Abstract
Poly(A) tails enhance the stability and translation of most eukaryotic messenger RNAs, but difficulties in globally measuring poly(A)-tail lengths have impeded greater understanding of poly(A)-tail function. Here we describe poly(A)-tail length profiling by sequencing (PAL-seq) and apply it to measure tail lengths of millions of individual RNAs isolated from yeasts, cell lines, Arabidopsis thaliana leaves, mouse liver, and zebrafish and frog embryos. Poly(A)-tail lengths were conserved between orthologous mRNAs, with mRNAs encoding ribosomal proteins and other ‘housekeeping’ proteins tending to have shorter tails. As expected, tail lengths were coupled to translational efficiencies in early zebrafish and frog embryos. However, this strong coupling diminished at gastrulation and was absent in non-embryonic samples, indicating a rapid developmental switch in the nature of translational control. This switch complements an earlier switch to zygotic transcriptional control and explains why the predominant effect of microRNA-mediated deadenylation concurrently shifts from translational repression to mRNA destabilization.
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Accession codes
Accessions
Gene Expression Omnibus
Data deposits
Sequencing data and the processed data for each gene are available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE52809.
References
Moore, M. J. & Proudfoot, N. J. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136, 688–700 (2009)
Goldstrohm, A. C. & Wickens, M. Multifunctional deadenylase complexes diversify mRNA control. Nature Rev. Mol. Cell Biol. 9, 337–344 (2008)
Chen, C. Y. & Shyu, A. B. Mechanisms of deadenylation-dependent decay. Wiley Interdiscip. Rev. RNA 2, 167–183 (2011)
Richter, J. D. Cytoplasmic polyadenylation in development and beyond. Microbiol. Mol. Biol. Rev. 63, 446–456 (1999)
Weill, L., Belloc, E., Bava, F. A. & Mendez, R. Translational control by changes in poly(A) tail length: recycling mRNAs. Nature Struct. Mol. Biol. 19, 577–585 (2012)
Eckmann, C. R., Rammelt, C. & Wahle, E. Control of poly(A) tail length. Wiley Interdiscip. Rev. RNA 2, 348–361 (2011)
Sallés, F. J., Lieberfarb, M. E., Wreden, C., Gergen, J. P. & Strickland, S. Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266, 1996–1999 (1994)
Barkoff, A., Ballantyne, S. & Wickens, M. Meiotic maturation in Xenopus requires polyadenylation of multiple mRNAs. EMBO J. 17, 3168–3175 (1998)
Preiss, T., Muckenthaler, M. & Hentze, M. W. Poly(A)-tail-promoted translation in yeast: implications for translational control. RNA 4, 1321–1331 (1998)
Beilharz, T. H. & Preiss, T. Widespread use of poly(A) tail length control to accentuate expression of the yeast transcriptome. RNA 13, 982–997 (2007)
Lackner, D. H. et al. A network of multiple regulatory layers shapes gene expression in fission yeast. Mol. Cell 26, 145–155 (2007)
Nutiu, R. et al. Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument. Nature Biotechnol. 29, 659–664 (2011)
Rosenthal, E. T., Tansey, T. R. & Ruderman, J. V. Sequence-specific adenylations and deadenylations accompany changes in the translation of maternal messenger RNA after fertilization of Spisula oocytes. J. Mol. Biol. 166, 309–327 (1983)
Palatnik, C. M., Wilkins, C. & Jacobson, A. Translational control during early Dictyostelium development: possible involvement of poly(A) sequences. Cell 36, 1017–1025 (1984)
Paynton, B. V., Rempel, R. & Bachvarova, R. Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Dev. Biol. 129, 304–314 (1988)
Kane, D. A. & Kimmel, C. B. The zebrafish midblastula transition. Development 119, 447–456 (1993)
Newport, J. & Kirschner, M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30, 687–696 (1982)
Decker, C. J. & Parker, R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev. 7, 1632–1643 (1993)
Aanes, H. et al. Zebrafish mRNA sequencing deciphers novelties in transcriptome dynamics during maternal to zygotic transition. Genome Res. 21, 1328–1338 (2011)
Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009)
McGrew, L. L., Dworkin-Rastl, E., Dworkin, M. B. & Richter, J. D. Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element. Genes Dev. 3, 803–815 (1989)
Paris, J. & Richter, J. D. Maturation-specific polyadenylation and translational control: diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes. Mol. Cell. Biol. 10, 5634–5645 (1990)
