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Large-scale tethered function assays identify factors that regulate mRNA stability and translation

Nature Structural & Molecular Biology volume 27, pages 989–1000 (2020)Cite this article

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Abstract

The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3ʹ-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR–Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.

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Fig. 1: A large-scale tethered function screen identifies RBPs regulating stability and translation.
Fig. 2: eCLIP identifies endogenous RNA targets of candidate stabilizers and destabilizers.
Fig. 3: Integration of eCLIP and RNA-seq data defines regulatory classes of RBPs and transcripts.
Fig. 4: UBAP2L is associated with translating ribosomes and promotes translation.
Fig. 5: UBAP2L binds directly to the ribosome.

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Data availability

Sequencing data are available at NCBI GEO (accession number GSE117294). Source data are provided with this paper.

Code availability

All code described in the Methods is publicly available and can be found at https://github.com/YeoLab/.

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Acknowledgements

We thank members of the Yeo lab, in particular E. Wheeler and F. Krach, for helpful discussions and A. Palazzo for advice on polysome fractionation. E.-C.L. was partly supported by a study-abroad graduate student fellowship from the Taiwanese government. J.C.S. was partially supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarships–Doctoral (PGS D-532649-2019). Y.H. was supported by the UCSD Frontiers of Innovation Scholars Program. G.A.P. was supported by a graduate fellowship from the National Science Foundation. F.E.T. was supported by a postdoctoral fellowship from the American Cancer Society (129547-PF-16-060-01-RMC). S.M. was supported by a postdoctoral fellowship from the Larry L. Hillblom Foundation (2014-A-027-FEL). This work was supported by grants from the NIH (R01HG004659, U19MH107367, R01NS103172 and U41HG009889) to G.W.Y.

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Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA

    En-Ching Luo, Jason L. Nathanson, Frederick E. Tan, Joshua L. Schwartz, Jonathan C. Schmok, Archana Shankar, Sebastian Markmiller, Brian A. Yee, Shashank Sathe, Gabriel A. Pratt, Duy B. Scaletta, Yuanchi Ha, Stefan Aigner & Gene W. Yeo

  2. Stem Cell Program, University of California, San Diego, La Jolla, CA, USA

    En-Ching Luo, Jason L. Nathanson, Frederick E. Tan, Joshua L. Schwartz, Jonathan C. Schmok, Archana Shankar, Sebastian Markmiller, Brian A. Yee, Shashank Sathe, Gabriel A. Pratt, Duy B. Scaletta, Yuanchi Ha, Stefan Aigner & Gene W. Yeo

  3. Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA, USA

    En-Ching Luo, Jason L. Nathanson, Frederick E. Tan, Joshua L. Schwartz, Jonathan C. Schmok, Archana Shankar, Sebastian Markmiller, Brian A. Yee, Shashank Sathe, Gabriel A. Pratt, Duy B. Scaletta, Yuanchi Ha, Stefan Aigner & Gene W. Yeo

  4. Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA

    David E. Hill

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Contributions

G.W.Y. designed the study; S.A., E.-C.L. and G.W.Y. wrote the manuscript. D.E.H. provided RBP ORF plasmids; J.L.N., D.B.S., J.C.S. and D.B.S. collected, built and validated the tagged RBP libraries. J.L.N. designed and validated the tethering reporters. E.-C.L. and J.L.N. performed RT–qPCR and luciferase assays. E.-C.L. performed knockdown and overexpression assays, western blots and polysome profiling experiments. E.-C.L. and A.S. performed eCLIP and immunofluorescence and microscopy. E.-C.L. generated RNA-seq and polysome profiling RNA-seq libraries. S.M. generated the UBAP2L-knockout cell line. F.E.T. and E.-C.L. performed the RCas9 reporter assay. J.L.S. performed the SUnSET assay. E.-C.L., B.A.Y., S.S., Y.H. and G.A.P. performed bioinformatics analyses.

Corresponding author

Correspondence to Gene W. Yeo.

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Competing interests

G.W.Y. is a cofounder and a member of the board of directors, is on the scientific advisory board and is an equity holder and paid consultant for Locana and Eclipse BioInnovations and a visiting professor at the National University of Singapore. The interests of G.W.Y. have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. The authors declare no other competing financial interests.

