RNA regulons in Hox 5′ UTRs confer ribosome specificity to gene regulation

Abstract

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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Figure 1: Select HoxA 5′ UTRs contain IRES elements regulated by RPL38.
Figure 2: The Hoxa9 IRES is an RNA structure that facilitates recruitment of the ribosome and is required for normal translation.
Figure 3: Identification of minimal IRES domains in all IRES-containing HoxA 5′ UTRs.
Figure 4: Identification of a common TIE that blocks cap-dependent translation of Hox IRES-mRNAs.
Figure 5: The Hoxa9 IRES is required for axial skeleton patterning but not for Hoxa9 mRNA expression.
Figure 6: The Hoxa9 IRES is critically required for Hoxa9 translation in vivo.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

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.

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Acknowledgements

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.).

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Authors

Contributions

M.B. conceived and supervised the project; S.X. and M.B. designed experiments; S.X. performed all experiments with help from K.F. for 5′ RACE and sucrose gradient fractionation; S.T. and R.D. designed the RNA structure experiments; S.T. and W.K. performed the RNA structure experiments and analysed results; S.X. and M.B. wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to Maria Barna.

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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.

Extended Data Figure 2 Disruption of Rpl38 in C3H10T1/2 by TALEN nucleases.

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.

Extended Data Figure 3 Alignment of the Hoxa9 IRES element between vertebrate species.

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).

Extended Data Figure 5 Chemical mapping and secondary structure model of full-length Hoxa5 5′ UTR.

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 ce 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 (ce), P3a (fk), P4b (lo), P4a (pu) and pk3-4 (vai). 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 (ce), P3a (fk), P4b (mo) and P4a (pq, 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 Hoxa9ΔIRESIRES embryos.

a, Overlay of A260 trace during fractionation showing no difference in polysome profiles between E11.5 Hoxa9+/+ and Hoxa9ΔIRESIRES embryos. b, qPCR from each fraction reveals no difference in β-actin mRNA accumulation between Hoxa9+/+ and Hoxa9ΔIRESIRES 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

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