Paris, J. & Philippe, M. Poly(A) metabolism and polysomal recruitment of maternal mRNAs during early Xenopus development. Dev. Biol. 140, 221–224 (1990)
Simon, R., Tassan, J. P. & Richter, J. D. Translational control by poly(A) elongation during Xenopus development: differential repression and enhancement by a novel cytoplasmic polyadenylation element. Genes Dev. 6, 2580–2591 (1992)
Vassalli, J. D. et al. Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes. Genes Dev. 3, 2163–2171 (1989)
Gebauer, F., Xu, W., Cooper, G. M. & Richter, J. D. Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse. EMBO J. 13, 5712–5720 (1994)
Wu, L. et al. CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of α-CaMKII mRNA at synapses. Neuron 21, 1129–1139 (1998)
Oh, B., Hwang, S., McLaughlin, J., Solter, D. & Knowles, B. B. Timely translation during the mouse oocyte-to-embryo transition. Development 127, 3795–3803 (2000)
Burns, D. M. & Richter, J. D. CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation. Genes Dev. 22, 3449–3460 (2008)
Novoa, I., Gallego, J., Ferreira, P. G. & Mendez, R. Mitotic cell-cycle progression is regulated by CPEB1 and CPEB4-dependent translational control. Nature Cell Biol. 12, 447–456 (2010)
Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011)
Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009)
Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008)
Hendrickson, D. G. et al. Concordant regulation of translation and mRNA abundance for hundreds of targets of a human microRNA. PLoS Biol. 7, e1000238 (2009)
Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010)
Bazzini, A. A., Lee, M. T. & Giraldez, A. J. Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish. Science 336, 233–237 (2012)
Braun, J. E., Huntzinger, E. & Izaurralde, E. A molecular link between miRISCs and deadenylases provides new insight into the mechanism of gene silencing by microRNAs. Cold Spring Harb. Perspect. Biol. 4, a012328 (2012)
Audic, Y., Omilli, F. & Osborne, H. B. Postfertilization deadenylation of mRNAs in Xenopus laevis embryos is sufficient to cause their degradation at the blastula stage. Mol. Cell. Biol. 17, 209–218 (1997)
Jan, C. H., Friedman, R. C., Ruby, J. G. & Bartel, D. P. Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs. Nature 469, 97–101 (2011)
Ulitsky, I. et al. Extensive alternative polyadenylation during zebrafish development. Genome Res. 22, 2054–2066 (2012)
Schürer, H., Lang, K., Schuster, J. & Morl, M. A universal method to produce in vitro transcripts with homogeneous 3′ ends. Nucleic Acids Res. 30, e56 (2002)
Ruby, J. G., Jan, C. H. & Bartel, D. P. Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83–86 (2007)
Sallés, F. J., Richards, W. G. & Strickland, S. Assaying the polyadenylation state of mRNAs. Methods 17, 38–45 (1999)
Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862 (2001)
Pall, G. S., Codony-Servat, C., Byrne, J., Ritchie, L. & Hamilton, A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 35, e60 (2007)
Meijer, H. A. et al. A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells. Nucleic Acids Res. 35, e132 (2007)
Djebali, S. et al. Landscape of transcription in human cells. Nature 489, 101–108 (2012)
Holstege, F. C. et al. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 (1998)
Wang, Y. et al. Precision and functional specificity in mRNA decay. Proc. Natl Acad. Sci. USA 99, 5860–5865 (2002)
Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M. D. & Hughes, T. R. Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol. Cell. Biol. 24, 5534–5547 (2004)
Shalem, O. et al. Transient transcriptional responses to stress are generated by opposing effects of mRNA production and degradation. Mol. Syst. Biol. 4, 223 (2008)
Munchel, S. E., Shultzaberger, R. K., Takizawa, N. & Weis, K. Dynamic profiling of mRNA turnover reveals gene-specific and system-wide regulation of mRNA decay. Mol. Biol. Cell 22, 2787–2795 (2011)
Sun, M. et al. Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation. Genome Res. 22, 1350–1359 (2012)
Haimovich, G. et al. Gene expression is circular: factors for mRNA degradation also foster mRNA synthesis. Cell 153, 1000–1011 (2013)
Sun, M. et al. Global analysis of eukaryotic mRNA degradation reveals Xrn1-dependent buffering of transcript levels. Mol. Cell 52, 52–62 (2013)