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Peer review information 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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Source of RBP ORFs, correlation between luciferase levels and RBP sizes, concordance between two luciferase systems, and correlation of reporter RNA and luciferase levels.

a, Western dot blot analysis of transiently expressed MCP-V5-tagged RBP ORFs in HeLa cells using a V5 antibody. Blue circles denote negative controls (no plasmid), red circles denote positive controls (CNOT7-V5-MCP). The order of wells and fold changes over negative controls are listed in Extended Data Table 1. b, Distribution of known classical and nonclassical RNA-binding domains in RBPs represented in our library. c, Distribution of molecular categories for RNA-related functions of RBPs represented in our library. d, Scatter plot of RBP size and luciferase effect. R, Pearson correlation coefficient. e, Luciferase activities from two different reporter constructs. Bar graphs showing log2-fold changes of the activity of Renilla (top) or firefly (bottom) luciferase reporters in presence of the MCP-fusion ORFs over FLAG control. Each vertical line represents a tethered ORF. f, Scatter plot of luciferase activities from the two reporter constructs. Values are expressed as log2-fold changes of the mean luciferase activity in the presence of MCP-fusion ORFs over FLAG controls. R, Pearson correlation coefficient.

Extended Data Fig. 2 IP validation from eCLIP experiments, correlation between eCLIP libraries, and de novo sequence motifs and metagene maps for candidate RBPs.

a,b, In-line western blots of eCLIP IPs of candidate RBPs. Extracts from HEK293T cells (a) or HEK293T cells transfected with the indicated V5-tagged RBP ORFs (b) immunoprecipitated with nonimmune (IgG) control antibodies, and western blot analysis using either RBP-specific (a) or anti-V5 (b) antibodies. The molecular weights (in kilodaltons) of standards are indicated on the right. Arrowheads indicate the calculated molecular weight for each RBP or RBP-V5 fusion protein. c, Heatmap of the Pearson correlation coefficients of fold enrichment of eCLIP peaks for the indicated 14 RBPs analyzed in duplicate. d, De novo sequence motifs in significant eCLIP peaks of the indicated RBP candidates enriched above background, with associated binomial P value. e–h, Metagene maps showing the distribution of eCLIP peak densities at target transcripts. The x axis indicates the relative length of each region. Dark red lines indicate the average number of significantly enriched peaks (≥4-fold enriched and P ≤ 10−2 versus SMInput) of eCLIP peak densities at all transcripts for BOLL, IFIT2, MEX3C, AIMP1 and CNOT7 (e), which show peak enrichment in 3ʹ UTR; DDX6, TOB1, NANOS3 and TOB2 (f), which show peak enrichment in 5ʹ UTR/3ʹ UTR; PARN and CLK3 (g), which show peak enrichment in 5ʹ UTR; and UBAP2L and MTDH (h), which show peak enrichment in CDS. Light shaded areas denote the 95% confidence interval.

Extended Data Fig. 3 Confirmation of RBP knockdown/overexpression, numbers of genes differentially regulated/unaffected by RBP perturbation, and region-level analysis of bound transcripts.

a,b, shRNA-mediated depletion of RBPs in HEK293T cells using 3-5 distinct shRNAs for each RBP, as indicated, compared to nontargeting shRNA control. a, Western blots with GAPDH or tubulin serving as loading controls, as indicated. b, Bar graphs indicating RBP transcript levels determined by RT–qPCR, normalized to levels of 18S rRNA. Data are shown as mean ± SD (n = 3 replicates). Asterisks denote significance at P < 0.05 determined by two-sided Student’s t-test. c, Overexpression of RBPs in HEK293T cells. Bar plots showing transcript levels (RPKM) for each RBP following transfection of RBP expression constructs or FLAG vector control. Data are shown as mean ± s.d. (n = 2 replicates). d–g, Numbers of up- or downregulated (log2-fold change ≥ 1.23 and FDR-corrected P ≤ 0.05) or unchanged genes for transcripts bound (≥4-fold enriched and P ≤ 10−2 versus eCLIP SMInput) or not bound by the indicated RBP for knockdown (d) and overexpression (e) of destabilizing RBPs and for knockdown (f) and overexpression (g) of stabilizing RBPs. h,i, Volcano plots showing the distribution of fold changes in transcript levels, with distribution histograms at the top, upon depletion of the destabilizer PARN (h) and depletion of the stabilizer CLK3 (left) and overexpression of the destabilizer IFIT2 (right) (i). Transcripts with log2(fold change) ≥1.23 and FDR-corrected P ≤ 0.05 are in color, with red and green denoting transcripts with or without at least one significant RBP binding peak (≥4-fold enriched and P ≤ 10−2 versus SMInput in eCLIP), respectively. j, Heatmap showing significance in differential expression of genes significantly differentially expressed (log2(fold change) ≥ 1.23 and FDR-corrected P ≤ 0.05) and significantly bound (≥4-fold enriched and P ≤ 10−2 versus SMInput in eCLIP) versus all unbound genes upon knockdown (KD) or overexpression (OE) of candidate RBPs in each region. Significance was calculated using a two-tailed Mann-Whitney U test. Uncropped images for a are available as source data online, and data for graphs in b, c are available as source data online.

