Abstract
High-throughput RNA sequencing (RNA-seq) has revealed an enormous complexity of alternative splicing (AS) across diverse cell and tissue types. However, it is currently unknown to what extent repertoires of splice-variant transcripts are translated into protein products. Here, we surveyed AS events engaged by the ribosome. Notably, at least 75% of human exon-skipping events detected in transcripts with medium-to-high abundance in RNA-seq data were also detected in ribosome profiling data. Furthermore, relatively small subsets of functionally related splice variants are engaged by ribosomes at levels that do not reflect their absolute abundance, thus indicating a role for AS in modulating translational output. This mode of regulation is associated with control of the mammalian cell cycle. Our results thus suggest that a major fraction of splice variants is translated and that specific cellular functions including cell-cycle control are subject to AS-dependent modulation of translation output.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Barbosa-Morais, N.L. et al. The evolutionary landscape of alternative splicing in vertebrate species. Science 338, 1587–1593 (2012).
Merkin, J., Russell, C., Chen, P. & Burge, C.B. Evolutionary dynamics of gene and isoform regulation in mammalian tissues. Science 338, 1593–1599 (2012).
Pan, Q., Shai, O., Lee, L.J., Frey, B.J. & Blencowe, B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat. Genet. 40, 1413–1415 (2008).
Wang, E.T. et al. Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 (2008).
Braunschweig, U. et al. Widespread intron retention in mammals functionally tunes transcriptomes. Genome Res. 24, 1774–1786 (2014).
Dominguez, D. et al. An extensive program of periodic alternative splicing linked to cell cycle progression. eLife 5, e10288 (2016).
Irimia, M. et al. A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell 159, 1511–1523 (2014).
Li, Q., Lee, J.A. & Black, D.L. Neuronal regulation of alternative pre-mRNA splicing. Nat. Rev. Neurosci. 8, 819–831 (2007).
Xing, Y. & Lee, C.J. Protein modularity of alternatively spliced exons is associated with tissue-specific regulation of alternative splicing. PLoS Genet. 1, e34 (2005).
Buljan, M. et al. Tissue-specific splicing of disordered segments that embed binding motifs rewires protein interaction networks. Mol. Cell 46, 871–883 (2012).
Jangi, M. & Sharp, P.A. Building robust transcriptomes with master splicing factors. Cell 159, 487–498 (2014).
Weatheritt, R.J., Davey, N.E. & Gibson, T.J. Linear motifs confer functional diversity onto splice variants. Nucleic Acids Res. 40, 7123–7131 (2012).
Wong, J.J. et al. Orchestrated intron retention regulates normal granulocyte differentiation. Cell 154, 583–595 (2013).
Abascal, F. et al. Alternatively spliced homologous exons have ancient origins and are highly expressed at the protein level. PLoS Comput. Biol. 11, e1004325 (2015).
Abascal, F., Tress, M.L. & Valencia, A. The evolutionary fate of alternatively spliced homologous exons after gene duplication. Genome Biol. Evol. 7, 1392–1403 (2015).
Weatheritt, R.J. & Gibson, T.J. Linear motifs: lost in (pre)translation. Trends Biochem. Sci. 37, 333–341 (2012).
Bensimon, A., Heck, A.J. & Aebersold, R. Mass spectrometry-based proteomics and network biology. Annu. Rev. Biochem. 81, 379–405 (2012).
Ezkurdia, I. et al. Most highly expressed protein-coding genes have a single dominant isoform. J. Proteome Res. 14, 1880–1887 (2015).
Battle, A. et al. Genomic variation: impact of regulatory variation from RNA to protein. Science 347, 664–667 (2015).
Ingolia, N.T., Ghaemmaghami, S., Newman, J.R. & Weissman, J.S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).
Ingolia, N.T. Ribosome footprint profiling of translation throughout the genome. Cell 165, 22–33 (2016).
Tanenbaum, M.E., Stern-Ginossar, N., Weissman, J.S. & Vale, R.D. Regulation of mRNA translation during mitosis. eLife 4, e07957 (2015).
Yap, K., Lim, Z.Q., Khandelia, P., Friedman, B. & Makeyev, E.V. Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention. Genes Dev. 26, 1209–1223 (2012).
Marquez, Y., Höpfler, M., Ayatollahi, Z., Barta, A. & Kalyna, M. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity. Genome Res. 25, 995–1007 (2015).
Irimia, M. et al. Origin of introns by 'intronization' of exonic sequences. Trends Genet. 24, 378–381 (2008).
Eisenberg, E. & Levanon, E.Y. Human housekeeping genes, revisited. Trends Genet. 29, 569–574 (2013).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Ly, T. et al. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells. eLife 3, e01630 (2014).
