Emerging evidence suggests that the ribosome has a regulatory function in directing how the genome is translated in time and space. However, how this regulation is encoded in the messenger RNA sequence remains largely unknown. Here we uncover unique RNA regulons embedded in homeobox (Hox) 5′ untranslated regions (UTRs) that confer ribosome-mediated control of gene expression. These structured RNA elements, resembling viral internal ribosome entry sites (IRESs), are found in subsets of Hox mRNAs. They facilitate ribosome recruitment and require the ribosomal protein RPL38 for their activity. Despite numerous layers of Hox gene regulation, these IRES elements are essential for converting Hox transcripts into proteins to pattern the mammalian body plan. This specialized mode of IRES-dependent translation is enabled by an additional regulatory element that we term the translation inhibitory element (TIE), which blocks cap-dependent translation of transcripts. Together, these data uncover a new paradigm for ribosome-mediated control of gene expression and organismal development.
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Redefining GBA gene structure unveils the ability of Cap-independent, IRES-dependent gene regulation
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Hoxa4 5′ UTR sequence has been deposited in GenBank under accession number KM596709. All chemical mapping datasets have been deposited at the RNA Mapping Database (http://rmdb.stanford.edu) under the following accession codes: (1) Full-length: HOXA5_STD_0000, HOXA9_STD_0000; (2) Hoxa9 TIE: HOXA9A_STD_0001; (3) Hoxa9 IRES: HOXA9D_STD_0001, HOXA9D_STD_0002, HOXA9D_1M7_0001, and HOXA9D_RSQ_0001.
Kondrashov, N. et al. Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell 145, 383–397 (2011)
Lee, A. S., Burdeinick-Kerr, R. & Whelan, S. A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs. Proc. Natl Acad. Sci. USA 110, 324–329 (2013)
Vesper, O. et al. Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli . Cell 147, 147–157 (2011)
Xue, S. & Barna, M. Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nature Rev. Mol. Cell Biol. 13, 355–369 (2012)
Dinman, J. D. The eukaryotic ribosome: current status and challenges. J. Biol. Chem. 284, 11761–11765 (2009)
Komili, S., Farny, N. G., Roth, F. P. & Silver, P. A. Functional specificity among ribosomal proteins regulates gene expression. Cell 131, 557–571 (2007)
Alexander, T., Nolte, C. & Krumlauf, R. Hox genes and segmentation of the hindbrain and axial skeleton. Annu. Rev. Cell Dev. Biol. 25, 431–456 (2009)
Sonenberg, N. & Hinnebusch, A. G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136, 731–745 (2009)
Livingstone, M., Atas, E., Meller, A. & Sonenberg, N. Mechanisms governing the control of mRNA translation. Phys. Biol. 7, 021001 (2010)
Plank, T.-D. M. & Kieft, J. S. The structures of nonprotein-coding RNAs that drive internal ribosome entry site function. Wiley Interdiscip. Rev. RNA 3, 195–212 (2012)
Stoneley, M., Paulin, F. E., Le Quesne, J. P., Chappell, S. A. & Willis, A. E. C-Myc 5′ untranslated region contains an internal ribosome entry segment. Oncogene 16, 423–428 (1998)
Riley, A., Jordan, L. E. & Holcik, M. Distinct 5′ UTRs regulate XIAP expression under normal growth conditions and during cellular stress. Nucleic Acids Res. 38, 4665–4674 (2010)
Ungureanu, N. H. et al. Internal ribosome entry site-mediated translation of Apaf-1, but not XIAP, is regulated during UV-induced cell death. J. Biol. Chem. 281, 15155–15163 (2006)
Yoon, A. et al. Impaired control of IRES-mediated translation in X-linked dyskeratosis congenita. Science 312, 902–906 (2006)
