Tho1 is necessary for recruitment of transcription elongation factors and nuclear mRNP assembly
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The formation of messenger ribonucleoprotein particles (mRNPs) is an essential step in gene expression. Already during transcription, RNA-binding proteins (RBPs) are recruited to the emerging mRNA; together they form the mRNP. RBPs orchestrate all important steps in the processing of mRNA. Additionally, they are necessary for mRNA stability, ... nuclear export and translation. The heterononameric TREX complex couples transcription to mRNA export. It consists of the pentameric THO sub-complex (Tho2, Hpr1, Mft1, Thp1 and Tex1), the SR-like proteins Gbp2 and Hrb1, the RNA helicase Sub2 and the export adaptor protein Yra1. The nuclear mRNA-binding protein Tho1 was identified based on its ability to supress the phenotype of THO mutants when overexpressed. Tho1 can bind RNA and its recruitment to the transcribed gene depends on the mRNA and the THO complex. The human orthologue of Tho1, CIP29, co-purifies with the TREX complex in the presence of ATP. The role of Tho1 in mRNP formation is still mainly unknown and a ∆tho1 strain has no known phenotype in S. cerevisiae. In a previous study, it could be shown that the phosphorylation of the C-terminal region (CTR) of Spt5 is important for the recruitment of Hpr1, Tho1 and Paf1. Paf1 is part of the PAF complex and functions in transcription elongation. The recruitment of Paf1 depends on the correct phosphorylation of the CTR. In CTR phosphorylation mutants, the recruitment of Tho1 resembles that of Paf1 and is anticorrelated with Hpr1 recruitment (Meinel, 2013). In this study, I was interested in the interplay between Tho1, Hpr1 and Paf1. First, growth assays with single and double mutants were performed. ∆tho1 suppresses the cold-sensitive growth defect of ∆hpr1 and ∆paf1. ChIP experiments showed that Tho1 regulates the recruitment of TREX to the transcribed gene in a negative manner. In contrast, Tho1 influences the recruitment of the PAF complex positively. Furthermore, we could show that Tho1 co-purifies with both the TREX/THO complex and the PAF-complex. Additionally, the deletion of hpr1 enhances the co-purification of Paf1 with Tho1. Tho1 is necessary for the correct assembly of nuclear mRNPs; changes in the level of Tho1 lead to changes in mRNP composition. No mRNA export defect was observed in cells where Tho1 was overexpressed or deleted. In sum, Tho1 is relevant for the recruitment of the elongation factor Paf1 and regulates the occupancy of Hpr1 at the transcribed gene. Moreover, changed intracellular Tho1 level leads to an altered mRNP assembly.