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mRNA quality control is bypassed for immediate export of stress-responsive transcripts

Abstract

Cells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated1. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67–Mtr2 (TAP–p15 in humans)2. Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.

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Figure 1: Stress induces the dissociation of Mex67 and its adaptor proteins from mRNAs and Mex67-mediated export of heat-shock mRNAs.
Figure 2: Mex67 directly binds and exports heat-shock mRNAs using the same domain as it uses to interact with the adaptor protein Npl3.
Figure 3: Stress induces direct recruitment of Mex67 to promoters of heat-shock genes.
Figure 4: Stress-specific mRNAs are not quality controlled.

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Acknowledgements

We thank H. Bastians for advice, L. Oldehaver for technical assistance, W. Kramer for discussion and R. Lill, P. A. Silver, C. Dargemont and E. Hurt for providing plasmids, strains or antibodies. This work was funded by grants from the Deutsche Forschungsgemeinschaft and the SFB860 to H.K.

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Authors

Contributions

Experiments were designed and data interpreted by all authors; experiments were performed by G.Z. (Figs 1b, c, 2d, e, 3c, 4b–d), A.H. (Figs 1c, 3a, b, 4a), L.B. (Figs 1a, 2a–c) D.B. (Fig. 1c) and T.L. and G.S. (Fig. 1c). The manuscript was written by H.K.; all authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Heike Krebber.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Mex67 dissociates from poly(A)+ RNA together with the adaptor proteins, which are dispensable for heat-shock mRNA export.

a, Stress leads to the dissociation of Mex67 and its adaptor proteins from bulk mRNAs. Example experiments of immunoprecipitations from Fig. 1a with the indicated GFP-tagged or Myc-tagged proteins are shown in western blots. The co-precipitated poly(A)+RNA was detected in northern blots with a DIG-labelled oligo d(T)50 probe. b, Gbp2 precipitates after extended heat stress. Western blot analysis for the presence of Gbp2 in the soluble supernatant fraction or the insoluble pellet fraction is shown. c, Mex67 dissociates from mRNA, but not from its adaptor protein. Immunoprecipitation analysis of Mex67 and the co-precipitated Npl3 is shown in western blots. Mex67-bound poly(A)+RNA was analysed in northern blots. d, FISH experiments of the SSA4 heat-shock mRNA reveal export defects in mutants of MEX67–MTR2, but not the Mex67 adaptor proteins, upon a 30-min temperature shift to 42 °C. A DIG-labelled SSA4 RNA probe (green) detects the heat-shock mRNA in the indicated strains. DNA was stained with Hoechst (blue) and poly(A)+RNA with an oligo d(T)50 probe (red). e, Overview of the experiment shown in d. The frame indicates the enlarged single cells in d. f, Control experiment that shows the experiment described in d at 25 °C, in which heat-shock RNAs are not produced in visible amounts. g, The experiment was performed as described in d, with the cells shifted to 37 °C for 30 min. h, The same experiment as described in d with a different heat-shock RNA probe against HSP12. i, The same experiment as in d with probes against a single mRNA, GPM1 (Cy3, red) and poly(A)+ (Atto488, green). j, Control experiment that shows the experiment described in i without the DIG-labelled probe (green) or a Cy3 probe (red). The circle indicates the contour of the cells. k, Deletion of all three SR-protein genes is lethal to cells. The indicated strains were spotted in serial dilution onto agar plates selecting either for a covering plasmid (plates lacking uracil, −URA) or for the loss of this plasmid (5-fluoroorotic acid plates, FOA).

Extended Data Figure 2 Genome-wide analyses of Mex67- and Npl3-associated RNAs.

Dissociation of Mex67 and Npl3 from highly expressed transcripts upon a 30-min heat shock at 42 °C. a, Genome-wide RNA-seq studies reflect the dissociation of Mex67 and Npl3 from bulk RNA. RIP experiments were performed with both proteins before and after a 30-min incubation at 42 °C. Shown are 20 highly expressed transcripts arranged into functional groups that were bound by Mex67 or Npl3 and released upon heat shock. b, The top 50 transcripts from which indicated proteins dissociate that were obtained in microarray analysis are displayed by functional group. c, d, Top 50 transcripts obtained in RNA-seq. experiments for Npl3 (c) and Mex67 (d) are displayed by functional group. e, Genome-wide analysis of microarray data showing functional groups of mRNAs that co-purify with Npl3 or Mex67 at 25 °C or 42 °C (log2 ≥ 0.5). f, Reduced binding of Npl3 and Mex67 to housekeeping mRNAs (transcripts shown in e) upon shift from 25 °C to 42 °C.

