Decoding mRNA translatability and stability from the 5′ UTR (2024)

Table of Contents
Data availability Code availability References Acknowledgements Author information Authors and Affiliations Contributions Corresponding author Ethics declarations Competing interests Additional information Extended data Extended Data Fig. 1 Characterization of uORF reporters. Extended Data Fig. 2 Establishing massively parallel uORF reporters. Extended Data Fig. 3 Characterizing massively parallel uORF reporters using flow cytometry. Extended Data Fig. 4 Identification of TIS sequence features in uORF reporters enriched in ribosome fractions. Extended Data Fig. 5 uORF translation triggers mRNA decay in a ribosome-dependent manner. Extended Data Fig. 6 Identification of inhibitory elements in 5’UTR that impair translation. Extended Data Fig. 7 RG4 in 5’UTR triggers mRNA decay in the P-body. Extended Data Fig. 8 RG4 in 5’UTR derived from NRAS triggers mRNA decay. Extended Data Fig. 9 An A-rich element in 5’UTR enables cap-independent translation. Extended Data Fig. 10 An A-rich element in 5’UTR promotes translation-independent decay. Supplementary information Reporting Summary Peer Review Information Supplementary Table 1 Source data Source Data Fig. 1 Source Data Fig. 2 Source Data Fig. 3 Source Data Fig. 4 Source Data Fig. 5 Source Data Fig. 6 Source Data Extended Data Fig. 1 Source Data Extended Data Fig. 3 Source Data Extended Data Fig. 4 Source Data Extended Data Fig. 5 Source Data Extended Data Fig. 6 Source Data Extended Data Fig. 7 Source Data Extended Data Fig. 8 Source Data Extended Data Fig. 9 Source Data Extended Data Fig. 10 Rights and permissions About this article Cite this article

Data availability

Deep sequencing data that support the findings of this study have been deposited in GEO database with the accession code GSE145046. Source data are provided with this paper.

Code availability

All the procedures but those for sequencing reads cleaning were completed using custom Perl and R scripts, which are available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the Grimson lab for providing us with several shRNAs targeting mammalian decay factors. We are grateful to Cornell University Life Sciences Core Laboratory Center for sequencing, FACS, and confocal microscope support. This work was supported by US National Institutes of Health (R01GM1222814 and R21CA227917) and HHMI Faculty Scholar (55108556) to S.-B.Q.

Author information

Author notes

  1. These authors contributed equally: Longfei Jia, Yuanhui Mao.

Authors and Affiliations

  1. Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA

    Longfei Jia,Yuanhui Mao,Quanquan Ji&Shu-Bing Qian

  2. Cellular Biology Section, Laboratory of Viral Diseases, NIAID, Bethesda, MD, USA

    Devin Dersh&Jonathan W. Yewdell

Authors

  1. Longfei Jia

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  2. Yuanhui Mao

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  3. Quanquan Ji

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  4. Devin Dersh

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  5. Jonathan W. Yewdell

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  6. Shu-Bing Qian

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Contributions

S.-B.Q. conceived the project and designed the experiments. L.J. performed the majority of experiments and Y.M. conducted the majority of data analysis. Q.J. contributed to the PABP1 knockdown experiments. D.D. and J.W.Y. helped with 25D1 reagents and HEK293-Kb cells. S.-B.Q. wrote the manuscript with comments from L.J. and Y.M. All authors discussed the results and edited the manuscript.

Corresponding author

Correspondence to Shu-Bing Qian.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review informaiton Peer reviewer reports are available. Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Characterization of uORF reporters.

a, Basic design of the uORF reporter with the SIINFEKL sequence highlighted. 5 additional amino acids (LEQLE) are present, which permits processing of SIINFEKL from the same flanking amino acids regardless of the TIS sequence. b, Bar graphs show the 25D1 or GFP mean fluorescence of HEK293-Kb cells transfected with plasmids of uORF reporters. n = 2 biological replicates, Error bars indicate SEM. c, Representative flow cytometry scatterplots of HEK 293-Kb cells transfected with synthetic mRNA reporters with optimal or suboptimal AUG codons. d, A bar graph shows the ratio of 25D1/GFP in HEK 293-Kb cells transfected with synthetic mRNA or plasmid DNA. Error bars, mean ± s.e.m; n = 3 biological replicates.

