Abstract
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 subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). Rpb4/7 has been proposed to dissociate from Pol II, enter the cytoplasm, and function there in mRNA translation and degradation. Here we provide evidence that Rpb4 mainly functions in nuclear mRNA synthesis by Pol II, as well as evidence arguing against an important cytoplasmic role in mRNA degradation. We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis 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 that functions mainly in mRNA synthesis.
Highlights
The RNA polymerase II (Pol II) subunit Rpb4 was reported to function in mRNA degradation
We used metabolic RNA labeling and comparative Dynamic Transcriptome Analysis 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
These results show that the fusion mutant can adopt its mRNA metabolism after heat shock at 37 °C and that this does not depend on free Rpb4, further arguing against a direct role of Rpb4 in mRNA degradation
Summary
The RNA polymerase II (Pol II) subunit Rpb was reported to function in mRNA degradation. Rpb has been proposed to function directly in translation initiation [18] These studies led to the conclusion that Rpb4/7 influence transcription in the nucleus and mRNA degradation in the cytoplasm and were crucial for deriving the model of “mRNA imprinting” [20, 23, 24]. Rpb Functions Mainly in mRNA Synthesis degradation, a mutant has to be used in which the putative functions in transcription and degradation are uncoupled We created such a mutant by covalently linking Rpb to the Pol II core subunit Rpb. We could not detect strong differences in mRNA degradation between the Rpb2-Rpb mutant and wild-type yeast These results demonstrate that Rpb is a bona fide Pol II subunit with functions in transcription and do argue against the proposed direct function of Rpb in mRNA degradation
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