Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

Abstract

mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs.

Keywords: 5′ untranslated region; N1-methylpseudouridine; RNA modification; high-throughput screening; ribosome; therapeutic mRNA; translation initiation.

Figure 1.. DART quantifies human 5′ UTR-mediated translational control over a 200-fold range

A) Schematic of the DART workflow. The DNA UTR library sequences contain a T7 promoter, >27nt of coding sequence, and an RT binding site for library preparation. Endogenous 5′ UTR sequences were derived from Ensembl annotations. B) Human DART reproducibly measures ribosome recruitment over a 200-fold range. C) Pearson correlations among six DART replicates. See also Figure S1.

Figure 2.. CCC motifs repress Vaccinia-capped 5′ UTR activity

A) Scanning deletion library design for systematic identification of regulatory elements. B) Scanning deletion analysis identifies hundreds of hexamers that significantly increase (red) or decrease (blue) RRS (padj < 0.01). C) Translational enhancer in the TMSB15 5′ UTR. Deletion of nucleotides 1–6 or 7–12 (red) reduces RRS by over 4-fold. D) Volcano plot of tetramers deleted ≥30 times covered in the scanning deletion library. Tetramers that significantly altered RRS are highlighted in red (padj < 0.01). E) Global trend of reduced RRS with increasing cytidine content. F) DREME analysis shows enrichment of C-rich sequence motifs in the bottom 10% of 5′ UTRs by RRS. G) The number of CCC trinucleotide motifs correlates with decreased RRS (n = 2,295 5′ UTRs containing 30–35% cytidine; mean ± 95% CI) *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001, one-way ANOVA and Tukey’s multiple comparisons test. H) Library design testing CCC motif dose-dependence using exogenous CCC additions. I) and J) CCC motifs repress RRS in a dose-dependent manner. I) Repressive effect for ZFPL1 and J) across all 5′ UTRs tested (n = 225 parent 5′ UTRs, 1,350 total variants). K) Adding CCC motifs represses translation, with a larger effect on vaccinia-capped than co-transcriptionally capped mRNAs (mean ± SD). L) Library design to test CCC motif dose-dependence by deleting endogenous CCC motifs. M) and N) Removal of CCC motifs from 5′ UTRs increases RRS. The effect of CCC removal for the POC1B 5′ UTR (M) and all UTRs tested (N, n = 225 parent 5′ UTRs, 1,350 total variants). O) Deleting CCC motifs within 5′ UTRs increases translation, with a larger effect on vaccinia-capped than co-transcriptionally capped mRNA (mean ± SD). See also Figure S2.

Figure 3.. Known regulatory elements explain little of the observed variation in 5′ UTR-specific translation activity

A) DART scores predict translational output over a 200-fold range. Correlation of RRS with protein production from transfected in vitro transcribed mRNAs (mean ± SD). B) UTR length modestly negatively correlates with RRS. C) Histogram of log2(RRS) scores for 5′ UTRs shorter than 15 nucleotides (red) or greater than 15 nucleotides (blue). D) Predicted minimum free energy modestly negatively correlates with RRS. E) Impact of GC content on RRS (mean ± 95% CI). F) Kozak sequence strength promotes ribosome recruitment to random 10-nucleotide 5′ UTRs. UTRs scored by conformity to the consensus human Kozak sequence. G) Pyrimidines at the −3 position correlate with decreased RRS. ****p < 0.0001, unpaired Welch’s t-test. H) A linear regression model incorporating length, predicted minimum free energy, GC content, and Kozak strength explains only 28% of the variance in DART. I) and J) Sequence motifs enriched in I) worst 10% and J) best 10% of 5′ UTRs by RRS. K) Volcano plot of tetramers deleted ≥30 times covered in the scanning deletion library. Tetramers that significantly altered RRS are highlighted in red. See also Figure S3.

