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Functional and structural basis of extreme conservation in vertebrate 5′ untranslated regions

Nature Genetics volume 53, pages 729–741 (2021)Cite this article

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Abstract

The lack of knowledge about extreme conservation in genomes remains a major gap in our understanding of the evolution of gene regulation. Here, we reveal an unexpected role of extremely conserved 5′ untranslated regions (UTRs) in noncanonical translational regulation that is linked to the emergence of essential developmental features in vertebrate species. Endogenous deletion of conserved elements within these 5′ UTRs decreased gene expression, and extremely conserved 5′ UTRs possess cis-regulatory elements that promote cell-type-specific regulation of translation. We further developed in-cell mutate-and-map (icM2), a new methodology that maps RNA structure inside cells. Using icM2, we determined that an extremely conserved 5′ UTR encodes multiple alternative structures and that each single nucleotide within the conserved element maintains the balance of alternative structures important to control the dynamic range of protein expression. These results explain how extreme sequence conservation can lead to RNA-level biological functions encoded in the untranslated regions of vertebrate genomes.

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Fig. 1: Hyperconserved 5′ UTRs in vertebrate genomes.
Fig. 2: Hyperconserved 5′ UTRs impact translation efficiency.
Fig. 3: Noncanonical translation enhancer in hyperconserved 5′ UTRs.
Fig. 4: Cellular remodeling hyperconserved 5′ UTR RNA structures.
Fig. 5: icM2 reveals structured elements in the hyperconserved Csde1 5′ UTR.
Fig. 6: Csde1 5′ UTR tunes translation efficiency by encoding multiple alternative structures that are actively maintained by RNA helicases.

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Data availability

Raw sequencing data (related to Figs. 4, 5 and 6) are deposited to GEO with accession code GSE155656. Processed reactivity data have been deposited in the RNA Mapping Database (RMDB) with accession codes CSDE1_DMS_0000 and CSDE1_DMS_0001. Sources for publicly available data are described in the Methods.

Code availability

All software used to analyze the study data are listed in the Methods and in the Nature Research Reporting Summary and are publicly available. All codes used to analyze icM2 data are available through a Github repository: github.com/barnalab/icm2p.

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Acknowledgements

We thank the members of the Barna laboratory for constructive criticism of the manuscript. This work was supported by New York Stem Cell Foundation grant NYSCF-R-I36 (M.B.), NIH grant 1R01HD086634 (M.B.), Alfred P. Sloan Research Fellowship (M.B.), Pew Scholars Award (M.B.), Mallinckrodt Foundation Award (M.B.), Benchmark Stanford Graduate Fellowship (G.W.B.) and Walter and Idun Berry Foundation (E.S.C.). M.B. is a New York Stem Cell Robertson Investigator.

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  1. Elif Sarinay Cenik

    Present address: Department of Molecular Biosciences, University of Texas Austin, Austin, TX, USA

Authors and Affiliations

  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Gun Woo Byeon, Elif Sarinay Cenik, Lihua Jiang, Hua Tang & Maria Barna

  2. Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA

    Gun Woo Byeon, Elif Sarinay Cenik & Maria Barna

  3. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA

    Rhiju Das

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Contributions

M.B., G.W.B. and E.S.C. conceived the project. M.B. supervised the project. L.J. and H.T. provided the GTEx data and critical feedback on its analysis. R.D. provided critical feedback on the development and analysis of icM2. E.S.C. carried out the large-scale reporter screens. G.W.B. performed all other experiments and data analysis. G.W.B. and M.B. wrote the manuscript in consultation with all authors.

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Correspondence to Maria Barna.

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The authors declare no competing interests.