Ray, P. S., Grover, R. & Das, S. Two internal ribosome entry sites mediate the translation of p53 isoforms. EMBO Rep. 7, 404–410 (2006)
Bellodi, C., Kopmar, N. & Ruggero, D. Deregulation of oncogene-induced senescence and p53 translational control in X-linked dyskeratosis congenita. EMBO J. 29, 1865–1876 (2010)
Pyronnet, S., Dostie, J. & Sonenberg, N. Suppression of cap-dependent translation in mitosis. Genes Dev. 15, 2083–2093 (2001)
Holcik, M., Sonenberg, N. & Korneluk, R. G. Internal ribosome initiation of translation and the control of cell death. Trends Genet. 16, 469–473 (2000)
Xia, X. & Holcik, M. Strong eukaryotic IRESs have weak secondary structure. PLoS ONE 4, e4136 (2009)
Phinney, D. G., Gray, A. J., Hill, K. & Pandey, A. Murine mesenchymal and embryonic stem cells express a similar Hox gene profile. Biochem. Biophys. Res. Commun. 338, 1759–1765 (2005)
Thompson, S. R. So you want to know if your message has an IRES? Wiley Interdiscip. Rev. RNA 3, 697–705 (2012)
Fraser, C. S. & Doudna, J. A. Structural and mechanistic insights into hepatitis C viral translation initiation. Nature Rev. Microbiol. 5, 29–38 (2007)
Kieft, J. S., Zhou, K., Jubin, R. & Doudna, J. A. Mechanism of ribosome recruitment by hepatitis C IRES RNA. RNA 7, 194–206 (2001)
Spahn, C. M. T. et al. Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor. Cell 118, 465–475 (2004)
Wilkinson, K. A., Merino, E. J. & Weeks, K. M. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nature Protocols 1, 1610–1616 (2006)
Lukavsky, P. J., Kim, I., Otto, G. A. & Puglisi, J. D. Structure of HCV IRES domain II determined by NMR. Nature Struct. Biol. 10, 1033–1038 (2003)
Kladwang, W., VanLang, C. C., Cordero, P. & Das, R. A two-dimensional mutate-and-map strategy for non-coding RNA structure. Nat. Chem. 3, 954–962 (2011)
Tian, S., Cordero, P., Kladwang, W. & Das, R. High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states. RNA 20, 1815–1826 (2014)
Berry, K. E., Waghray, S. & Doudna, J. A. The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit. RNA 16, 1559–1569 (2010)
Jan, E. & Sarnow, P. Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus. J. Mol. Biol. 324, 889–902 (2002)
Stupina, V. A., Yuan, X., Meskauskas, A., Dinman, J. D. & Simon, A. E. Ribosome binding to a 5′ translational enhancer is altered in the presence of the 3′ untranslated region in cap-independent translation of turnip crinkle virus. J. Virol. 85, 4638–4653 (2011)
Calvo, S. E., Pagliarini, D. J. & Mootha, V. K. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl Acad. Sci. USA 106, 7507–7512 (2009)
Fletcher, L., Corbin, S. D., Browning, K. S. & Ravel, J. M. The absence of a m7G cap on β-globin mRNA and alfalfa mosaic virus RNA 4 increases the amounts of initiation factor 4F required for translation. J. Biol. Chem. 265, 19582–19587 (1990)
Coulombe, Y. et al. Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo. PLoS ONE 5, e10600 (2010)
Fromental-Ramain, C. et al. Specific and redundant functions of the paralogous Hoxa-9 and Hoxd-9 genes in forelimb and axial skeleton patterning. Development 122, 461–472 (1996)
Chen, F. & Capecchi, M. R. Targeted mutations in Hoxa-9 and Hoxb-9 reveal synergistic interactions. Dev. Biol. 181, 186–196 (1997)
Boulet, A. M. & Capecchi, M. R. Targeted disruption of hoxc-4 causes esophageal defects and vertebral transformations. Dev. Biol. 177, 232–249 (1996)
Komar, A. A. & Hatzoglou, M. Internal ribosome entry sites in cellular mRNAs: mystery of their existence. J. Biol. Chem. 280, 23425–23428 (2005)
