ABSTRACT
RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.
Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Active Transport, Cell Nucleus , Binding Sites , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/genetics , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Stress, Physiological , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Termination of Saccharomyces cerevisiae RNA polymerase II (Pol II) transcripts occurs through two alternative pathways. Termination of mRNAs is coupled to cleavage and polyadenylation while noncoding transcripts are terminated through the Nrd1-Nab3-Sen1 (NNS) pathway in a process that is linked to RNA degradation by the nuclear exosome. Some mRNA transcripts are also attenuated through premature termination directed by the NNS complex. In this paper we present the results of nuclear depletion of the NNS component Nab3. As expected, many noncoding RNAs fail to terminate properly. In addition, we observe that nitrogen catabolite-repressed genes are upregulated by Nab3 depletion.
Subject(s)
Nitrogen/metabolism , Nuclear Proteins/metabolism , RNA Polymerase II/genetics , RNA, Messenger/genetics , RNA, Small Nucleolar/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Termination, Genetic/physiology , Catabolite Repression/genetics , Codon, Nonsense/genetics , Glutamate Plasma Membrane Transport Proteins/genetics , Glutamate-Ammonia Ligase/antagonists & inhibitors , Glutamate-Ammonia Ligase/metabolism , IMP Dehydrogenase/biosynthesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/biosynthesisABSTRACT
The fidelity of RNA splicing is maintained by a network of factors, but the molecular mechanisms that govern this process have yet to be fully elucidated. We previously found that TDP-43, an RNA-binding protein implicated in neurodegenerative disease, utilizes UG microsatellites to repress nonconserved cryptic exons and prevent their incorporation into mRNA. Here, we report that two well-characterized splicing factors, polypyrimidine tract-binding protein 1 (PTBP1) and polypyrimidine tract-binding protein 2 (PTBP2), are also nonconserved cryptic exon repressors. In contrast to TDP-43, PTBP1 and PTBP2 utilize CU microsatellites to repress both conserved tissue-specific exons and nonconserved cryptic exons. Analysis of these conserved splicing events suggests that PTBP1 and PTBP2 repression is titrated to generate the transcriptome diversity required for neuronal differentiation. We establish that PTBP1 and PTBP2 are members of a family of cryptic exon repressors.
Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/genetics , Nerve Tissue Proteins/genetics , Neurons/metabolism , Polypyrimidine Tract-Binding Protein/genetics , RNA Splicing , RNA, Messenger/genetics , Transcriptome , Animals , Base Sequence , Brain/cytology , Brain/metabolism , Cell Differentiation , Exons , HeLa Cells , Heterogeneous-Nuclear Ribonucleoproteins/antagonists & inhibitors , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Mice , Microsatellite Repeats , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Neurons/cytology , Polypyrimidine Tract-Binding Protein/antagonists & inhibitors , Polypyrimidine Tract-Binding Protein/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Yeast RNA polymerase II (Pol II) terminates transcription of coding transcripts through the polyadenylation (pA) pathway and non-coding transcripts through the non-polyadenylation (non-pA) pathway. We have used PAR-CLIP to map the position of Pol II genome-wide in living yeast cells after depletion of components of either the pA or non-pA termination complexes. We show here that Ysh1, responsible for cleavage at the pA site, is required for efficient removal of Pol II from the template. Depletion of Ysh1 from the nucleus does not, however, lead to readthrough transcription. In contrast, depletion of the termination factor Nrd1 leads to widespread runaway elongation of non-pA transcripts. Depletion of Sen1 also leads to readthrough at non-pA terminators, but in contrast to Nrd1, this readthrough is less processive, or more susceptible to pausing. The data presented here provide delineation of in vivo Pol II termination regions and highlight differences in the sequences that signal termination of different classes of non-pA transcripts.
