Abstract
Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1–decaysome and Ccr4–Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.
Highlights
Gene expression was thought to be a linear process where each step came after the without interfering in each other’s paths
We studied the response of GAL1 to the genetic perturbation of mRNA decay and transcription elongation machineries by measuring parameters that define the different levels of the gene expression pathway
We studied mutants lacking Xrn1, the main cytoplasmic 5 -3 exoribonuclease [33]; Ccr4, the main cytoplasmic deadenylase and a subunit of the Ccr4–Not complex [10]; Not4, another component of this complex [34] that controls de ubiquitylation and degradation of arrested RNA polymerase II (RNAPII) [35]; Dhh1, an RNA helicase involved in several steps of the mRNA cycle that is tightly associated with both de decaysome [9,22] and Ccr4– Not complexes [36,37]; Dst1/Transcription factor IIS (TFIIS), a transcription factor able to stimulate the resolution of RNAPII backtracking events [38]; Sfp1, a transcription factor that stimulates expression of most growth-related genes and that functionally interacts with TFIIS [39]; and Pfd1, a subunit of the prefoldin complex that contributes to chromatin dynamics during elongation and displays a strong functional interaction with TFIIS [40]
Summary
Gene expression was thought to be a linear process where each step came after the without interfering in each other’s paths. A few transcription factors have already been found to be implicated in this cross-talk since they affect the fate of nascent mRNA This process has been named mRNA imprinting [5], and the most prominent factors involved are Rpb4/7, components of the RNA polymerase II (RNAPII) [6] and the multifunctional Ccr4–Not complex [7]. These factors bind to the nascent mRNA in the nucleus, influencing its export into the cytoplasm, translation and degradation.
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