Source data

Extended Data Fig. 4 Translation monitoring in a UBAP2L knockout replicate line, replicate concordance, and validation of polysome analyses in UBAP2L knockouts.

a, Translation monitoring using puromycin incorporation. Anti-puromycin western blot of extracts from puromycin-treated UBAP2L knockout (KO1) and parental (WT) HEK293T cell lines. GAPDH served as loading control. b,c, Polysome profile of UBAP2L after treatment of cells with 0.5 mM puromycin (b) and treatment of lysates with 30 mM EDTA (c). Top, absorbance (at 260 nm) plot of a HEK293T cell lysate fractionated through a 10-50% sucrose gradient. Bottom, western blots of UBAP2L from the corresponding fractions. d, Polysome profiles of HEK293T cells (WT, n = 2) and UBAP2L knockout HEK293T cells (KO, n = 4) fractionated through 10-50% sucrose gradients. Light-colored lines indicate means from each set (WT, light blue; KO, pink), and darkly shaded areas denote s.d. (WT, blue; KO, red). e, Bar graphs showing percentages of transcripts with RPKM ≥ 1 of all transcripts with ≥10 reads per transcript for two UBAP2L knockout lines (KO, 2 replicates each) and control samples (WT, two replicates). f, Scatter plots showing correlation of log2-transformed ratios of input-normalized polysome transcript levels (RPKM) between the two UBAP2L knockout HEK293T lines. R, Pearson correlation coefficient. g, Bar graph showing the percentage of regulated transcripts in UBAP2L targets and nontargets. *P < 0.0001 (χ2 test with Yates’s correction). h, RT–qPCR validation of reduced polysome association for the indicated transcripts. Transcript levels in inputs and polysome fractions were measured for KO and WT samples. KO/WT ratios of input-normalized polysome association of transcripts were then calculated. i, Western blots of DDX54, EIF4G1, EIF3B, and EEF2 in UBAP2L knockout cells (KO1, KO2). GAPDH served as a loading control. j,k, Quantitative flow cytometry reporter assay for mRNA translation using RCas9-fused 4EBP1. j, Plasmid design for the RCas9-4EBP1 experiment. k, Bar graph showing mean YFP levels in RCas9-4EBP1-expressing cells, normalized to RCas9-expressing cells, on each targeting site. Error bars denote s.d. from n > 5,000 RCas9-4EBP1-expressing and n > 5,000 rCas9-expressing cells per site. *P < 0.005; n.s., not significant (P > 0.5); two-tailed Student’s t-test. Uncropped images for a–c and i and data for graphs in h and k are available as source data online.

Source data

Extended Data Fig. 5 Repetitive element analysis of UBAP2L eCLIP data.

a, Immunofluorescence images showing UBAP2L (green) in HEK293T cells. DAPI (blue) marks nuclei. Scale bar, 10 μm. b, Pie chart showing fractions of UBAP2L replicate 2 eCLIP reads unambiguously mapping to repeat families in HEK293T cells. c, Line plot of UBAP2L binding sites on rRNAs. Fold enrichment of reads for IP over SMInput is plotted against the nucleotide positions of 18S and 28S rRNAs. Asterisk (*) denotes relative entropy ≥0.01. d–g, Location of UBAP2L binding sites on rRNA. d, ES15L; e, ES7S; f, ES27L; g, ES31L. Nucleotides with significant binding are highlighted in yellow. h, RIP of UBAP2L-RIP and RT–PCR in HEK293T cell lysates. The RIP assay was performed using anti-UBAP2L antibody or rabbit nonimmune IgG. RT–PCR was performed using primer sets within UBAP2L target regions ES7S, ES7L, ES15L, and ES31L. Uncropped images for h are available as source data online.

