RNAP II CTD tyrosine 1 performs diverse functions in vertebrate cells

The RNA polymerase II largest subunit (Rpb1) contains a unique C-terminal domain (CTD) that plays multiple roles during transcription. The CTD is composed of consensus Y1S2P3T4S5P6S7 repeats, in which Ser, Thr and Tyr residues can all be phosphorylated. Here we report analysis of CTD Tyr1 using genetically tractable chicken DT40 cells. Cells expressing an Rpb1 derivative with all Tyr residues mutated to Phe (Rpb1-Y1F) were inviable. Remarkably, Rpb1-Y1F was unstable, degraded to a CTD-less form; however stability, but not cell viability, was fully rescued by restoration of a single C-terminal Tyr (Rpb1-25F+Y). Cytoplasmic and nucleoplasmic Rpb1 was phosphorylated exclusively on Tyr1, and phosphorylation specifically of Tyr1 prevented CTD degradation by the proteasome in vitro. Tyr1 phosphorylation was also detected on chromatin-associated, hyperphosphorylated Rpb1, consistent with a role in transcription. Indeed, we detected accumulation of upstream antisense (ua) RNAs in Rpb1-25F+Y cells, indicating a role for Tyr1 in uaRNA expression. DOI: http://dx.doi.org/10.7554/eLife.02112.001
eLife digest When a gene is expressed, the DNA is first transcribed to produce an intermediate molecule called a messenger RNA (mRNA), which is then translated to produce a protein. RNA Polymerase II is an enzyme that makes mRNA molecules in organisms as diverse as plants, animals and yeast. RNA Polymerase II is a complex made of a number of proteins. The largest protein in this complex includes a ‘carboxy-terminal domain’ that has multiple repeats of seven amino acids one after the other. The first amino acid in each repeat, a tyrosine, is referred to as tyrosine-1. Adding various chemical tags to the amino acids in these repeats co-ordinates the steps involved in the transcription of genes. In yeast, for example, adding a phosphate groups to tyrosine-1 seems to help the polymerase to proceed to make long mRNA molecules. However, it is not known what these chemical tags do in humans or other animals. Now Hsin et al. (and independently Descostes, Heidemann et al.) have shown that the same phosphate groups on tyrosine-1 perform functions in vertebrates (animals with backbones) that are different to those performed in yeast. These functions include protecting the carboxy-terminal domain from being broken down inside cells, and transcribing the DNA that is upstream of genes. Hsin et al. replaced tyrosine-1 in RNA Polymerase II from chicken cells with a related amino acid that cannot have phosphate groups added to it. This mutant RNA Polymerase II was unstable and degraded by the molecular machinery in cells that breaks down damaged or unneeded proteins back into amino acids. Hsin et al. also compared the mRNA molecules that are made by the wild-type RNA Polymerase II with those produced by a related mutant. This comparison revealed an unexpected accumulation of RNA molecules that are transcribed in the opposite direction from mRNAs. These RNA molecules, known as ‘upstream antisense RNAs’, have been described only recently. And while the function of these RNAs remains mysterious, the results of Hsin et al. suggest that tyrosine-1 helps to ensure that these RNA molecules are rapidly broken down. The results of Hsin et al. raise a number of important questions, and foremost among these questions is: how do these newly discovered properties of tyrosine-1 contribute to the control of gene expression in animals? Further work is needed to answer this question. DOI: http://dx.doi.org/10.7554/eLife.02112.002