Larsson, E., Sander, C. & Marks, D. mRNA turnover rate limits siRNA and microRNA efficacy. Mol. Syst. Biol. 6, 433 (2010)
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005)
Acknowledgements
We thank D. Weinberg, V. Auyeung, I. Ulitsky, C. Jan, J.-W. Nam, A. Shkumatava, S.-J. Hong, Y. Erlich and the Whitehead Genome Technology Core (V. Dhanapal, L. Francis, S. Gupta and T. Volkert) for discussions; J.-W. Nam, I. Ulitsky and D. Weinberg for assistance with transcript annotation; C. Bresilla, X. Guo, S.-J. Hong and A. Rothman for experimental assistance; and D. Weinberg for comments on the manuscript. Supported by NIH grant GM067031 (D.P.B.) and NIH Medical Scientist Training Program fellowship T32GM007753 (A.O.S.). D.P.B. is an investigator of the Howard Hughes Medical Institute.
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A.O.S. developed PAL-seq, generated tail-length measurements, and performed associated analyses. S.W.E. performed ribosome profiling, RNA-seq and associated analyses. G.R.C. performed zebrafish injections and assisted with staging. D.P.B. supervised with help from H.S. All authors helped to design the study and write the manuscript.
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Extended data figures and tables
Extended Data Figure 1 Development and characterization of the PAL-seq method.
a, Optimization of the primer-extension reaction. A 5′-radiolabelled primer was annealed to a single-stranded DNA template containing a (dA)25 tract immediately upstream of the primer-binding site (top schematic). Two templates (I and II), which differed at the segment immediately 5′ of the poly(dA) tract, were used in the experiments shown. Primer extension was performed with either Klenow fragment (K, NEB), Klenow fragment lacking 3′-to-5′ exonuclease activity (K−, NEB), or T4 DNA polymerase (T4, NEB). Reactions contained the recommended buffer and enzyme concentrations and a 50:1 molar mixture of dTTP:biotin-16-dUTP at the dTTP concentrations indicated. In one experiment (centre left), the dTTP concentration was kept constant, and the concentration of the primer-template duplex was varied instead. Reactions were incubated for 5 min, unless stated otherwise (bottom two panels), at the indicated temperature (temp; room temperature, r.t.), then stopped and in most cases supplemented with a gel-mobility standard (St), which was a 32P-labelled synthetic oligonucleotide that had four extra dT residues appended to the intended full-length primer-extension product (P). Products were resolved on denaturing polyacrylamide gels, alongside a size ladder (L), which was a mixture of 32P-labelled oligonucleotides that differed from the full-length primer-extension product by −1, 0, +1, +2, +3 and +4 dTs (three of these are indicated as +1, +2, +3), and visualized using a phosphorimager. Full-length extension without additional untemplated nucleotides was favoured by using Klenow fragment at 37 °C with very low dTTP concentrations (upper right panel and bottom two panels). Under these conditions the product did not change with prolonged reaction times (bottom). b, Poly(A)-tail lengths of the synthetic standards. Poly(A) tails >10 nucleotides retained some length heterogeneity generated during their enzymatic synthesis. To determine the actual poly(A) lengths of the barcode-poly(A) RNAs used to generate the standards, each RNA was 33P-labelled at its 5′ terminus and analysed on denaturing polyacrylamide gels under conditions that enabled single-nucleotide resolution. The values to the right of each panel indicate the modes and approximate ranges of the poly(A) tail lengths (after accounting for the 10-nucleotide barcodes). Also shown are marker lanes with 33P-labelled Century Plus ladder (C, Ambion), 33P-labelled Decade ladder (D, Ambion) and a partial base-hydrolysis ladder of the labelled barcode-poly(A) RNA used to make the 324-nucleotide standard of mix 2 (OH). c, The relative PAL-seq yield of each poly(A)-length standard. For each standard in the indicated mix, the yield of poly(A) tags relative to that of the A10 standard is plotted, after normalizing to the starting ratio determined from analysis of 5′-labelled mix on a denaturing polyacrylamide gel. Box plots show the distribution of yields for 32 PAL-seq libraries (line, median; box, 25th and 75th percentiles; whiskers, 10th and 90th percentiles).
