Introns are mediators of cell response to starvation

Article metrics

Abstract

Introns are ubiquitous features of all eukaryotic cells. Introns need to be removed from nascent messenger RNA through the process of splicing to produce functional proteins. Here we show that the physical presence of introns in the genome promotes cell survival under starvation conditions. A systematic deletion set of all known introns in budding yeast genes indicates that, in most cases, cells with an intron deletion are impaired when nutrients are depleted. This effect of introns on growth is not linked to the expression of the host gene, and was reproduced even when translation of the host mRNA was blocked. Transcriptomic and genetic analyses indicate that introns promote resistance to starvation by enhancing the repression of ribosomal protein genes that are downstream of the nutrient-sensing TORC1 and PKA pathways. Our results reveal functions of introns that may help to explain their evolutionary preservation in genes, and uncover regulatory mechanisms of cell adaptations to starvation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Introns are required for cell survival under starvation conditions.
Fig. 2: Intron deletions repress cell growth by promoting entry into the stationary phase.
Fig. 3: Introns promote cell survival independently of host-gene expression.
Fig. 4: The 5′ UTR is required for intron functions.
Fig. 5: Intron deletion increases the expression of genes associated with translation and respiration.
Fig. 6: Introns promote nutrient-dependent repression of ribosome biogenesis.

Data availability

Supplementary Data are available online. Additional data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo) under the accession number GSE111056. All strains are available upon request.

References

  1. 1.

    Irimia, M. & Roy, S. W. Origin of spliceosomal introns and alternative splicing. Cold Spring Harb. Perspect. Biol. 6, a016071 (2014).

  2. 2.

    Jo, B. S. & Choi, S. S. Introns: the functional benefits of introns in genomes. Genomics Inform. 13, 112–118 (2015).

  3. 3.

    Neuvéglise, C., Marck, C. & Gaillardin, C. The intronome of budding yeasts. C. R. Biol. 334, 662–670 (2011).

  4. 4.

    Hooks, K. B., Delneri, D. & Griffiths-Jones, S. Intron evolution in Saccharomycetaceae. Genome Biol. Evol. 6, 2543–2556 (2014).

  5. 5.

    Hartung, F., Blattner, F. R. & Puchta, H. Intron gain and loss in the evolution of the conserved eukaryotic recombination machinery. Nucleic Acids Res. 30, 5175–5181 (2002).

  6. 6.

    Spingola, M., Grate, L., Haussler, D. & Ares, M. Jr. Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae. RNA 5, 221–234 (1999).

  7. 7.

    Parenteau, J. et al. Deletion of many yeast introns reveals a minority of genes that require splicing for function. Mol. Biol. Cell 19, 1932–1941 (2008).

  8. 8.

    Cox, J. S. & Walter, P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391–404 (1996).

  9. 9.

    Gray, J. V. et al. “Sleeping beauty”: quiescence in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 68, 187–206 (2004).

  10. 10.

    Munding, E. M., Shiue, L., Katzman, S., Donohue, J. P. & Ares, M. Jr. Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing. Mol. Cell 51, 338–348 (2013).

  11. 11.

    González, A. & Hall, M. N. Nutrient sensing and TOR signaling in yeast and mammals. EMBO J. 36, 397–408 (2017).

  12. 12.

    Martin, D. E., Soulard, A. & Hall, M. N. TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell 119, 969–979 (2004).

  13. 13.

    Peter, G. J., Düring, L. & Ahmed, A. Carbon catabolite repression regulates amino acid permeases in Saccharomyces cerevisiae via the TOR signaling pathway. J. Biol. Chem. 281, 5546–5552 (2006).

  14. 14.

    de Souza, S. J., Long, M. & Gilbert, W. Introns and gene evolution. Genes Cells 1, 493–505 (1996).

  15. 15.

    Pleiss, J. A., Whitworth, G. B., Bergkessel, M. & Guthrie, C. Rapid, transcript-specific changes in splicing in response to environmental stress. Mol. Cell 27, 928–937 (2007).

  16. 16.

    Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27 (1989).

  17. 17.

    Boeke, J. D., Trueheart, J., Natsoulis, G. & Fink, G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 154, 164–175 (1987).

  18. 18.

    Yofe, I. et al. Accurate, model-based tuning of synthetic gene expression using introns in S. cerevisiae. PLoS Genet. 10, e1004407 (2014).

  19. 19.

    Gietz, R. D. & Woods, R. A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96 (2002).

  20. 20.

    Ito, H., Fukuda, Y., Murata, K. & Kimura, A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163–168 (1983).

  21. 21.

    Zakian, V. A. & Scott, J. F. Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol. Cell. Biol. 2, 221–232 (1982).