Extended Data Figure 3 Analyses of Mex67 and Npl3 binding to mRNAs of Hsf1 controlled genes.

Mex67 binds preferentially to mRNAs controlled by Hsf1 at 42 °C. a, Detailed view with the bound Hsf1 gene names depicted shown in Fig. 1c. b, Heat map showing the change in binding from 25 °C to 42 °C for Npl3 and Mex67 for the genes depicted in Fig. 1c. c, qRT–PCR experiments for selected transcripts confirm the simultaneous binding of Mex67 and Npl3 to regular transcripts at 25 °C and the selective binding of Mex67 to heat-shock transcripts upon incubation for 30 min at 42 °C.

Extended Data Figure 4 The loop-domain of Mex67 is important for binding and export of heat-shock mRNAs.

a, b, Western blot showing purified recombinant His6–Mtr2 and the co-precipitated recombinant Mex67 with Mex67-specific antibodies (top) and anti-His antibodies (bottom) used for Fig. 2a, b (a and b, respectively). c, Scheme of the heterodimer Mex67–Mtr2 which indicates the loop domain that is mutated in the experiments shown in Fig. 2 (adapted from refs 9, 48). d, The loop mutants of Mex67 are not degraded in vivo. Western blots are shown from cells cultivated at 25 °C and from cells that were shifted to 42 °C for 30 min. e, f, FISH experiments reveal defects in the export of the heat-shock mRNAs SSA4 (e) or HSP12 (f) (green) in mutants of MEX67 with defects in the loop-domain upon 30-min heat shock at 42 °C. The framed areas in e indicate the enlarged cells shown in Fig. 2c. g, FISH experiments as performed in e and f, with probes against the single housekeeping mRNA GPM1, show accumulation in all strains. h, Quantification of four different experiments shown in Fig. 2d. i, In vitro interaction study of Npl3 and the indicated Mex67 proteins. Recombinant Mex67 or mutant mex67 were incubated with recombinant GST–Npl3. Subsequent co-immunoprecipitations of Npl3 with Mex67 are shown. j, Quantification of five different experiments shown in i. k, Increasing amounts of RNA, but not DNA, substantially reduce the interaction of Npl3 and Mex67. l, Full version of the western blot shown in Fig. 3b. The blot was cut into three pieces (black frame) and probed with the indicated antibodies. The green frame indicates the parts that are shown in Fig. 3b.

Extended Data Figure 5 Mex67 adaptor proteins have no mRNA export defects when deleted, but induce defects when overexpressed or mutated.

a, c, Deletion of the adaptor proteins results in no mRNA export defects and the nuclear accumulation seen in rrp6∆ cells alone is reduced when adaptor proteins are also deleted. Cells were grown at 30 °C (a) or at 25 °C and shifted to 37 °C for 3 h (c) before FISH experiments with an oligo d(T)50 probe (red, top), with a Cy3-labelled probe against an ADH1 promoter driven GFP RNA and the U3 snoRNA (red, bottom) were carried out (a). DNA was stained with Hoechst (blue). Framed areas reflect the enlarged cells. b, Quantification of FISH experiments shown in Fig. 4a. d, The deletion of NAB2 is lethal, but the simultaneous deletion of RRP6 supresses lethality and allows growth at 25 °C. The indicated strains are shown in tenfold serial dilution on plates that retain a covering plasmid or an empty vector (−URA) and on plates that select for the loss of the covering wild-type plasmid (FOA). e, The combination of rrp6mex67-5 is synthetically lethal. Growth of the indicated strains with (−URA) or without (FOA) a covering plasmid is shown. f, Overexpression of mex67-5 in the indicated rrp6adaptor protein∆ double mutants decreases the leakage of poly(A)+RNA to the cytoplasm as shown by FISH experiments. g, Overexpression of quality control factors from the strong GAL1 promoter is toxic to cells. The indicated strains are shown in tenfold serial dilution on the indicated plates. h, Overexpression of quality control factors lead to mRNA export defects and identifies them as mRNA retention factors. FISH with an oligo d(T)50 probe is shown in the indicated strains. i, Mutation in NPL3 is dominant and toxic to cells, as seen in serial drop tests. j, Mutation in NPL3 is dominant and causes mRNA export defects as shown in FISH experiments with an oligo d(T)50 probe.