Extended Data Figure 2 Validation of PAL-seq performance.
a, Evidence against non-specific RNA degradation. Plotted are nucleotide identities at the positions immediately upstream of poly(A) tags that both mapped uniquely to the genome (or standards) and ranged from 22–30 nucleotides in length (a range chosen to be long enough to enable mostly unique mapping to the genome, yet short enough to include enough 5′ adaptor nucleotides in a 36-nucleotide read to clearly identify the 5′ end of the tag). Frequencies were normalized to the aggregate nucleotide composition of positions 23–31 in either uniquely genome- or standard-mapping tags that extended the full length of the reads (36 nucleotides). Because RNase T1 cuts after Gs, the nucleotide preceding each 22–30-nucleotide tag was expected to be G, unless the mRNA had been cut for some other reason. The high frequency of G indicated that most mRNA fragments had not been cut for other reasons, which also implied that for these samples the poly(A) tails had also remained intact. We are unable to explain the high signal for an upstream U or C in some samples. Nonetheless, the frequency of an upstream A was low, which indicated that there had been little cleavage after As, again implying that the poly(A) tails had remained intact. In the A. thaliana leaf analysis, for which the raw reads had the first base removed, estimation of RNA integrity was performed with length ranges shortened by one nucleotide (for example, informative poly(A) tags were 21–29 nucleotides long). b, Consistent results from similar samples or biological replicates. Plotted are the relationships between average poly(A)-tail lengths generated using HeLa total RNA or RNA from a cytoplasmically enriched lysate (left), between average poly(A)-tail lengths generated using S. cerevisiae total RNA or RNA from a cytoplasmically enriched lysate (sample 1 and 2, respectively; middle), and between average poly(A)-tail lengths generated using cytoplasmically enriched lysates from two different 3T3 cell lines (right). Although the 3T3 lines were each engineered to express a miRNA (either miR-1 or miR-155), the miRNA was not induced in the cells used for this comparison. NM_001007026 fell outside the plot for HeLa, and YDL080C fell outside the plot for yeast.
Extended Data Figure 3 Discrepancies between the results of PAL-seq and those of previous methods.