Source data

Extended Data Fig. 2 Establishing massively parallel uORF reporters.

a, Schematic of generating a library of RNA-based uORF reporters by PCR-amplification using primers composed of random 10-nt sequences upstream of the uORF. Pooled PCR products were utilized as templates for in vitro RNA synthesis followed by 5’ capping and 3’ polyadenylation. b, Comparison of sequence randomness for nucleotide oligos synthesized by different vendors. c, A histogram shows the distribution of read count on individual unique random sequences. d, A scatter plot shows the correlation of read counts between two biological replicates of original oligo sequences (Rho = 0.79, P < 2.2 × 10-16).

Extended Data Fig. 3 Characterizing massively parallel uORF reporters using flow cytometry.

a, Representative flow cytometry scatterplots of HEK 293-Kb cells transfected with the GFP mRNA reporter or pooled uORF reporters. Relative GFP and 25D1 fluorescence intensity between GFP and uORF-GFP reporters are shown in histograms as well as bar graphs. Error bars, mean ± s.e.m. n = 3 biological replicates. b, HEK293-Kb cells were transfected with mRNA reporters followed by FACS soring into GFPH and 25D1H populations. A bar plot (top) shows the ratio of triplet frequency within the random sequences enriched in the 25D1H population over the GFPH population. Only the top 10% sequence variants ranked in 25D1H and GFPH populations are used. The original frequency of triplets in different populations is shown as a heat map (bottom). c, Correlation of triplet frequencies within the sequence variants enriched in 25D1H or GFPH populations. All points are color-encoded based on the similarity to ATG.

Source data

Extended Data Fig. 4 Identification of TIS sequence features in uORF reporters enriched in ribosome fractions.

a, A total of 14,381 sequences with all permutations of NNNNAUGNNN are ranked based on the ratio of frequency between monosome and polysome. Both the top and bottom hits are highlighted. b, Sequence logo of 10 nt random sequences with high (top) or low (bottom) M/P ratio. Note that the high M/P sequence is consistent with the Kozak consensus sequence. c, A violin plot shows the ratio of relative frequencies of inserted sequences in monosome over polysome, when ATG triplet is followed by different nucleotides. d, Representative flow cytometry scatterplots of HEK 293-Kb cells transfected with mRNA reporters with sequence variants chosen from monosome (M1 - M4) or polysome (P1 - P4).

Source data

Extended Data Fig. 5 uORF translation triggers mRNA decay in a ribosome-dependent manner.

a, A scatter plot shows the correlation of triplet frequencies enriched in polysome and GFPH population from HEK293-Kb cells transfected with mRNA reporters. All points are color-encoded based on the similarity to ATG. b, A scatter plot shows the correlation of in vivo and in vitro half-lives of mRNA reporters. Half-life values were centered to medians. c, A boxplot shows positive correlation between GFP intensities and half-lives of mRNA reporters. All random sequences were divided into five groups based GFP intensity measured by flow cytometry. d, HEK293-Kb cells were transfected with DNA plasmids with or without optimal ATG codons (left) or representative hits from M and P fractions (right), followed by RT-qPCR at indicated time points (n = 3 biological replicates; t test). Error bars indicate SEM. ** P < 0.01; * P < 0.05. e, Representative flow cytometry scatterplots of HEK 293-Kb cells transfected with mRNA reporters capped with ApppG with sequence variants chosen from monosome (M1) or polysome (P1).

Source data

Extended Data Fig. 6 Identification of inhibitory elements in 5’UTR that impair translation.

a, Flow cytometry scatterplots of HEK 293-Kb cells transfected with mRNA reporters enriched in the ribosome-free fractions (N1 – N8). b, HEK293-Kb cells were transfected with mRNA reporters or plasmids with sequence variants chosen from the ribosome-free fractions (N1 – N8), followed by RT-qPCR at indicated time points (n = 3 biological replicates; t test). Error bars indicate SEM. ** P < 0.01. c, Flow cytometry scatterplots of HEK 293-Kb cells transfected with mRNA reporters enriched in the ribosome-free fractions (N1 – N8) and stained with QUMA-1.