Oh, S. K., Scott, M. P. & Sarnow, P. Homeotic gene Antennapedia mRNA contains 5′-noncoding sequences that confer translational initiation by internal ribosome binding. Genes Dev. 6, 1643–1653 (1992)
Johnston, I. A. Environment and plasticity of myogenesis in teleost fish. J. Exp. Biol. 209, 2249–2264 (2006)
Landry, D. M., Hertz, M. I. & Thompson, S. R. RPS25 is essential for translation initiation by the Dicistroviridae and hepatitis C viral IRESs. Genes Dev. 23, 2753–2764 (2009)
Bellodi, C. et al. Loss of function of the tumor suppressor DKC1 perturbs p27 translation control and contributes to pituitary tumorigenesis. Cancer Res. 70, 6026–6035 (2010)
Chaudhuri, S. et al. Human ribosomal protein L13a is dispensable for canonical ribosome function but indispensable for efficient rRNA methylation. RNA 13, 2224–2237 (2007)
Flicek, P. et al. Ensembl 2014. Nucleic Acids Res. 42, D749–D755 (2014)
Carlson, D. F. et al. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl Acad. Sci. USA 109, 17382–17387 (2012)
Kladwang, W., Cordero, P. & Das, R. A mutate-and-map strategy accurately infers the base pairs of a 35-nucleotide model RNA. RNA 17, 522–534 (2011)
Mortimer, S. A. & Weeks, K. M. A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129, 4144–4145 (2007)
Talkish, J., May, G., Lin, Y., WoolFord J. L. Jr & McManus, C. J. Mod-seq : high-throughput sequencing for chemical probing of RNA structure. RNA 20, 713–730 (2014)
Harris, D. A. & Walter, N. G. Probing RNA structure and metal-binding sites using Terbium (III) footprinting. Curr. Protoc. Nucleic Acid Chem. http://dx.doi.org/10.1002/0471142700.nc0608s13 (2003)
Cheng, C., Chou, F. & Kladwang, W. MOHCA-seq : RNA 3D models from single multiplexed proximity-mapping experiments. Preprint at bioRxivhttp://dx.doi.org/10.1101/004556 (2014)
Yoon, S. et al. HiTRACE: high-throughput robust analysis for capillary electrophoresis. Bioinformatics 27, 1798–1805 (2011)
Kim, H., Cordero, P., Das, R. & Yoon, S. HiTRACE-Web: an online tool for robust analysis of high-throughput capillary electrophoresis. Nucleic Acids Res. 41, W492–W498 (2013)
Kim, J. et al. A robust peak detection method for RNA structure inference by high-throughput contact mapping. Bioinformatics 25, 1137–1144 (2009)
Seetin, M., Kladwang, W., Bida, J. & Das, R. in RNA Folding: Methods and Protocols (ed. Waldsich, C. ) 1086, 95–117 (Humana Press, 2014)
Kladwang, W. et al. Standardization of RNA chemical mapping experiments. Biochemistry 53, 3063–3065 (2014)
Deigan, K. E., Li, T. W., Mathews, D. H. & Weeks, K. M. Accurate SHAPE-directed RNA structure. Proc. Natl Acad. Sci. USA 106, 97–102 (2009)
Reuter, J. S. & Mathews, D. H. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11, 129 (2010)
Hajdin, C. E. et al. Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots. Proc. Natl Acad. Sci. USA 110, 5498–5503 (2013)
Darty, K., Denise, A. & Ponty, Y. VARNA: interactive drawing and editing of the RNA secondary structure. Bioinformatics 25, 1974–1975 (2009)
We would like to thank members of the Barna laboratory and D. Ruggero for discussion and critical reading of the manuscript. We thank E. Sarinay Cenik for advice with RNA pull-down experiments; and Y. Rim, A. Sapiro and A. Mateo for technical assistance. This work was supported by the Agency of Science, Technology and Research of Singapore (S.X.), Stanford Graduate Fellowship (S.T.), Human Frontier Science Program Fellowship (K.F.), NIH R01 GM102519 (R.D.), NIH Director’s New Innovator Award, 7DP2OD00850902 (M.B.), Alfred P. Sloan Research Fellowship (M.B.) and Pew Scholars Award (M.B.).