Park, J.E., Yi, H., Kim, Y., Chang, H. & Kim, V.N. Regulation of Poly(A) tail and translation during the somatic cell cycle. Mol. Cell 62, 462–471 (2016).
Sterne-Weiler, T. et al. Frac-seq reveals isoform-specific recruitment to polyribosomes. Genome Res. 23, 1615–1623 (2013).
Floor, S.N. & Doudna, J.A. Tunable protein synthesis by transcript isoforms in human cells. eLife 5, e10921 (2016).
Kutchko, K.M. et al. Multiple conformations are a conserved and regulatory feature of the RB1 5′ UTR. RNA 21, 1274–1285 (2015).
Chang, L. & Barford, D. Insights into the anaphase-promoting complex: a molecular machine that regulates mitosis. Curr. Opin. Struct. Biol. 29, 1–9 (2014).
Boutz, P.L., Bhutkar, A. & Sharp, P.A. Detained introns are a novel, widespread class of post-transcriptionally spliced introns. Genes Dev. 29, 63–80 (2015).
Olsen, J.V. & Mann, M. Status of large-scale analysis of post-translational modifications by mass spectrometry. Mol. Cell. Proteomics 12, 3444–3452 (2013).
Juntawong, P., Girke, T., Bazin, J. & Bailey-Serres, J. Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis. Proc. Natl. Acad. Sci. USA 111, E203–E212 (2014).
Newton, D.C. et al. Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5′-untranslated region leader exon. J. Biol. Chem. 278, 636–644 (2003).
Remy, E. et al. Intron retention in the 5'UTR of the novel ZIF2 transporter enhances translation to promote zinc tolerance in Arabidopsis. PLoS Genet. 10, e1004375 (2014).
Zhang, Y. et al. Translational control of the rat angiotensin type 1a receptor by alternative splicing. Gene 341, 93–100 (2004).
Vogel, C. & Marcotte, E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat. Rev. Genet. 13, 227–232 (2012).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).
Cunningham, F. et al. Ensembl 2015. Nucleic Acids Res. 43, D662–D669 (2015).
Andreev, D.E. et al. Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression. eLife 4, e03971 (2015).
Guo, H., Ingolia, N.T., Weissman, J.S. & Bartel, D.P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).
Rooijers, K., Loayza-Puch, F., Nijtmans, L.G. & Agami, R. Ribosome profiling reveals features of normal and disease-associated mitochondrial translation. Nat. Commun. 4, 2886 (2013).
Rouskin, S., Zubradt, M., Washietl, S., Kellis, M. & Weissman, J.S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705 (2014).
Merico, D., Isserlin, R., Stueker, O., Emili, A. & Bader, G.D. Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One 5, e13984 (2010).
Stumpf, C.R., Moreno, M.V., Olshen, A.B., Taylor, B.S. & Ruggero, D. The translational landscape of the mammalian cell cycle. Mol. Cell 52, 574–582 (2013).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Labbé, R.M. et al. A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals. Stem Cells 30, 1734–1745 (2012).
Bray, N. et al. Near-optimal RNA-seq quantification. Preprint at http://arxiv.org/abs/1505.02710 (2015).
Ingolia, N.T., Lareau, L.F. & Weissman, J.S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).
Whitfield, M.L. et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell 13, 1977–2000 (2002).
Santos, A., Wernersson, R. & Jensen, L.J. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes. Nucleic Acids Res. 43, D1140–D1144 (2015).
Lee, S. et al. Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proc. Natl. Acad. Sci. USA 109, E2424–E2432 (2012).
Ingolia, N.T. et al. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell Rep. 8, 1365–1379 (2014).
Acknowledgements
We thank U. Braunschweig, J. Ellis, T. Gonatopoulos-Pournatzis, S. Gueroussov, K. Ha, M. Irimia and J. Roth for helpful comments on the manuscript and technical assistance. We thank M. Irimia for providing annotations for ORF-disrupting and ORF-preserving AS events. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) to B.J.B., by CIHR postdoctoral and Marie Curie IOF fellowships to R.J.W. and by CIHR and Charles H. Best postdoctoral fellowships to T.S.-W. B.J.B. holds the Banbury Chair in Medical Research at the University of Toronto.
Author information
Authors and Affiliations
Contributions
R.J.W. conceived the study and designed and performed analyses with input from B.J.B. T.S.-W. contributed to methods for analyzing ribosome profiling data. R.J.W. and B.J.B. wrote the manuscript with input from T.S.-W.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Frequency of alternative-cassette-exon engagement with the ribosome is consistent across data from multiple human cell types and from mouse stem cells.