Source data

Extended Data Fig. 7 RG4 in 5’UTR triggers mRNA decay in the P-body.

a, HEK293-Kb cells with specific decay factor knockdown were transfected with the N1 mRNA reporter, followed by RT-qPCR at indicated time points. (n = 3 biological replicates; t test). Error bars indicate SEM. * P < 0.05. b, Representative confocal images of HEK 293-Kb cells transfected with P1 or N1 mRNA reporters. The mRNA reporters were synthesized in the presence of Alexa Fluor-UTP and the transfected cells were co-stained with a DCP2 antibody. DNA was counter-stained with Hoechst 33342. Arrowheads indicate typical mRNA foci. Bar, 10 μm. Images are representative of at least 50 cells.

Source data

Extended Data Fig. 8 RG4 in 5’UTR derived from NRAS triggers mRNA decay.

a, The left panel shows the schematic of mRNA reporter with 5’UTR derived from NRAS with (WT) or without (MT) RG4. The right panel shows the decay of mRNA reporters in transfected HEK293-Kb cells. (n = 3 biological replicates; t test). Error bars indicate SEM. ** P < 0.01. b, The stability of endogenous NRAS was measured in HEK293-Kb cells with or without DHX36 knockdown. (n = 3 biological replicates; t test). Error bars indicate SEM. * P < 0.05.

Source data

Extended Data Fig. 9 An A-rich element in 5’UTR enables cap-independent translation.

a, A scatter plot shows the distribution of quadruplets between ribosome free and ribosome-bound fractions. Each point refers a 4-mers sequence, and all points are color-encoded based on the ratio of A/C. b, Representative flow cytometry of HEK 293-Kb cells transfected with 10C, 10A, 5A, or 4A mRNA reporters capped with non-functional ApppG. c, Representative flow cytometry of HEK 293-Kb cells transfected with mRNA reporters containing AAAAAUGAAA capped with m7G or ApppG.

Source data

Extended Data Fig. 10 An A-rich element in 5’UTR promotes translation-independent decay.

a, HEK293-Kb cells were transfected with 10A or 10C mRNA reporters capped with m7G (right) or ApppG (left), followed by RT-qPCR at indicated time points. (n = 3 biological replicates; t test). Error bars indicate SEM. b, The left panel shows the distribution of in vitro half-lives of mRNA reporters. The most stable (top 10%) sequences are highlighted in red, and the most unstable sequences are highlighted in light blue. The right panel shows the heat map of base frequency at different positions of random sequences. c, A violin plot shows half-life of mRNAs groups with different number of continuous As in random sequences. d, A heat map shows the effect of A-cluster length and position on the in vitro half-life of mRNA reporters. e, The in vitro decay of mRNA reporters (10A, 5A, and 4A) in the lysates of HEK293-Kb cells was determined by RT-qPCR at indicated time points. (n = 3 biological replicates; t test). Error bars indicate SEM. f, The in vitro decay of 10A mRNA reporters in the lysates of HEK293-Kb cells with or without UPF1 knockdown was determined by RT-qPCR at indicated time points (left). For the in vivo stability, HEK293-Kb cells with or without UPF1 knockdown were transfected with 10A mRNA reporters followed by RT-qPCR at indicated time points (right). (n = 3 biological replicates; t test). Error bars indicate SEM. g, The in vitro stability of mRNA reporters (10A, M1, and P1) in the lysates of HEK293-Kb cells with CNOT1, PARN, or PAN3 knockdown was determined by RT-qPCR at indicated time points. (n = 3 biological replicates; t test). Error bars indicate SEM. ** P < 0.01.

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Supplementary information

Supplementary Table 1

List of all oligonucleotide sequences used

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Decoding mRNA translatability and stability from the 5′ UTR (1)

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Jia, L., Mao, Y., Ji, Q. et al. Decoding mRNA translatability and stability from the 5′ UTR. Nat Struct Mol Biol 27, 814–821 (2020). https://doi.org/10.1038/s41594-020-0465-x

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Decoding mRNA translatability and stability from the 5′ UTR (2024)
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