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Short poly(A) tails are a conserved feature of highly expressed genes

Abstract

Poly(A) tails are important elements in mRNA translation and stability, although recent genome-wide studies have concluded that poly(A) tail length is generally not associated with translational efficiency in nonembryonic cells. To investigate whether poly(A) tail size might be coupled to gene expression in an intact organism, we used an adapted TAIL-seq protocol to measure poly(A) tails in Caenorhabditis elegans. Surprisingly, we found that well-expressed transcripts contain relatively short, well-defined tails. This attribute appears to be dependent on translational efficiency, as transcripts enriched for optimal codons and ribosome association had the shortest tail sizes, whereas noncoding RNAs retained long tails. Across eukaryotes, short tails were a feature of abundant and well-translated mRNAs. This seems to contradict the dogma that deadenylation induces translational inhibition and mRNA decay and suggests that well-expressed mRNAs accumulate with pruned tails that accommodate a minimal number of poly(A)-binding proteins, which may be ideal for protective and translational functions.

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Figure 1: The C. elegans poly(A) profile.
Figure 2: Highly expressed mRNAs have short poly(A) tails.
Figure 3: Efficient translation is associated with short poly(A) tails.
Figure 4: Short poly(A) tails are features of highly expressed mRNAs in yeast and mouse.
Figure 5: Model for short poly(A) tails on highly expressed mRNAs.

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Acknowledgements

We thank V.N. Kim, J. Lim, and H. Chang for providing a detailed TAIL-seq protocol, their algorithm (tailseeker2), and technical assistance, E. Van Nostrand and members of the Yeo lab for assistance with the Illumina MiSeq platform, J. Chen and J. Broughton for programming support, and J. Lykke-Andersen, H. Cook-Andersen, M. Wilkinson, and members of the Pasquinelli lab for suggestions and critical reading of the manuscript. L.B.C. and A.L.N. were supported by the UCSD Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine (T32 GM007240) and NSF Graduate Research Fellowships DGE-1650112 (L.B.C.) and DGE-1650112 (A.L.N.). This work was supported by grants from the NIH (GM071654) and UCSD Academic Senate to A.E.P., NIH (GM118018) to J.C., and NIH (HG004659) to G.W.Y. S.A.L. was supported by an international predoctoral fellowship from the HHMI.

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A.E.P. and S.A.L. designed the project and wrote the paper. S.A.L. conducted the experiments and data analysis with help from L.B.C., A.L.N., B.A.Y. and G.W.Y. Y.-H.C. and J.C. designed and performed experiments for Supplementary Figure 5.

Corresponding author

Correspondence to Amy E Pasquinelli.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of mTAIL-seq.

(A) Cumulative frequency plot of poly(A) measurements of spike in controls of varying tail lengths. (B) Pearson correlation between median tail-length per gene in two biological replicates (n = 12,017 genes). (C) Pearson correlation of measured transcript abundance between two biological replicates (n = 12,017 genes). (D) Functional annotations and (E) tissue enrichment profiles for genes with median tail sizes predicted to accommodate 1–2 (29–60 median tail sizes), 2–3 (61–90), 3–4 (91–120), 4–5 (121–150) and 5 or more (>151) PABPs.

Supplementary Figure 2 Range of poly(A) tails.

(A to C) Heat maps show the median maximal tail length reported for each box (A), the median minimal tail length (B) and the tail range (C) (n = 13,421 protein coding genes). Poly(A) tail measurements and frequency of optimal codons (Fop) for C. elegans transcripts are available in Supplementary Data Set 1.

Supplementary Figure 3 Short poly(A) tails are associated with well-expressed genes in Drosophila S2 cells.

(A and B) Cumulative distribution plots showing the relationship between codon optimization, poly(A) length (A) and transcript abundance (B) (n = 4,904 genes). Source data is from Subtelny et al., Nature. 508, 66–71, 2014, and summarized in Supplementary Table 2.

Supplementary Figure 4 Poly(A) tails of ribosomal protein transcripts.

(A) Cumulative distribution plots of median poly(A) tail length for the mRNAs of ribosomal proteins in S. cerevisiae (n = 56 RP and 3,526 all genes), C. elegans (n = 71 RP and 13,601 all genes), NIH3T3 (n = 67 RP and 10,107 all genes) and HeLa (n = 58 RP and 10,213 all genes). (B) Violin distribution plots with inlaid box-plots (white dot represents the median) of all tail-length measurements for transcripts of 8 ribosomal proteins in C. elegans. (C) Northern blot for poly(A) length analysis of rps-5 mRNA. The expected length of the poly(A)-fragment and an RNA marker were used to determine the poly(A) tail size in the three biological replicates. Poly(A) tail measurements and frequency of optimal codons (Fop) for C. elegans transcripts are available in Supplementary Data Set 1. References and details for the source data used in (A) are in Supplementary Table 2.

Supplementary Figure 5 Codon optimality affects poly(A) tail size.

Northern blot for poly(A) length analysis of steady state HIS3 reporter mRNAs with varying percentages of optimal codons (Radhakrishnan et al., Cell. 167, 122–132, 2016). The normalized signal intensity plotted for the 0%, 50% and 100% codon optimality lanes shows an inverse relationship between poly(A) tail size and codon optimality. Results are representative of 3 independent biological replicates.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 824 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Supplementary Table 2

Summary and references for datasets used in the analyses (XLSX 11 kb)

Supplementary Data Set 1

Poly(A) tail measurements, abundance, Fop, DAVID and tissue enrichment analyses - Data related to Fig. 1, 2, 3; Supplementary Fig. 1, 2, 4; Supplementary Table 1 (XLSX 3784 kb)

Supplementary Data Set 2

Ribosome enrichment calculations after removing the first 50nt of ORF - Data related to Fig. 2d, 2h; Supp Table 1 (XLSX 1249 kb)

Supplementary Data Set 3

WT (L4) C. elegans RNA-seq - Data related to Supplementary Table 1 (XLSX 1156 kb)

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Lima, S., Chipman, L., Nicholson, A. et al. Short poly(A) tails are a conserved feature of highly expressed genes. Nat Struct Mol Biol 24, 1057–1063 (2017). https://doi.org/10.1038/nsmb.3499

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