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 HoxA IRES controls confirming that Fluc activity from bicistronic vector is due to IRES activity.
a, qPCR of both Rluc and Fluc from transfected cells shows that Rluc and Fluc are produced at the same ratio. All Rluc and Fluc values are normalized to that of HCV (set to 1). n = 3, individual experiments performed in duplicate. b, c, shRNA against Rluc decreased reporter activity of both Rluc (a) and Fluc (b), confirming that Rluc and Fluc were transcribed on the same mRNA. n = 3, individual experiments performed in triplicate. d, RT–PCR using primers in Rluc and Fluc show that there is no cryptic splice site in the cloned Hox 5′ UTR. Primer locations are shown as arrows in the diagram. e, Inserting a strong hairpin (−67 kcal mol−1) after the Rluc reporter did not affect Fluc activity, suggesting that Fluc activity was not due to ribosome read-through.
a, Location of TALEN pairs. Two pairs of TALENs were designed to bind at the end of exon 2 and the beginning of exon 3 to make a genomic break close to the ATG. Sequencing of a positive clone shows a deletion of the ATG and most of the intron after it. Coding sequence is highlighted in green. b, Rpl38 knockdown does not change cap-dependent translation (Rluc) but decreases IRES-dependent translation (Fluc) from specific Hox 5′ UTRs. Luciferase activity was normalized to amount of Fluc RNA in the cells as quantified by qPCR. *P < 0.05 (t-test compared to control). n = 2, individual experiments performed in duplicate.
Nucleotides 945 to 1,266 of the mouse Hoxa9 5′ UTR were aligned with sequences from other vertebrates and show high sequence homology. Nucleotides are coloured based on their homology, with darker colours representing higher conservation.
Extended Data Figure 4 Chemical mapping and secondary structure prediction of full-length Hoxa9 5′ UTR.
a, Secondary structure modelling of full-length Hoxa9 using ligSHAPE data. The Hoxa9 IRES element (nt 957–1,132, shaded in green) is modelled as the same secondary structure shown in Fig. 2a. Confidence values from bootstrapping (bulge percentages) exceed 90% for this element, suggesting a well-determined subdomain, but are generally low outside this region, indicating poor certainty in other regions. b, Normalized SHAPE reactivity of Hoxa9 IRES (nt 957–1,132 and 944–1,266 from one-dimensional SHAPE read out through capillary electrophoresis (CE), full-length 1–1,266 from MiSeq-based ligSHAPE). c, Normalized SHAPE reactivity of Hoxa9 TIE (nt 1–342 from CE-based one-dimensional SHAPE, full-length 1–1,266 from MiSeq-based ligSHAPE).
a, Secondary structure modelling of Hoxa5 using one-dimensional SHAPE data. Nucleotides are coloured with SHAPE reactivities. Percentage labels give bootstrap support values for each helix. The feature highlighted in blue resembles P3 in Hoxa9 and the tip highlighted in pink is deleted in b. b, The deletion of the tip identified in Hoxa5 IRES structure shown in a decreases IRES activity in bicistronic reporter assays. IRES activity was normalized to full length Hoxa5 5′ UTR (A5, set to 1). **P < 0.01 (t-test as compared to A5). n = 2 experiments, performed in triplicate. c, Both Hoxa9 and Hoxa5 contain an asymmetric bulge in a region important for IRES activity. d, e, Normalized SHAPE (d) and DMS (e) reactivity of Hoxa5 (CE-based and MiSeq-based).
Extended Data Figure 6 Secondary structure model and mutate-and-map (M2) data set of Hoxa9 IRES element.
a, b, Entire M2 data set and Z-score contact-map of Hoxa9 nt 957–1,132 across 177 single mutants probed by 1M7. c, Secondary structure model of Hoxa9 nucleotides 957–1,132 using M2 data alone. d, Secondary structure model of Hoxa9 nt 957–1,132 using one-dimensional SHAPE data alone. Nucleotides are coloured with SHAPE reactivity. e, Secondary structure model of Hoxa9 nt 944–1,266 using one-dimensional SHAPE data. The models in c–e contain the same helices as the model from combined SHAPE/M2 analysis in Fig. 2a, up to register shifts and edge base pairs; the small rearrangements are labelled P3b’, P3c’, P3d, P4b’ and P4c’.
