Nuclear mRNA degradation tunes the gain of the unfolded protein response in Saccharomyces cerevisiae.

Unfolded protein response (UPR) is triggered by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which is accomplished by a dramatic induction of genes encoding ER chaperones. Activation of these genes involves their rapid transcription by Hac1p, encoded by the HAC1 precursor transcript harboring an intron and a bipartite element (3'-BE) in the 3'-UTR. ER stress facilitates intracellular targeting and recruitment of HAC1 pre-mRNA to Ire1p foci (requiring 3'-BE), leading to its non-spliceosomal splicing mediated by Ire1p/Rlg1p. A critical concentration of the pre-HAC1 harboring a functional 3'-BE element is governed by its 3'→5' decay by the nuclear exosome/DRN. In the absence of stress, pre-HAC1 mRNA undergoes a rapid and kinetic 3'→5' decay leading to a precursor pool, the majority of which lack the BE element. Stress, in contrast, causes a diminished decay, thus resulting in the production of a population with an increased abundance of pre-HAC1 mRNA carrying an intact BE, which facilitates its more efficient recruitment to Ire1p foci. This mechanism plays a crucial role in the timely activation of UPR and its prompt attenuation following the accomplishment of homeostasis. Thus, a kinetic mRNA decay provides a novel paradigm for mRNA targeting and regulation of gene expression.

RRM1 and PAB domains of translation initiation factor eIF4G (Tif4631p) play a crucial role in the nuclear degradation of export-defective mRNAs in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the CBC-Tif4631p-dependent exosomal targeting (CTEXT) complex consisting of Cbc1/2p, Tif4631p and Upf3p promotes the exosomal degradation of aberrantly long 3'-extended, export-defective transcripts and a small group of normal (termed 'special') mRNAs. We carried out a systematic analysis of all previously characterized functional domains of the major CTEXT component Tif4631p by deleting each of them and interrogating their involvement in the nuclear surveillance of abnormally long 3'-extended and export-defective messages. Our analyses show that the N-terminal RNA recognition motif 1 (RRM1) and poly(A)-binding protein (PAB) domains of Tif4631p, spanning amino acid residues, 1-82 and 188-299 in its primary structure, respectively, play a crucial role in degrading these aberrant messages. Furthermore, the physical association of the nuclear exosome with the altered/variant CTEXT complex harboring any of the mutant Tif4631p proteins lacking either the RRM1 or PAB domain becomes abolished. This finding indicates that the association between CTEXT and the exosome is accomplished via interaction between these Tif4631p domains with the major exosome component, Rrp6p. Abolition of interaction between altered CTEXT (harboring any of the RRM1/PAB-deleted versions of Tif4631p) and the exosome further leads to the impaired recruitment of the RNA targets to the Rrp6p subunit of the exosome carried out by the RRM1/PAB domains of Tif4631p. When analyzing the Tif4631p-interacting proteins, we identified a DEAD-box RNA helicase (Dbp2p), as an interacting partner that turned out to be a previously unknown component of CTEXT. The present study provides a more complete description of the CTEXT complex and offers insight into the functional relationship of this complex with the nuclear exosome.

The Sequential Recruitments of Rab-GTPase Ypt1p and the NNS Complex onto pre-HAC1 mRNA Promote Its Nuclear Degradation in Baker's Yeast.

Induction of unfolded protein response involves activation of transcription factor Hac1p that is encoded by HAC1 pre-mRNA harboring an intron and a bipartite element (BE), which is subjected to nuclear mRNA decay by the nuclear exosome/Cbc1p-Tif4631p-dependent Exosome Targeting (CTEXT) complex. Using a combination of genetic and biochemical approaches, we demonstrate that a Rab-GTPase Ypt1p controls unfolded protein response signaling dynamics. This regulation relies on the nuclear localization of a small fraction of the cellular Ypt1p pool in the absence of endoplasmic reticulum (ER)-stress causing a strong association of the nuclear Ypt1p with pre-HAC1 mRNA that eventually promotes sequential recruitments of NNS, CTEXT, and the nuclear exosome onto this pre-mRNA. Recruitment of these decay factors onto pre-HAC1 mRNA is accompanied by its rapid nuclear decay that produces a precursor RNA pool lacking functional BE thereby causing its inefficient targeting to Ire1p foci leading to their diminished splicing and translation. ER stress triggers rapid relocalization of the nuclear pool of Ypt1p to the cytoplasm leading to its dissociation from pre-HAC1 mRNA thereby causing decreased recruitment of these decay factors to precursor HAC1 RNA leading to its diminished degradation. Reduced decay results in an increased abundance of pre-HAC1 mRNA with intact functional BE leading to its enhanced recruitment to Ire1p foci.

Determination of the Stability and Intracellular (Intra-Nuclear) Targeting and Recruitment of Pre-HAC1 mRNA in the Saccharomyces cerevisiae During the Activation of UPR.

Nuclear degradation of pre-HAC1 mRNA and its subsequent targeting plays a vital role in the activation as well as attenuation of Unfolded Protein Response (UPR) in Saccharomyces cerevisiae. Accurate measurement of the degradation of precursor HAC1 mRNA therefore appears vital to determine the phase of activation or attenuation of this important intracellular signaling pathway. Typically, pre-HAC1 mRNA degradation is measured by the transcription shut-off experiment in which RNA Polymerase II transcription is inhibited by a potent transcription inhibitor to prevent the de novo synthesis of all Polymerase II transcripts followed by the measurement of the steady-state levels of a specific (e.g., pre-HAC1) mRNA at different times after the inhibition of the transcription. The rate of the decay is subsequently determined from the slope of the decay curve and is expressed as half-life (T1/2). Estimation of the half-life values and comparison of this parameter determined under different physiological cues (such as in absence or presence of redox/ER/heat stress) gives a good estimate of the stability of the mRNA under these conditions and helps gaining an insight into the mechanism of the biological process such as activation or attenuation of UPR.Intra-nuclear targeting of the pre-HAC1 mRNA from the site of its transcription to the site of non-canonical splicing, where the kinase-endonuclease Ire1p clusters into the oligomeric structures constitutes an important aspect of the activation of Unfolded Protein Response pathway. These oligomeric structures are detectable as the Ire1p foci/spot in distinct locations across the nuclear-ER membrane under confocal micrograph using immunofluorescence procedure. Extent of the targeting of the pre-HAC1 mRNA is measurable in a quantified manner by co-expressing fluorescent-labeled pre-HAC1 mRNA and Ire1p protein followed by estimating their co-localization using FACS (Fluorescence-Activated Cell Sorter) analysis. Here, we describe detailed protocol of both determination of intra-nuclear decay rate and targeting-frequency of pre-HAC1 mRNA that were optimized in our laboratory.

Nuclear mRNA Surveillance Mechanisms: Function and Links to Human Disease.

Production of export-competent mRNAs involves transcription and a series of dynamic processing and modification events of pre-messenger RNAs in the nucleus. Mutations in the genes encoding the transcription and mRNP processing machinery and the complexities involved in the biogenesis events lead to the formation of aberrant messages. These faulty transcripts are promptly eliminated by the nuclear RNA exosome and its cofactors to safeguard the cells and organisms from genetic catastrophe. Mutations in the components of the core nuclear exosome and its cofactors lead to the tissue-specific dysfunction of exosomal activities, which are linked to diverse human diseases and disorders. In this article, we examine the structure and function of both the yeast and human RNA exosome complex and its cofactors, discuss the nature of the various altered amino acid residues implicated in these diseases with the speculative mechanisms of the mutation-induced disorders and project the frontier and prospective avenues of the future research in this field.