Figure 4.. Cap-proximal nucleotide composition affects ribosome recruitment

A) Deletions of nucleotides 1–6 or 7–12 more often caused a significant change (padj < 0.01) and B) caused larger magnitudes of change in RRS (mean ± 95% CI). C) Gain of uridines and loss of guanosines within the first 6 nucleotides increases RRS in a dose-dependent manner. 5′ UTRs were binned based on the change in the number of each nucleotide. Change in RRS relative to parent sequences is plotted on the y-axis (mean ± SEM). D) Cap-proximal uridines or guanosines are enriched in highly or minimally active 5′ UTRs, respectively. UTRs binned as the top 10% (solid lines) or bottom 10% (dashed lines) by RRS. Plot displays the percent of UTRs containing uridine (top) or guanosine (bottom) at each position. E) Illustration of 5′ UTR isoform types. F) 5′ extension is the most prevalent isoform type in DART library. G) Most 5′ extension isoform pairs differ significantly in RRS. H) Example of 5′ extension isoforms of ADHFE1 that exhibit significantly different RRS. Adding 1–7 nucleotides at the 5′ end is sufficient to alter ribosome recruitment. The changes in the 5′ UTR sequences are labeled below. I) Gain of uridines and loss of guanosines in the first 6 nucleotides between 5′ extension pairs increases RRS in a dose-dependent manner (mean ± SEM). J) and K) An isoform of DNASE2 that is poorly translated in HeLa extracts is preferentially expressed in neurons. J) The RNA expression level for each transcript isoform of gene DNASE2 in kidney, liver, neuron and T cells. K) RRS scores for two major isoforms of DNASE2. See also Figure S4.

Figure 5.. Coding sequence significantly affects ribosome recruitment to many 5′ UTRs

A) Library design to test the impact of coding sequences on ribosome recruitment. B) Coding sequence significantly affects ribosome recruitment. Each 5′ UTR is plotted according to its RRS with EGFP and endogenous coding sequence. C) The impact of coding sequence on RRS is context-dependent. 5′ UTRs with significantly altered RRS with endogenous compared to EGFP coding sequence (fold change > 2, padj < 0.01) are color labelled. D) Changes in uridine, cytidine, guanosine, and GC content within the coding sequence correlate with altered RRS. P-values determined by Wilcoxon rank test and Benjamini-Hochberg adjustment. E and F) nucleotide content in the coding sequence influences recruitment. E) Uridine, F) cytosine. ****p < 0.0001 unpaired Welch’s t-test. See also Figure S5.

Figure 6.. DART assay can be miniaturized to save input material and increase throughput

A) DART can be scaled down 25-fold without losing sequence representation. Cell lysate input is shown as a bar plot (right y-axis), and the number of sequences > 1 CPM is shown as a line plot (left y-axis). Plates indicate the cell culture area required for each lysate volume. B) DART reproducibility is preserved across volumes. RRS from each input volume are plotted against each other. C) RRS from miniaturized DART predict 5′ UTR-driven translational activity in luciferase reporter mRNAs (mean ± SD). See also Figure S6.

Figure 7.. Global and 5′ UTR-specific effects of N1-methylpseudouridine on ribosome recruitment

A) Uridine-containing and m1Ψ-containing RNAs were barcoded and combined in the same reaction for direct comparison of activity. B) Identical 5′ UTR sequences containing uridine versus m1Ψ (middle) show widespread differences in RRS (n = 6) compared to replicate reproducibility with uridine (U, left) or m1Ψ (right). C) Volcano plot of RRS differences with m1Ψ substitution. 5′ UTRs exhibiting significantly changes in RRS (fold change > 2, padj < 0.01) are color labelled D) and E) Cumulative distribution plots of D) RRS and E) stability scores from UTRs containing uridine (green) or m1Ψ (purple). F) The effect of m1Ψ on ribosome recruitment is 5′ UTR-specific. Individual examples of 5′ UTRs that are strongly stimulated (top) or repressed (bottom) by m1Ψ substitution (n = 6, mean ± SD). G) Scanning deletion analysis identifies tetramers that significantly alter ribosome recruitment when deleted in m1Ψ-substituted RNAs (right). H) Increasing numbers of (A/U)UUU motifs and I) poly(m1Ψ) stretch length correlate with increased ribosome recruitment in m1Ψ-substituted RNAs, (mean ± 95% CI, p < 2.2e⁻¹⁶ by two-way ANOVA test considering the interaction between modifications and number/length of U/m1Ψ stretches. J) Optimal m1Ψ-substituted mRNAs produce more protein from luciferase reporter mRNAs than current commercial vaccine 5′ UTRs (mean ± SD, p < 0.05 two-tailed Student’s t-test). See also Figure S7.