  22. 22.

    Rose, M. D., Winston, F. & Hieter, P. Methods in Yeast Genetics: A Laboratory Course Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1990).

  23. 23.

    Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989).

  24. 24.

    Brosseau, J. P. et al. High-throughput quantification of splicing isoforms. RNA 16, 442–449 (2010).

  25. 25.

    Klinck, R. et al. Multiple alternative splicing markers for ovarian cancer. Cancer Res. 68, 657–663 (2008).

  26. 26.

    González, A. et al. TORC1 promotes phosphorylation of ribosomal protein S6 via the AGC kinase Ypk3 in Saccharomyces cerevisiae. PLoS ONE 10, e0120250 (2015).

  27. 27.

    Rubin, G. M. Preparation of RNA and ribosomes from yeast. Methods Cell Biol. 12, 45–64 (1975).

  28. 28.

    Toussaint, M. et al. A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures. Mut. Res. 606, 92–105 (2006).

  29. 29.

    Huberman, J. A., Spotila, L. D., Nawotka, K. A., el-Assouli, S. M. & Davis, L. R. The in vivo replication origin of the yeast 2μm plasmid. Cell 51, 473–481 (1987).

  30. 30.

    Wellinger, R. J., Wolf, A. J. & Zakian, V. A. Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase. Cell 72, 51–60 (1993).

  31. 31.

    Boudreault, S. et al. Global profiling of the cellular alternative RNA splicing landscape during virus–host interactions. PLoS ONE 11, e0161914 (2016).

  32. 32.

    Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotechnol. 31, 46–53 (2013).

  33. 33.

    Deschamps-Francoeur, G., Boivin, V., Abou Elela, S. & Scott, M. S. CoCo: RNA-seq read assignment correction for nested genes and multimapped reads. Preprint at https://www.biorxiv.org/content/early/2018/11/29/477869 (2018).

  34. 34.

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

  35. 35.

    Ferreira, J. A. The Benjamini–Hochberg method in the case of discrete test statistics. Int. J. Biostat. 3, 11 (2007).

  36. 36.

    Liu, Y. et al. XBSeq2: a fast and accurate quantification of differential expression and differential polyadenylation. BMC Bioinformatics 18, 384 (2017).

  37. 37.

    Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).

  38. 38.

    Chaowanathikhom, M., Nuchnoi, P. & Palasuwan, D. Significance of 3′ UTR and pathogenic haplotype in glucose-6-phosphate deficiency. Lab. Med. 48, 73–88 (2017).

  39. 39.

    Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

  40. 40.

    Agresti, A. A survey of exact inference for contingency tables. Stat. Sci. 7, 131–153 (1992).

Download references

Acknowledgements

This work was supported by NSERC and a Research Chair in RNA Biology and Cancer Genomics (S.A.E.). We thank M. Ares Jr for discussion and for providing the yeast strains used in Extended Data Fig. 3e; R. Wellinger, B. Chabot and M. Scott for critical reading of the manuscript; and C. Nour Abou Chakra for reviewing the statistical analyses. Sequencing libraries were prepared by the Université de Sherbrooke RNomics Platform and sequenced in the Centre of Applied Genomics (Toronto).

Reviewer information

Nature thanks S. Montgomery and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

J.P. designed and performed experiments, analysed data, produced figures and participated in the writing of the paper. L.M., M.C. and V.G performed experiments, M.B. performed RNA sequencing data analysis. S.A.E. planned the work, proposed and designed experiments, analysed data and wrote the paper.

Correspondence to Sherif Abou Elela.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Strategy for examining the effect of introns on cell growth and starvation.

a, Summary of the pipeline used for the identification of intron functions. b, Validation of the BSP PCR assay to detect variation in cell number. Intron-deletion cells were diluted with wild-type cells and the BSP PCR was performed on each dilution; the amplicon amount was plotted as function of dilution. The mean value of two independent experiments is presented and the s.d. is indicated by error bars. The correlation value (Spearman correlation coefficient) is indicated. This figure is related to Fig. 1.

