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bioRxiv · 10.64898/2026.10.26.761550

Inhibition of CDK7, CDK9 or CDK12 affects phosphorylation of human SPT5.

Murphy, S.·Maudlin, I.·Barker, S.·Mali, G. R.·Riepsaame, J.·Sheldon, S.
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Abstract

SPT5, complexed with SPT4 in the DRB-sensitivity-inducing factor, DSIF, is a critical regulator of transcription elongation that acts negatively in the unphosphorylated form and positively when phosphorylated. In vivo, phosphorylation of multiple sites in the largely unstructured linker and C-terminal regions of the protein facilitates release from an early elongation checkpoint and processive transcription elongation. Transcription elongation factors, including the PAF complex (PAF1C), interact with phosphorylated SPT5 and phosphorylation of residues S666 and T806 are associated with the positive elongation functions of SPT5. Here, using in vitro phosphorylation of regions of the SPT5 C-terminus followed by GST-mediated pull-down we confirm that the RTF1 subunit of PAF1C interacts with residues 750-850 only when phosphorylated. Antibodies we raised to phospho-S666 or phospho-T806 are specific for these sites phosphorylated in vitro and in vivo. Western analysis of endogenous SPT5 in HEK293 cells using these antibodies before and after inhibition of analog-sensitive versions of CDK7, CDK9 and CDK12 indicate that all three kinases play roles in the phosphorylation of SPT5, although inhibition of CDK9 has the most marked effect. CDK9 inhibition also inhibits the association of PAF1C subunits with SPT5 in vivo and affects phosphorylation of chromatin-associated SPT5 as measured by chromatin immunoprecipitation (ChIP).

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Murphy, S., Maudlin, I., Barker, S., Mali, G. R., Riepsaame, J., Sheldon, S.. 2026-10-28. Inhibition of CDK7, CDK9 or CDK12 affects phosphorylation of human SPT5.. https://doi.org/10.64898/2026.10.26.761550

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