von Hippel–Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination

doi:10.1073/pnas.0436037100

von Hippel–Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination

^*Department of Molecular and Cellular Physiology, ^‡Pediatric Informatics, Children's Hospital Research Foundation, ^¶Department of Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576; ^§Department of Informatics, Nicholas Copernicus University, 87-100, Torun, Poland; ^‖Stowers Institute for Medical Research, Kansas City, MO 64110; and ^**Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160

Edited by Robert G. Roeder, The Rockefeller University, New York, NY, and approved January 7, 2003 (received for review October 7, 2002)

Abstract

The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1α (HIF-1α) that binds von Hippel–Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-α and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

Footnotes

↵ † A.V.K. and J.M. contributed equally to this work.
↵ ‡‡ To whom correspondence should be addressed at: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, P.O. Box 670576, Cincinnati, OH 45267-0576. E-mail: Maria.Czyzykkrzeska{at}uc.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

pVHL,

von Hippel–Lindau protein;

TH,

tyrosine hydroxylase;

PC12,

phoechromocytoma cell line;

CTD,

carboxyl-terminal domain;

Rpb1 or Rpb6,

subunit 1 or 6 of RNA polymerase II;

HA,

hemagglutinin;

CbzLLn,

N-Cbz-l-Leu-l-Leu-l-norvalinal;

HIF,

hypoxia-inducible factor;

RCC,

renal cell carcinoma;

ODDD,

oxygen-dependent degradation domain