Extended Data Fig. 2 Intron deletions inhibit cell maintenance during starvation independently of the expression pattern and function of their host gene.

af, The growth curves of wild-type cells and ∆i strains of genes with unrelated functions were determined in minimal medium with dextrose (a, f) or in minimal medium with low dextrose (be). The experiments were repeated independently three times with similar results. The expression profile of each host gene was determined by RT–qPCR in wild-type (dark grey) and ∆i (light grey) cells in log phase or stationary phase of growth, and is presented in the form of a bar graph. The gene name is indicated in each panel. The mean value of three (two for wild type) biologically independent strains is presented and the s.d. is indicated by error bars. This figure is related to Figs. 2, 3. Source Data

Extended Data Fig. 3 Introns affect growth during starvation through modification of splicing.

a, Introns that accumulate in the stationary phase (increased), and those that did not increase or decrease (not increased), are indicated. Intron abundance was considered to be increased when the transcripts per million (TPM) of introns at 48 h increased by more than 1.5× the TPM detected in log phase. b, The ratio of spliced and unspliced mRNA was calculated in cells that lack the MMS2 or YSF3 intron, in the log phase and stationary phase of growth; the per cent of introns is shown. c, The number of introns that accumulate or decrease upon the deletion of either MMS2 or YSF3 introns (or with both deletions) is indicated, for the log phase and stationary phase of growth. The pie charts shown are a descriptive representation of the data obtained by RNA sequencing, and the data that were validated using RT–qPCR (for example, see Fig. 3c and Supplementary Table 7). d, Intron deletion increases the splicing of RPGs. The intron accumulation of eight RPGs that display enhanced splicing upon the deletion of MMS2 or YSF3 introns is shown. Relative intron accumulation was determined between Δi and wild-type strains in stationary phase of growth as described in b. e, MMS2 introns were deleted in wild-type cells, and in cells that express temperature-sensitive alleles of the splicing factors PRP4 or PRP11 or express the RPG transcription factor IFH1 from an inducible promoter; these cells were tested for growth in low-dextrose medium at the semi-permissive temperature. The relative growth was calculated by subtracting the optical density at 660 nm (OD660 nm) after 96 h of growth of the Δi or double-mutant strains from that of the wild-type or the single-mutant strains, respectively. The growth assays were repeated independently three times with similar results. Differences between groups were calculated using a two-sided t-test assuming unequal variances. **P = 0.0044. This figure is related to Figs. 3, 4. Source Data

Extended Data Fig. 4 The accumulation of the unspliced RNA and not the mature mRNA is required for cell maintenance under starvation conditions.

Growth profiles of ∆i cells that carry different plasmids or mutations. a, b, Expression of the host gene is not required for growth under starvation conditions. Cells that lack introns were transformed with plasmids that express the host gene of the respective intron (mms2∆i + pMMS2 or ysf3Δi + pYSF3) and the growth profile was compared to wild-type (WT + p-empty) or ∆i strains (mms2∆i + p-empty or ysf3Δi + p-empty) containing empty plasmids. The experiments were repeated independently nine times with similar results. c, d, Stop-codon and branch-point mutations increase intron abundance. The splicing index (calculated by dividing the amount of unspliced over total RNA, ×100) was detected using end-point PCR, and the relative abundance of cDNA (dark grey), intronic RNA (grey) and exonic RNA (light grey) was determined using RT–qPCR. The average value of three (two for wild type) biologically independent replicates is presented and the s.d. is indicated by error bars; for c, differences between wild type and mms2∆i were calculated using two-sided t-test. *P = 0.013, **P < 0.0072, ***P = 9.8 × 10−8. e, f, Increasing the number of introns and not the host cDNA inhibits cell maintenance under starvation conditions. Growth profiles of wild-type cells transformed with empty plasmids (p-empty), plasmids expressing MMS2 RNA (p-MMS2), MMS2 RNA carrying a stop codon (p-MMS2-stop) and plasmid expressing MMS2 cDNA (p-mms2∆i). The experiments were repeated independently four times with similar results. The position of the log phase and stationary phase of the growth are indicated on the growth curves. This figure is related to Fig. 3. Source Data

Extended Data Fig. 5 Effect of different introns and intronic mutations on the growth of wild-type and ∆i cells.

a, Increasing the number of genes that contain nutrient-independent introns does not affect growth. Growth profile of wild-type cells transformed with empty plasmids or a plasmid expressing the TUB1 gene (which contains an intron that has no effect on cell growth under starvation conditions). The experiments were repeated independently four times with similar results. bd, Genes that contain nutrient-independent introns do not rescue the intron-deletion phenotype. Growth profile of mms2∆i, ysf3∆i or glc7∆i cells transformed with plasmid expressing the intron-containing TUB1 gene. For bd, the growth assays were repeated independently six times with similar results. e, f, Intron deletions are genetically epistatic. The growth profiles of wild-type (black), single (red) and double (blue) ∆i strains were monitored for 48 h in minimal medium that is low in dextrose. The experiments were repeated independently eight times with similar results. The position of the log phase and stationary phase of the growth are indicated on the growth curves. This figure is related to Figs. 3, 4. Source Data

Extended Data Fig. 6 Introns are required for cell maintenance under starvation conditions in the context of the host gene.