Box plots showing AS frequency (scored as the fraction of annotated exons in canonical transcripts that show skipping) for genes with different levels of RNA-Seq or ribosome-profiling read coverage in (a) mouse ES cells, (b) human BJ cells and (c) human HEK293 cells. Simple, complex and microexon (i.e. 3-27 nt) cassette events were analyzed and only genes with detected AS events in RNA-Seq data were included. See Figure 2 and Online Methods for description of boxplots.
Supplementary Figure 2 Features of cassette exons detected in ribosome profiling and RNA-seq data.
a) Bar plot showing the percentage change in detection of AS events in different transcript locations using ribosome profiling and RNA-Seq data. Transcript locations are mapped based on Ensembl GTF annotations37. b) Bar plot comparing fractions of total alternative 3´-and 5´ splicing events identified in RNA-Seq data that are also identified as alternative in ribosome profiling (RP) data at increasing expression levels. Events were only analyzed if adjacent constitutive exons were detected in ribosome profiling data. c) Bar plots showing the percentage of alternative exons predicted to be contribute to open reading frames at different expression levels in RNA-Seq data. d) Bar plots showing the percent of exons identified as alternative in both the full dataset and the subsampled datasets. Only reads from genes with cRPKM > 250 included. Statistical test used: Fisher’s exact test.
Supplementary Figure 3 Fraction of IR events identified in ribosome profiling data varies depending on transcript location.
a) Bar plot showing fraction of total intron retention events identified in RNA-Seq data that are also detected as retained in ribosome profiling (RP) data, for events in locations other than the 5´-UTR and not divisible by 3. Only retention events with supporting evidence in ribosome profiling data are shown. b) Bar plot showing fraction of total intron retention events identified in RNA-Seq data that are also detected as retained in ribosome profiling (RP) data, only for events within 5´-UTR or divisible by 3. Only retention events with supporting evidence in ribosome profiling data are shown. c) Bar plot showing the fraction of genes with 5´-UTR IR events that have annotated upstream open reading frames (uORFs).
Supplementary Figure 4 Features of periodic AS events.
a) Heat map showing the maximum percentage-spliced in (PSI) or percent intron retained (PIR) change between tissues or conditions highlighting events that are tissue-specific and cell-cycle stage-specific. For the analysis, cell-type specific events (Cell Type AS) defined using RNA-Seq7 that were also detected within ribosome-profiling data and periodic events showed differential cyclic changes as identified by fourier transform analysis. Individual columns were sorted and events matched from right to left to ensure direct comparison of events across cell stages or cell types (see Methods) b) Bar plots comparing the overlap of exons with annotated ATG start sites from Ensembl v71. c) An annotated version of the Enrichment map in Figure 4e. d) Percent of 5´-UTR events that influence upstream open reading frames (uORFs) and Terminal oligopyrimidine tract (TOP) motifs, respectively, for different types (un)regulated events e) Heatmap of PSI/PIR values of Aurora Kinase A (AURKA) and Cell division cycle protein 20 homolog (CDC20) during two rounds of cell cycle. f) Bar plots showing the location of periodic AS events within transcripts based on Ensembl GTF annotation36, as compared to all detected events from ribosomal profiling. Occurrences in CDS cede to occurrences in UTR. See Figure 2 and Online Methods for description of boxplots. Statistical test used: Fisher’s exact test.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4 (PDF 821 kb)
Supplementary Table 1
Description of the publically available datasets used in this paper (XLSX 52 kb)
Supplementary Data Set 1
All cassette events with coordinates used in Figure 1. (XLSX 471 kb)
Supplementary Data Set 2
All intron events detected in RiboSeq with coordinates used in Figure 2; intron events within or overlapping 5′-UTR: Figure 2d,e. (XLS 216 kb)
Supplementary Data Set 3
Intron events within or overlapping 5′-UTR: Figures 2d,e and 3a. (XLSX 59 kb)
Rights and permissions
About this article
Cite this article
Weatheritt, R., Sterne-Weiler, T. & Blencowe, B. The ribosome-engaged landscape of alternative splicing. Nat Struct Mol Biol 23, 1117–1123 (2016). https://doi.org/10.1038/nsmb.3317
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3317
This article is cited by
-
The status of the human gene catalogue
Nature (2023)
-
The physiology of alternative splicing
Nature Reviews Molecular Cell Biology (2023)
-
Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity
Military Medical Research (2022)
-
Enhanced protein isoform characterization through long-read proteogenomics
Genome Biology (2022)
-
Differential fates of introns in gene expression due to global alternative splicing
Human Genetics (2022)