Extended Data Figure 7 Mutation/rescue results of Hoxa9 IRES structure (nt 944–1,266) probed by 1M7.
Electropherograms of mutation/rescue to test base-pairings in P3c (a, b), P3b (c–e), P3a (f–k), P4b (l–o), P4a (p–u) and pk3-4 (v–ai). Perturbation of the chemical mapping reactivities by mutations of one strand and restoration by mutations in the other strand provide strong evidence for the tested pairings in P3c (a, b), P3b (c–e), P3a (f–k), P4b (m–o) and P4a (p–q, t). Near-perfect restoration by compensatory mutations in (x) and (ad) support pseudoknot pk3-4. Lack of rescue in other tested pairings is consistent with either absence of those pairings or higher-order structure (for example, base triples) interacting with those pairings.
Extended Data Figure 8 Putative uORFs within the 5′ UTRs of Hoxa9 and Hoxa5 do not inhibit cap-dependent translation and Hoxa9ΔIRES targeting strategy.
uORFs are marked by black circles on the diagram of monocistronic reporter for the Hoxa9 (a) and Hoxa5 (b) 5′ UTR. All the ATGs in the 5′ UTR were mutated to TTG in A9ΔuORF construct and GTG in A5ΔuORF. The IRES element (944–1,266) was removed in A9ΔIRES construct. The IRES element was removed from the A9ΔuORF construct in A9ΔIRESΔuORF. n = 3 individual experiments in duplicates. Data represent mean ± s.d. c, Diagrams of the Hoxa9 locus and the targeting vector. Boxes represent exons, grey boxes represent UTRs, and black boxes represent the coding sequence. Nucleotides 944–1,145 were replaced by a floxed Neo cassette in the targeting vector. Locations of Southern blot probes, restriction enzymes used for Southern analysis and expected sizes are marked on the diagrams. d, Southern blot analysis of targeted cells using both the 5′ and 3′ probes showing that both arms integrated correctly into the Hoxa9 locus. Mice were generated from clone P3A5.
Extended Data Figure 9 The presence of a Neo cassette in the Hoxa9 locus is linked to the presence of an L1 → T13 homeotic transformation.
a, Diagram of the Hoxa9 locus (top) and axial skeleton phenotype (bottom) in different Hoxa9 mouse mutants. The original Hoxa9−/− was made by replacing the homeodomain with a Neo cassette. Vertebra with homeotic transformation is coloured red. b, Representative skeletons of Hoxa9+/+, Hoxa9Neo/+ and Hoxa9Neo/Neo . Arrows point to the additional rib(s) on L1, revealing a homeotic transformation to T13. These results show that it is the presence of Neo in the targeting locus, which may affect the expression of neighbouring Hox gene, that is sufficient to cause the L1 → T13 phenotype. When the Neo cassette is removed from the targeting locus by crossing the Hoxa9Neo/+ mouse with a CMV Cre line, the L1 → T13 phenotype is no longer present. n = 3 skeletons of each genotype.
Extended Data Figure 10 Sucrose gradient fractionation shows no difference in β-actin association with polysomes in Hoxa9+/+ and Hoxa9ΔIRES/ΔIRES embryos.
a, Overlay of A260 trace during fractionation showing no difference in polysome profiles between E11.5 Hoxa9+/+ and Hoxa9ΔIRES/ΔIRES embryos. b, qPCR from each fraction reveals no difference in β-actin mRNA accumulation between Hoxa9+/+ and Hoxa9ΔIRES/ΔIRES embryos. c, Quantification of β-actin mRNA in fractions. Fractions 1–8 are pre-polysomes and 9–16 are polysome fractions. n = 3 embryos of each genotype.
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Xue, S., Tian, S., Fujii, K. et al. RNA regulons in Hox 5′ UTRs confer ribosome specificity to gene regulation. Nature 517, 33–38 (2015). https://doi.org/10.1038/nature14010
Redefining GBA gene structure unveils the ability of Cap-independent, IRES-dependent gene regulation
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