a, b, Growth profile of mms2∆i and ysf3∆i cells expressing MMS2 or YSF3 introns from a heterologous gene (YFP-MMS2I and YFP-YSF3I). c, Growth profile of mms2∆i cells transformed with plasmid expressing a version of MMS2 carrying the YSF3 intron. d, Growth profile of mms2∆i cells transformed with plasmid expressing a version of the MMS2 gene terminating with a heterologous 3′ UTR and transcription termination sequence (ADH1t). e, Growth profile of ysf3∆i cells transformed with plasmid expressing a version of MMS2 carrying the exon 2 of YSF3. f, Growth profile of mms2∆i cells transformed with plasmid expressing a version of MMS2 carrying the exon 2 of TUB1. For af, the experiments were repeated independently six times with similar results. The position of the log phase and stationary phase of the growth are indicated on the growth curves. This figure is related to Figs. 3, 4. Source Data

Extended Data Fig. 7 Predicted structure of introns with different effects on growth under starvation conditions.

a, The structure of the 5′ UTR, exon 1 and the intron that affects growth under starvation conditions was calculated using the mfold default setting, and the average of 15 suboptimal structures is presented. b, Structure of the 5′ UTR, exon 1 and the intron of the TUB1 gene, which does not affect growth under starvation conditions. c, Structures of the mutated constructs tested in Fig. 4c. The substitution of the first half (SWAP1) or the second half (SWAP2) of the intron with sequence of ACT1 intron is indicated in red. d, Structure of the mutated constructs tested in Fig. 4e, f. The position of the mutations that disrupt (left) or restore (right) the structure is shown in red. 5′S indicates the position of the 5′ splice site. The 5′ UTR and exon 1 are indicated in pale and dark blue, respectively. This figure is related to Fig. 4.

Extended Data Fig. 8 Effect of starvation and intron deletion on gene expression.

a, Comparison between the expression profiles of wild-type cells in log (WT LP) and stationary (WT SP) phases of growth. b, GO analysis of genes that are downregulated after the deletion of introns from MMS2 and YSF3 in the stationary phase of growth. The per cent of genes in each process or activity is indicated in form of a pie chart. c, Comparison between the expression profiles of wild-type and ∆i strains in the log phase. d, Comparison between the expression profiles of ∆i strains in log phase and stationary phase. e, Comparison between the expression profiles of the two ∆i strains in the log phase (left) and stationary phase (right) of growth. Blue, red, grey and black dots indicate the number of genes that are upregulated, downregulated, not affected and upregulated, respectively, in ∆i strains in the stationary phase. For a, ce, the mean value from two biologically independent replicates is presented and the P value (t-test; one-sided) of the difference between the different strains and comparison was calculated as described in Methods, and is shown on each graph. This figure is related to Fig. 5. Source Data

Extended Data Fig. 9 Introns promote cell growth in a TORC1-dependent, and not TORC2-dependent, manner.

The growth profile of wild-type cells, cells that lack introns or cells that lack both introns and either a component of the TORC1 (tco89∆ or tor1∆) or TORC2 (avo2∆, slm1∆, slm2∆, bit61∆ or bit2∆) pathways was examined as function of the optical density of the culture in minimal medium with low dextrose. Deletions of the YSF3 intron are shown on the left and those of MMS2 intron are shown on the right. All these experiments were repeated independently three times with similar results. This figure is related to Fig. 6. Source Data

Extended Data Fig. 10 Effect of intron deletion on the PKA pathway and the expression of TORC1 targets.

a, Intron deletions do not alter the expression of TORC1 targets under starvation conditions. The expression profile of 12 regulatory targets of TORC1. The fold change (expressed as log2(mRNA abundance in SP/mRNA abundance in LP)) in RNA abundance of genes regulated by TORC1 pathway, in the log phase and stationary phase of growth, was determined using RNA sequencing in wild-type cells and cells that lack MMS2 (mms2∆i) or YSF3 (ysf3∆i) introns. The mean value and s.d. from two biologically independent replicates are presented. Genes are up- or downregulated with a P value < 0.001 (t-test; one-sided). b, c, Introns promote cell growth in a PKA-dependent manner. Growth profile of cells that lack different components of the PKA regulatory pathway (TPK1, TPK2 and TPK3) in the presence or the absence of MMS2 and YSF3 introns. In b, c, experiments were repeated independently three times with similar results. This figure is related to Fig. 6. Source Data

Supplementary information

Supplementary Information

This file contains gel source data for Figure 6d

Reporting Summary

Supplementary Tables

This file contains Supplementary Tables S1-S7.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Parenteau, J., Maignon, L., Berthoumieux, M. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019) doi:10.1038/s41586-018-0859-7

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.