Extended Data Figure 6 Heat-shock transcripts are not retained in the nuclei of cells defective in nuclear quality control factors.

a, FISH experiments with a DIG-labelled SSA4 probe (green) and an oligo d(T)50 probe (red) are shown in the indicated strains. Nuclear accumulation of mRNA was quantified (bottom). b, c, FISH experiments with DIG-labelled SSA4 and HSP12 probes (green) are shown in the indicated strains that had been shifted to 42 °C for 30 min (b) or 1 h (c). The frames in b indicate the enlarged cells that are also shown in a.

Extended Data Figure 7 Transcripts expressed from heat-shock promoters are not quality-controlled.

a, Representative expression analysis upon heat stress via qRT–PCR. Expression of GPM1 and HSP12, controlled by the indicated promoters, is shown upon 30-min heat stress at 42 °C. The endogenous SSA1 RNA served as a positive control. b, DNA staining of the cells shown in Fig. 4b. c, Experiment as shown in a but with CYC1 and HSP12 transcripts. d, Promoter-dependent accumulation of transcripts in quality-control-factor mutants. All strains carrying plasmids with the indicated gene constructs were analysed in FISH experiments with oligonucleotides targeting GFP mRNA (red) and poly(A)+ RNA (green). The DNA is stained with Hoechst (blue). Examples of typical cells (top) and quantification of the nuclear accumulation (bottom) is shown. e, The intron containing RPL23B RNA is expressed upon heat stress, when driven from the HSP12 promoter. qRT–PCR was carried out using lysates of log-phase cells that were subjected to 42 °C for 30 min. f, Schematic model of the retention of regular mRNAs under stress and the selective export of heat-shock mRNAs to the cytoplasm. Under normal conditions, adaptor proteins control the maturation of the transcripts and prevent the early association of Mex67 with Mtr2, avoiding premature nuclear export. Upon maturation, the adaptor proteins recruit Mex67–Mtr2, which allows subsequent nuclear export. Proper Mex67–Mtr2 coverage of the adaptor proteins is controlled at the NPC by the gatekeeper Mlp1 (top). During stress (bottom), regular mRNAs are retained in the nucleus by dissociation of Mex67–Mtr2 through its adaptor proteins. By contrast, heat-shock mRNAs circumvent an adaptor-mediated quality control by instant loading of Mex67–Mtr2 through Hsf1, resulting in an immediate nuclear export. Proper RNP formation is not controlled by Mlp1 at the NPC because Mlp1 is detached and accumulates in nuclear foci. This mechanism allows a quick switch between the controlled export of correct mRNAs and the immediate and uncontrolled export of stress-specific transcripts.

Extended Data Table 1 Yeast strains used in this study
Extended Data Table 2 Oligonucleotides used in this study
Extended Data Table 3 Plasmids used in this study

Supplementary information

Supplementary Information

This file contains Supplementary Methods for RIP-Microarray experiments and Supplementary Tables for Microarray. (PDF 159 kb)

Supplementary Information

This file contains Supplementary Methods for RNA-Seq experiments and Supplementary Tables for RNA-sequencing. (PDF 183 kb)

Supplementary Information

This file contains uncropped gel source data with size marker indications and Supplementary Figures 1-4. (PDF 8206 kb)

Supplementary Data

This file contains raw data of the Microarray with functional groups for each gene, genes sorted for binding >0,5 log2 fold change for Mex67 and Npl3 at 42°C and 25°C and raw data for the total dissociation of mRNAs at 42°C. (XLSX 3605 kb)

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Zander, G., Hackmann, A., Bender, L. et al. mRNA quality control is bypassed for immediate export of stress-responsive transcripts. Nature 540, 593–596 (2016). https://doi.org/10.1038/nature20572

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