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Dissociation of Rpb4 from RNA polymerase II is important for yeast functionality.

ABSTRACT: Rpb4 is an RNA polymerase II (Pol II) subunit that binds Pol II transcripts co-transcriptionally, accompanies them to the cytoplasm and modulates mRNA export, translation and decay by interacting with cytoplasmic RNA modulators. The importance of the cytoplasmic roles of Rpb4 was challenged by a study reporting that the phenotype of rpb2Δ rpb4Δ cells can be rescued by an Rpb2-Rpb4 fusion protein, assuming that its Rpb4 moiety cannot dissociate from Pol II and functions in the cytoplasm. Here we demonstrate that although the fusion protein supports normal transcription, it adversely affects mRNA decay, cell proliferation and adaptability-e.g., response to stress. These defects are similar, albeit milder, than the defects that characterize rpb4Δ cells. At least two mechanisms alleviate the deleterious effect of the fusion protein. First, a portion of this fusion protein is cleaved into free Rpb2 and Rpb4. The free Rpb4 is functional, as it binds mRNAs and polysomes, like WT Rpb4. Second, the fusion protein is also capable of binding poly(A)+ mRNAs in the cytoplasm, in an Rpb7-mediated manner, probably complementing the functions of the diminished Rpb4. Collectively, normal coupling between mRNA synthesis and decay requires wild-type configuration of Rpb4, and fusing Rpb4 to Rpb2 compromises this coupling.

SUBMITTER: Duek L

PROVIDER: S-EPMC6201915 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation.

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The Rpb4 subunit of RNA polymerase II contributes to cotranscriptional recruitment of 3' processing factors.

Project description:The RNA polymerase II enzyme from the yeast Saccharomyces cerevisiae is a complex of 12 subunits, Rpb1 to Rpb12. Crystal structures of the full complex show that the polymerase consists of two separable components, a 10-subunit core including the catalytic active site and a heterodimer of the Rpb4 and Rpb7 subunits. To characterize the role of the Rpb4/7 heterodimer during transcription in vivo, chromatin immunoprecipitation was used to examine an rpb4Delta strain for effects on the behavior of the core polymerase as well as recruitment of other protein factors involved in transcription. Rpb4/7 cross-links throughout transcribed regions. Loss of Rpb4 results in a reduction of RNA polymerase II levels near 3' ends of multiple mRNA genes as well as a decreased association of 3'-end processing factors. Furthermore, loss of Rpb4 results in altered polyadenylation site usage at the RNA14 gene. Together, these results indicate that Rpb4 contributes to proper cotranscriptional 3'-end processing in vivo.

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An Rpb4/Rpb7-like complex in yeast RNA polymerase III contains the orthologue of mammalian CGRP-RCP.

Project description:The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Deltarpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases.

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Rpb4 functions mainly in mRNA synthesis by RNA polymerase II

Project description:RNA polymerase II (Pol II) is the central enzyme that carries out eukaryotic mRNA transcription and consists of a 10-subunit catalytic core and a heterodimeric subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to shuttle from the nucleus to the cytoplasm, and to function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, and evidence arguing against an important cytoplasmic role. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis (cDTA) to show that Rpb4 deletion in Saccharomyces cerevisiae causes a drastic defect in mRNA synthesis that is compensated by down-regulation of mRNA degradation, resulting in mRNA level buffering. Deletion of Rpb4 can be rescued by covalent fusion of Rpb4 to the Pol II core subunit Rpb2, which largely restores mRNA synthesis and degradation defects caused by Rpb4 deletion. Thus Rpb4 is a bona fide Pol II core subunit which functions mainly in mRNA synthesis.

2014-05-09 | E-MTAB-2539 | biostudies-arrayexpress

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