Abstract
Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner—mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.
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Data availability
The raw sequencing data generated in this study were deposited in China National Center for Bioinformation with accession PRJCA007575 and in NCBI with accession PRJNA788163. A read_info file and a bam file for each FLEP-seq2 library were recorded in China National Center for Bioinformation with accession OMIX881. The bam file contains sequence and alignment information; the read-info file includes all the reads mapping to protein-coding regions and stored the corresponding gene, intron status and poly(A) tail length of each read. The median poly(A) lengths of genes in each library were recorded in Supplementary Table 4. Source data are provided with this paper.
Code availability
The jupyter notebook (jupter core v.4.6.1, python v.3.7.4) that contains examples and scripts for analysis and visualization was uploaded to GitHub (https://github.com/ZhaiLab-SUSTech/flep_seq2_polya_analysis).
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Acknowledgements
The group of J.Z. is supported by the National Key R&D Programme of China Grant (2019YFA0903903); the Programme for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S172); the Shenzhen Sci-Tech Fund (KYTDPT20181011104005); the Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes (2019KSYS006); and the Stable Support Plan Programme of Shenzhen Natural Science Fund Grant (20200925153345004). J.J. is supported by the National Natural Science Foundation of China (32100444) and the Shenzhen Fundamental Research Programme (JCYJ20210324105202007).
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J.Z., J.J., W.L. and Y.P. designed the experiments. W.L., J.J., B.L., H.F., X.J., Y.S. and Y.L. performed the experiments. J.J., W.L., Y.Y., W.M. and H.Z. analysed the data. J.Z. oversaw the study. J.J., W.L. and J.Z. wrote the manuscript and all authors revised the manuscript.
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Nature Plants thanks Dominique Gagliardi, Qingshun Quinn Li, Sureshkumar Balasubramanian and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1
The schematic diagram of FLEP-seq and FLEP-seq2.
Extended Data Fig. 2 Comparison of FLEP-seq2 PacBio and Nanopore data.
a, The distribution of global poly(A) tail lengths of transcripts/reads in FELP-seq2 seedling Nanopore and PacBio libraries. b, the median poly(A) tail length of genes (5 nt bin) measured based on PacBio and Nanopore data. Only genes with at least 20 reads were used. c, The correlation of the median poly(A) tail lengths measured based on PacBio and Nanopore data. Only genes with at least 50 reads in both dataset were used. The two-tailed p value for testing non-correlation was indicated in the figure.
Extended Data Fig. 3
The poly(A) tail length distribution of total RNA and nuclear RNA in different species.
Extended Data Fig. 4 Comparison of the poly(A) tail length of intron-containing transcripts and fully spliced transcripts.
a, The comparison of median poly(A) tail lengths between fully spliced transcripts and intron-containing (with introns) transcripts in total RNA samples. Only transcripts spanning all annotated introns were used, and only genes with at least 20 fully spliced reads and 20 intron-containing reads were used. b, The comparison of median poly(A) tail lengths of intron-containing transcripts between nuclear and total RNA samples in different species. Only the transcripts spanning all annotated introns were used, and only genes with at least 20 intron-containing transcripts in both nuclear and total RNA libraries were used. N: gene number. The Pearson’s r values were labelled upper each figure. All the two-sided p-values for testing non-correlation were less than 2.2e-16.
Extended Data Fig. 5
Example of alternative-splicing isoforms showing differential poly(A) tail lengths.
Extended Data Fig. 6 Boxplot showing the distribution of Δ minor isoform ratio (minor/[minor+major]) between nuclear RNAs and total RNAs.
The sums of the number of minor and major isoform reads in both nuclear and total RNA samples are required to be more than 10. The p-values of one-sided Mann–Whitney U-tests are labelled in the figure. For the boxplots, the centre lines show the median, the box limits show the interquartile range and the whiskers extend to the furthest value within 1.5× the interquartile range from the quartiles. N: the number of isoforms.
Extended Data Fig. 7 The poly(A) tail lengths of minor isoforms with longer tail than major isoforms are highly consistent with the poly(A) tail of intron-containing transcripts.
The comparison of median poly(A) tail lengths between intron-containing transcripts with minor isoforms (upper) or major isoforms (below) in total RNA samples of different species. Only genes with at least 20 minor isoform reads, 20 major isoform reads and 20 intron-containing reads were used. The numbers of isoforms showing no differential, longer, and shorter poly(A) tails were separated with ‘:’ and labelled above each figure. N: the number of isoforms.
Extended Data Fig. 8 Tissue-specific and evolutionarily conserved regulation of poly(A) tail length in Arabidopsis.
a, The median poly(A) tail length correlation matrix of different samples. All the two-sided p-values for testing non-correlation were less than 2.2e-16. b, Heatmap plot showing the relative median poly(A) tail length of genes among different tissues. c, The number of genes identified to show differential poly(A) tail length distribution in different tissues (Real, bottom panel) and in random data (Control, upper panel). Inflo.: Inflorescence. r1: biological replicate 1; r2: biological replicate 2.
Extended Data Fig. 9 Internal and 3’ end non-A bases of Poly(A) tails.
a, The ratio of poly(A) tails with internal non-A bases or 3’ end non-A bases. Two biological replicates were used. Data are presented as mean values±SD. b, The length distribution of poly(A) tail with or without 3’ end non-A bases. 1 nt each bin.
Extended Data Fig. 10 Tissue-specific expression of Arabidopsis genes coding Poly(A) binding proteins (PAB).
The gene expression values (Fragments per kilobase per million mapped fragments, FPKM value) of PABs in public RNA-seq data are downloaded from http://ipf.sustech.edu.cn/pub/athrna/. N: the number of RNA-seq libraries.
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Jia, J., Lu, W., Liu, B. et al. An atlas of plant full-length RNA reveals tissue-specific and monocots–dicots conserved regulation of poly(A) tail length. Nat. Plants 8, 1118–1126 (2022). https://doi.org/10.1038/s41477-022-01224-9
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DOI: https://doi.org/10.1038/s41477-022-01224-9
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