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Peer review information Nature Genetics thanks Jean-Denis Beaudoin, Philip Bevilacqua and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Hyperconserved 5’UTRs in vertebrate genomes.

a, Left: heatmap of the positions of LOD ≥ 500 PhastCons elements in each h5UTR. Middle: heatmap of the relative positions (calculated in 100 bins across the h5UTRs) of the elements. Right: plot of average element overlap across the 100 bins to illustrate the positional preference. b, Histogram of the length of h5UTRs. Average length is 674nt. c, Histogram of the number of nucleotides overlap between LOD ≥ 500 PhastCons elements and h5UTRs. Average overlap is 389nt. d, Distributions of cross-tissue transcriptome-proteome correlations for all genes, genes with h5UTRs, or genes with variance-matched non-conserved 5’UTRs. Indicated p-values are from two-sided Wilcoxon rank sum tests for cross-tissue correlation values between h5UTR genes and all genes or between h5UTR genes and variance-matched non-conserved controls. e, Distributions of the number of annotated alternative 5’UTRs for all genes, genes with h5UTRs, or genes with size-matched non-conserved 5’UTRs. Indicated p-values are from two-sided Wilcoxon rank sum tests for the number of alternative 5’UTRs between h5UTR genes and all genes or between h5UTR genes and size-matched non-conserved controls. f, Scatter plot illustrating the lack of significant term enrichments for a size-matched set of non-conserved 5’ UTRs. X-axis and y-axis plots expected and the observed number of genes for each term. Blue dashed line indicates the minimum observed/expected ratio cutoff of 3. Green line indicates expected and observed counts where Fisher’s test p-value (pf) is estimated to have FDR = 0.05. Neighbor-weighted test p-value (pfw) ≤0.05 is further used as an additional cutoff. The final set of enriched terms passing filter is colored by pf and sized by pfw.

Extended Data Fig. 2 Non-canonical translation activation by hyperconserved 5’UTRs across cell types.

a, Density plots of non-canonical translation initiation activities from h5UTRs by bicistronic reporter assay. X-axis is the luciferase reporter activity ratios. Jittered dots mark individual reporter ratios for each h5UTR in each cell type. b, Summarized plot of ribosome load (sum of % mRNA times the ribosome number for each fraction) differential ratio between NSCs and ESCs calculated from polysome profiles for each gene shown in Extended Data Fig. 2c-l. Red indicates significant increase in NSCs and black indicates significant decrease (two-sided t-test p ≤ 0.05, n = 3, marked by asterisk). c-l, Endogenous polysome profiles of NSCs versus ESCs for genes with h5UTRs that show high non-canonical translation reporter activities in NSCs compared to ESCs. Distribution of mRNAs across sucrose gradient fractions are plotted. Y-axis plots the mean percent mRNA. Error bars indicate standard error. Asterisk indicates two-sided t-test p ≤ 0.05 for each fraction between the two cell types. n = 3 for each cell type. Indicated p-value (pf) is calculated by Fisher’s method across all fractions. Note that Extended Data Fig. 2c shows the profile of 18 S rRNA, which indicates lower global translation in NSCs compared to ESCs.

Extended Data Fig. 3 Non-canonical activation by hyperconserved 5’UTRs substantially contributes to translation.