a, Comparison of S. cerevisiae poly(A)-tail lengths measured by PAL-seq on total RNA to the previous results from PASTA analysis10. Plotted are mean poly(A)-tail lengths measured by PAL-seq for genes previously classified as having either short or long tails (PASTA-short and PASTA-long, respectively)10. The vertical dashed lines indicate the mean for each group as measured by PAL-seq. b, Comparison between PAL-seq measurements and either PASTA-derived poly(A)-tail ranks in fission yeast11 (left), or results of a related method reporting log ratios of short- and long-tail fractions in actively dividing 3T3 cells46 (right). c, Schematic of tail-length measurements using RNA blots. A DNA oligonucleotide or a gapmer (chimaeric oligonucleotide with DNA flanked by 2′-O-methyl-RNA) was designed to pair near the 3′ end of the mRNA. This oligonucleotide directed RNase H cleavage, thereby generating 3′-terminal mRNA fragments with lengths suitable for high-resolution analysis on RNA blots. Some of each sample was also incubated with oligo(dT), which directed RNase H removal of most of the poly(A) tail. Cleavage fragments were resolved on RNA blots and detected by probing for the inter-oligo region of the mRNA. The average poly(A)-tail length was calculated as the difference in the average sizes of the oligo(dT)-minus and oligo(dT)-plus fragments, plus the average number of residual adenosine residues that remained because of incomplete digestion of the poly(A) tail (residual As). For each reaction guided by a gene-specific DNA oligo, the average number of residual adenosines was estimated as half the difference between the known length of the inter-oligo region and the observed length of the oligo(dT)-plus fragment. For the two reactions guided by a gene-specific gapmer (RPL28 and RPS9B), the inter-oligo region extended through the residues pairing to one of the 2′-O-methyl-RNA segments, and cleavage was assumed to occur across from the most poly(A)-proximal DNA residue. Thus, the average number of residual adenosines was estimated as the difference between the length of the inter-oligo region and the observed length of the oligo(dT)-plus fragment. d, RNA blots used to measure poly(A)-tail lengths, as described in panel c, with the length information determined by PAL-seq (on total RNA) and PASTA10 indicated below each blot for comparison. For each lane, the range of high signal predicted based on PAL-seq results (Extended Data Fig. 4) is shown as a line next to the blot (with and without oligo(dT), red and blue, respectively). These predicted sizes took into account the residual nucleotides flanking the inter-oligo region, using the migration of the oligo(dT)-plus fragment to estimate the residual nucleotides on one or both ends as described in panel c. Genes chosen for analysis were required to be adequately expressed and to have a relatively homogeneous cleavage and poly(A) site, as determined by 3P-seq (data not shown). Nonetheless, some genes, such as RPL28, had frequently used alternative cleavage and poly(A) sites, as reflected by the two ranges marked in red. A preference was also given to ribosomal protein genes and genes with contradictory poly(A)-tail lengths when comparing the results of PAL-seq and PASTA.
Extended Data Figure 4 The signal distributions for the RNA blots (Extended Data Fig. 3d) compared with those predicted using PAL-seq.
Predicted traces from PAL-seq accounted for the estimated number of residual nucleotides flanking the inter-oligo region after RNase H cleavage, as described (Extended Data Fig. 3c). The offsets added to account for these residual nucleotides are indicated below each plot. The horizontal dashed lines above each plot indicate the range of the signal determined by visual inspection of the RNA blots in Extended Data Fig. 3d (oligo(dT)-plus and minus, red and blue, respectively). Vertical dashed lines indicate the migration of Decade markers (Ambion). The vertical axes are in arbitrary units (a.u.). The range of the high signal predicted based on PAL-seq data (signal exceeding 33% of the maximum) was determined using these plots and shown on Extended Data Fig. 3d as vertical lines next to the RNA blots. For some genes, poly(A)-site heterogeneity caused the signal exceeding 33% to map to noncontiguous segments.
Extended Data Figure 5 Relationship between poly(A)-tail length and changes in gene expression during zebrafish embryogenesis.
Changes in gene expression between the indicated embryonic stages, as measured by RNA-seq, are plotted in relation to the mean poly(A)-tail length at the latter stage.
Extended Data Figure 6 Poly(A)-tail lengths of tandem alternative 3′-UTR isoforms.