Source data

Supplementary information

Supplementary Information

Supplementary Note.

Reporting Summary

Supplementary Table 1

For each RBP isoform in the tethered function ORF library, this table lists GenBank gene symbol and accession number(s), calculated molecular weight, prioritized GO term, whether the RBP was identified in Baltz et al. (2012) and/or Castello et al. (2012), prioritized known RNA-binding domain, the source of the ORF construct and expression levels in overexpression assays relative to the negative control.

Supplementary Table 2

For each RBP isoform (ORF) tested, this table lists RLuc–RBP and corresponding RLuc–FLAG luciferase levels of triplicate measurements and means for both; FLuc–RBP and corresponding FLuc–FLAG luciferase levels of triplicate measurements and means for both; mean fold changes (FLuc–RBP/RLuc–FLAG and RLuc–RBP/FLuc–FLAG ratios); P values; and whether the criterion of FDR < 0.01 was fulfilled.

Supplementary Table 3

This table lists the means of the log2-transformed normalized fold changes of luciferase levels of the FLuc reporter in the presence of the indicated tethered RBP and corresponding means of the reporter transcript levels as measured by RT–qPCR.

Supplementary Table 4

This table lists the oligonucleotide primer sequences for RT–qPCR of the luciferase reporter transcripts (RT–qPCR_luciferase assay), validation of shRNA-mediated RBP knockdown for RNA-seq assays (RT–qPCR_shRNA), validation of polysome profiling results (RT–qPCR_polysome) and RT–PCR assays used for RIP analysis on rRNAs (RIP-RT–PCR).

Supplementary Table 5

This table lists the manually curated functional categories and the direction of regulation.

Supplementary Table 6

This table lists the sources of the antibodies used for western blot (WB) and immunofluorescence staining (IF) with dilutions used and for IP in eCLIP (IP).

Supplementary Table 7

This table lists the numbers of raw reads, uniquely mapped reads and usable reads (that is, after PCR duplicate removal) as well as peak numbers (called peaks and significant peaks at thresholds ≥4-fold enrichment and P ≤ 10−2; χ2 test) for the eCLIP libraries generated. Each eCLIP experiment consists of a SMInput (Input) sample and an IP sample. For CNOT7 and AIMP1, the same input library was used for both RBPs due to their similar apparent molecular weight on SDS gel. OE, overexpression.

Supplementary Table 8

This table lists the numbers of raw reads, and numbers and fractions of uniquely mapping reads, for the RNA-seq libraries generated from lentiviral shRNA knockdown and overexpression (OE) samples.

Supplementary Table 9

This table lists the sources and target sequences for the lentiviral shRNA constructs used. TRC, The RNAi Consortium.

Supplementary Table 10

This table lists the numbers of raw reads and the numbers and fractions of uniquely mapping reads for the RNA-seq libraries generated in the polysome profiling studies.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1k,l.

Source Data Fig. 4

Uncropped western blots for Fig. 4a–c,i.

Source Data Fig. 4

Statistical source data for Fig. 4b,i,k,l.

Source Data Fig. 5

Uncropped western blots for Fig. 5b,c.

Source Data Extended Data Fig. 3

Uncropped western blots for Extended Data Fig. 3a.

Source Data Extended Data Fig. 3

Statistical source data for Extended Data Fig. 3b,c.

Source Data Extended Data Fig. 4

Uncropped western blots for Extended Data Fig. 4a–c.

Source Data Extended Data Fig. 4

Statistical source data for Extended Data Fig. 4h,k.

Source Data Extended Data Fig. 5

Uncropped gels for Extended Data Fig. 5h.

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Luo, EC., Nathanson, J.L., Tan, F.E. et al. Large-scale tethered function assays identify factors that regulate mRNA stability and translation. Nat Struct Mol Biol 27, 989–1000 (2020). https://doi.org/10.1038/s41594-020-0477-6

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