Summary The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation and while recent studies show inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is exquisitely length-dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.

Summary Most human genes are loaded with promoter proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, a protein that renders Pol II responsive to mediator, is involved in Pol II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb and suggest that mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.

U1 snRNP (U1) functions in splicing introns and telescripting, which suppresses premature cleavage and polyadenylation (PCPA). Using U1 inhibition in human cells, we show that U1 telescripting is selectively required for sustaining long-distance transcription elongation in introns of large genes (median 39 kb). Evidence of widespread PCPA in the same locations in normal tissues reveals that large genes incur natural transcription attrition. Underscoring the importance of U1 telescripting as a gene-size-based mRNA-regulation mechanism, small genes were not sensitive to PCPA, and the spliced-mRNA productivity of ~1,000 small genes (median 6.8 kb) increased upon U1 inhibition. Notably, these small, upregulated genes were enriched in functions related to acute stimuli and cell-survival response, whereas genes subject to PCPA were enriched in cell-cycle progression and developmental functions. This gene size–function polarization increased in metazoan evolution by enormous intron expansion. We propose that telescripting adds an overarching layer of regulation to size–function-stratified genomes, leveraged by selective intron expansion to rapidly shift gene expression priorities.

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