a, Scatter plot of luciferase activity versus RNA level ratios (mean from n = 3) observed for the bicistronic reporters of 90 h5UTRs measured in 10T1/2 cells. Dashed line marks the 10% FDR used in Fig. 3a. Spearman correlation indicated on top left. b, The effect of various truncations of the h5UTRs on non-canonical initiation and total translation efficiency (also see Fig. 3d). Left: positions of truncations. Dashed lines indicate truncations. Purple horizontal lines indicate uORFs; yellow and red lines indicate in-frame and out-of-frame uAUGs, respectively. Middle: non-canonical initiation efficiency. Right: total translation efficiency. X-axis indicates the mean of luciferase reporter ratios relative to the wild-type. Error bars indicate standard error. Dashed line marks the wild-type 5’UTR activity. Asterisk indicates two-sided t-test p ≤ 0.05 for each truncation versus the full-length. The numbers to the left of the bars indicate n and p-values. c, Comparison of translational activities between the full-length long, non-conserved 5’UTRs versus the only first 300nt truncation. 11 different pairs are tested. X-axis indicates the mean log2 luciferase reporter ratios of each truncation relative to its full-length wild-type. Error bars indicate standard error. Bars colored in red indicate significantly reduced translation in the shorter, truncated 300nt fragment; black indicates significant increase (two-sided t-test, paired n = 3, p ≤ 0.05, marked by asterisk). The numbers to the left of the bars indicate p-values. d, Violin plot of full-length/truncated reporter activity ratios (log2) from hyperconserved and non-conserved 5’UTRs. p indicates two-sided Wilcoxon rank sum test p-value. Box hinges: 25% quantile, median, 75% quantile, respectively from left to right. Whiskers: lower or upper hinge ±1.5*IQR. e, Scatter plot of change in translation efficiency between full-length and truncated h5UTRs shown in Fig. 3e versus change in uAUG density (change in number of AUGs / change in length between each pair of full-length and truncated h5UTRs). r indicates pearson’s correlation coefficient and p indicates two-tailed p-value.

Extended Data Fig. 4 Cellular remodeling of hyperconserved 5’UTR RNA structures.

a, Stacked bar plots showing proportions of significant (FDR ≤ 0.05) or not significant windows that overlap uAUG in black versus that do not overlap uAUG in red. OR indicates odds ratio for overlaps uAUG / does not overlap uAUG, and p indicates Fisher’s test p-value (one-sided, Ha = odds ratio>0). b, Stacked bar plots showing proportions of significant (FDR ≤ 0.05) or not significant windows that overlap uORF in black versus that do not overlap uORF in red. OR indicates odds ratio for overlaps uORF / does not overlap uORF, and p indicates Fisher’s test p-value (one-sided, Ha = odds ratio>0). c, Zoomed-in view of differential accessibilities along h5UTRs with one or more significantly different windows under ATP depletion. Top plot shows -log10 p-value for each window. Highlighted boxes mark significantly different windows, above the dashed line indicating 5% FDR. Middle plot shows differential accessibility on the y-axis, where greater than zero indicates increased accessibility upon ATP depletion and less than zero indicates decreased accessibility. Bottom plot shows differential accessibility for in vitro refolded RNA. Error bars in each plot show standard error, n = 3. The three profiled regions shown on the left side exhibit discordant profiles between accessibility changes observed in cells following ATP depletion and accessibility changes observed for in cell versus in vitro refolded RNA. The other three on the right side exhibit concordant profiles.

Extended Data Fig. 5 icM2 reveals structured elements in the hyperconserved Csde1 5’UTR.

a, Boxplot of average PhastCons scores in significant windows of ATP-dependent remodeling versus all windows shown in Fig. 4c. p indicates two-sided Wilcoxon rank sum test p-value. b, Same as Extended Data Fig. 5a, but showing the distribution of average PhyloP scores.

Extended Data Fig. 6 In-vitro M2 analysis of Csde1 5’UTR.

a, Heatmap of in-vitro M2 accessibility matrix for Csde1 5’UTR from position 190 to 386. For each row, the chemical mapping profile of a single-nucleotide variant of the RNA is plotted across the columns, where the colors indicate z-scaled accessibility change values from the wild-type RNA. 1D data from each mutant are vertically stacked to display a 2D matrix. White boxes mark perturbation signals that support the model shown in Extended Data Fig. 6b; color bars at the bottom indicate the nucleotide positions of the stems that match the same color in the model. b, The model for the in-vitro structure of Csde1 5’UTR from position 190 to 386. Also see Extended Data Fig. 6a.

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Byeon, G.W., Cenik, E.S., Jiang, L. et al. Functional and structural basis of extreme conservation in vertebrate 5′ untranslated regions. Nat Genet 53, 729–741 (2021). https://doi.org/10.1038/s41588-021-00830-1

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