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Unbiased mapping of Thr4 phosphorylation on RNA Pol II CTD indicates role in elongation and 3'-end processing

ABSTRACT: RNA polymerase II relies on a repetitive sequence domain (YSPTSPS) within its largest subunit to orchestrate transcription. While the significance of phosphorylation on Ser2/Ser5 is well-established, the role of Thr4 remains enigmatic. Thr4 phosphorylation has been implicated in elongation, termination, and mRNA processing, yet it has only been detected after transcription end sites, presenting a paradox. Our investigation revealed that Thr4 phosphorylation is initially obscured by flanking Ser5 phosphorylation at the onset of transcription. By selectively removing this masking effect using a phosphatase, we unveiled the true genomic location of this evolutionarily conserved phosphorylation mark, shedding light on Thr4 phosphorylation cellular functions. Subsequent proteomic analyses unearthed that many proteins previously associated with Ser2 are actually recruited to transcription via Thr4. Crucially, Thr4 phosphorylation primes Ser2 phosphorylation, leading to lethal defects in 3'-end processing. Our findings delineate the true genomic location and function of this "phantom" mark, prompting a reassessment of functional assignments of the CTD heptad.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Edward Marcotte

PROVIDER: MSV000094312 | MassIVE | Wed Mar 13 10:57:00 GMT 2024

SECONDARY ACCESSION(S): PXD050595

REPOSITORIES: MassIVE

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Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3’-end processing

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