Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast

Abstract

Gene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analyzed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts.A group of these mRNAs was also the target of the RNA binding protein, Puf3.We demonstrated that Rpb4 and Puf3 physically, genetically, and functionally interact and also affect mRNA stability, and likely the imprinting, of a common group of mRNAs. Furthermore, the Rpb4 and Puf3 association with mRNAs depends on one another. We also demonstrated, for the first time, that Puf3 associates with chromatin in an Rpb4-dependent manner. Our data also suggest that Rpb4 could be a key element of the RNA pol II that coordinates mRNA synthesis, imprinting and stability in cooperation with RBPs.

The Association of Rpb4 with RNA Polymerase II Depends on CTD Ser5P Phosphatase Rtr1 and Influences mRNA Decay in Saccharomyces cerevisiae

“…In agreement, RPB4/7 overexpression partially suppresses the mRNA stability increase provoked by the rpb6 Q100R mutation that affects the Rpb4/7 association with the rest of the enzyme [17]. Our data also point out that Rpb4-bound to chromatin-associated RNA pol II is a key element in Rpb4-mRNA imprinting, which coincides with previous propositions [14], as lack of Rtr1 does not alter the global Rpb4 amount, but increases the fraction of the free Rpb4 subunit.…”

“…As in S. cerevisiae, but also in other species, thiolutin affects multiple cellular pathways, such as proteasome activity, glucose metabolism or oxydative stress response, among others [82,[87][88][89], we cannot rule out side effects due to thiolutine addition. However, it should not significantly alter mRNA stability in our analyses, as the results obtained corroborated the described increase in mRNA stability caused by the absence of Rpb4, since the lack of Rtr1 affects the association of Rpb4 to the RNA pol II [13][14][15]17,68,71].…”

“…Previous results demonstrated a role for Rpb4 (and also for dimer Rpb4/7) in the life cycle of mRNA by imprinting it cotranscriptionally, and later accompanying it during the export to the cytoplasm, translation, mRNA decay or cytoplasmic accumulation. Furthermore, lack of Rpb4 increases mRNA stability [13][14][15]17,68,71]. Similarly, increased mRNA stability by RTR1 deletion was observed herein.…”

“…Rpb4/7 participates in different steps of transcription, such as initiation, elongation, termination, and polyadenylation, as well as gene looping and dephosphorylation of the CTD of Rpb1 [4][5][6][7][8][9][10]. Furthermore, Rpb4/7 binds mRNA cotranscriptionally, influencing mRNA export, translation, and decay [11][12][13][14][15][16][17][18]. Rpb4 and Rpb7 act as RNA binding proteins (RBP) and associate with regulatory factors such as Pat1, Lsm2, Puf3 or Not5 to link mRNA synthesis and decay [14][15][16]19,20].…”

“…Furthermore, Rpb4/7 binds mRNA cotranscriptionally, influencing mRNA export, translation, and decay [11][12][13][14][15][16][17][18]. Rpb4 and Rpb7 act as RNA binding proteins (RBP) and associate with regulatory factors such as Pat1, Lsm2, Puf3 or Not5 to link mRNA synthesis and decay [14][15][16]19,20]. Interestingly, post-translational modifications of Rpb4/7 are required to connect transcription with post-transcriptional mechanisms [21,22].…”