracted by CDK9, which can phosphorylate SPT5 in vitro . To test this model, in C. elegans we have inhibited
spt-4 and
spt-5 by RNAi simultaneously with
cdk-9 . We find that for the most part, lack of
spt-4 and
spt-5 does not overcome the requirement for CDK9/Cyclin T for transcription. Strikingly, however, inhibition of
spt-4 and
spt-5 can relieve this requirement at heat shock genes, which in yeast are regulated distinctly in that they can be transcribed in the absence of the CTD. We conclude that CDK9/CyclinT can overcome the inhibitory effects of SPT4/SPT5 in vivo , but that this aspect of its function is insufficient at most genes, which depend also upon its phosphorylation of the CTD. Efforts are now underway to determine whether CDK9/Cyclin T is required in vivo not only for transcription elongation, but also for efficient mRNA splicing.