1 Department of Molecular Biology, Faculty of Science, Aarhus University, Aarhus University2 Department of Molecular Biology and Genetics - Gene Expression and Gene Medicine, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University3 Department of Molecular Biology and Genetics - Gene Expression and Gene Medicine, Department of Molecular Biology and Genetics, Science and Technology, Aarhus University
In a variety of S. cerevisiae mutants with defective mRNP maturation and/or export, heat shock (hs) mRNPs are retained at or near their sites of transcription. For example, mutants of the THO complex display an intense hs-mRNA FISH signal, which co-localizes with the hs-gene after transcriptional induction. The THO complex is implicated in co-transcriptional mRNP assembly, but its precise role is still unclear. Transcriptional run-on analysis as well as genetic interaction data suggest that the function of the THO complex is linked to 3’-end processing. Chromatin immunoprecipitation (ChIP) assays were used to compare the density of the transcription machinery at hs-loci in THO mutant versus wild type cells. Surprisingly, there is a bias in the composition of chromatin fragments recovered after in vivo crosslinking and high speed centrifugation: The 3’-ends of hs-loci are greatly underrepresented in recovered fractions from mutant cells. This bias is abolished when a THO mutation is combined with a second site mutation alleviating the mRNA export block. Thus, the bias parallels transcription-site retention of the mRNP and suggests the existence of a complex specifically formed at the 3’-end of hs-genes in THO complex mutants. Current experiments focus on locating the precise position of this complex relative to the hs-gene ORF as well as to identify the constituents of the retained material.In all cases examined so far, mRNP retention is dependent on Rrp6p, a component of the nuclear exosome of 3’-5’ exonucleases. We have analyzed retention capabilities of different Rrp6p mutants. We find that mutants which are deficient in 3’-5 exonucleolytic decay are also deficient in mRNP retention. This implies that retained mRNAs are undergoing 3’-5’ degradation. Other data supports this hypothesis. We discuss our data in the context of a model for Rrp6p-mediated retention of aberrant mRNP.