a, Comparison of average poly(A)-tail lengths for proximal (short) and distal (long) isoforms in the indicated cell lines. Results are plotted for isoforms that were each represented by ≥ 25 poly(A) tags and had alternative poly(A) sites ≥ 500 nucleotides apart. For genes with more than one isoform pair meeting these criteria, the pair with poly(A) sites farthest apart was selected. Points for NM_001007026 and NM_003913 fell outside the boundaries of the plot for HeLa. P values, χ2 test evaluating whether the relationship between isoform length and tail length differs from that expected by chance. b, Average poly(A)-tail lengths for proximal and distal 3′-UTR isoforms in 2 hpf zebrafish embryos, comparing results for genes that either contain (red circles), or do not contain (open circles), a CPE anywhere within the region unique to the distal isoform. A CPE was defined as U12, permitting a single non-U anywhere within the 12 nucleotides19. For a CPE found in the unique region to be counted as present, a canonical poly(A) signal (AAUAAA) also had to exist in the last 30 nucleotides of the distal isoform19,24. For each gene with a CPE within the region unique to the distal isoform, five genes with unique distal regions of comparable length ( ± 10%) but lacking a CPE are also shown. Poly(A) tags from three zebrafish 2 hpf PAL-seq libraries (mock-, miR-132–, and miR-155–injected) were combined before calculating average tail length for each isoform. Tandem isoform pairs with a target site for miR-132 or miR-155 in the region unique to the distal isoform were not considered. Only genes for which both tandem isoforms had ≥ 25 poly(A) tags, and for which the alternative poly(A) sites were 50–500 nucleotides apart, are plotted. For genes for which isoform choice affected inclusion of a CPE, the isoform pair representing that gene was chosen as the two isoforms with the most 5′-proximal poly(A) sites that flanked a CPE and satisfied the above criteria. For the pool of genes from which controls were chosen, two adjacent isoforms were picked randomly. P value, Fisher’s exact test, comparing genes with a CPE in the unique region to controls.
Extended Data Figure 7 Relationship between poly(A)-tail length and translational efficiency, classifying genes based on CPE content, tail length or translational efficiency.
a, The same data as in Fig. 3a, except genes were classified based on whether their 3′ UTR contained no CPE (grey), one CPE (blue), or two or more non-overlapping CPEs (red). b, Evidence that the more restricted tail-length range observed at gastrulation did not substantially impact the coupling between tail length and translational efficiency. The zebrafish 4 hpf data from Fig. 3a were sampled with replacement so as to have the same distribution of tail lengths observed at 6 hpf (left). Likewise, the X. laevis stage 9 data were sampled with replacement so as to have the same distribution of tail lengths observed at stage 12–12.5 (right). c, Box plot as in Fig. 4b for the same set of genes, with slopes calculated omitting data from the fraction without bound ribosomes. d, Box plots as in Fig. 4b, creating four equal bins of genes based on either overall mean poly(A)-tail length (left) or translational efficiency (right). The same slopes were used as in Fig. 4b, but considering only genes with a determined translational efficiency value and ≥ 100 poly(A) tags in the actively dividing 3T3 sample.
Extended Data Figure 8 The influence of miRNAs on ribosomes, mRNA abundance and tails in the early zebrafish embryo.
a, The relationship between changes in tail length at 4 hpf (as determined by PAL-seq) and changes in mRNA abundance at 6 hpf (as determined by RNA-seq), after injecting miR-155 (left) or miR-132 (right). Changes observed between miRNA- and mock-injected embryos are plotted for predicted miRNA target genes (red, genes with ≥ 1 cognate miRNA site in their 3′ UTR) and control genes (grey, genes that have no cognate miRNA site yet resemble the targets with respect to 3′-UTR length). Lines indicate mean changes for the respective gene sets; statistically significant differences between the gene sets for each of the two parameters are indicated (*P ≤ 0.05; **P < 10−4, one-tailed Kolmogorov–Smirnov test). Because injected miRNAs partially inhibited miR-430–mediated repression, genes with a site complementary to nucleotides 2–7 of miR-430 were not considered. All data were normalized to the median changes observed for the controls. b, The relationship between changes in ribosome-protected fragments (RPFs) and changes in mRNA levels (top), and between changes in RPFs and changes in tail lengths (bottom) after injecting miR-132. At 2, 4 and 6 hpf, embryos were analysed using ribosome profiling, RNA-seq and PAL-seq. Plots are as in Fig. 5a.
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Subtelny, A., Eichhorn, S., Chen, G. et al. Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature 508, 66–71 (2014). https://doi.org/10.1038/nature13007
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DOI: https://doi.org/10.1038/nature13007
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