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Alternative polyadenylation by sequential activation of distal and proximal PolyA sites

Abstract

Analogous to alternative splicing, alternative polyadenylation (APA) has long been thought to occur independently at proximal and distal polyA sites. Using fractionation-seq, we unexpectedly identified several hundred APA genes in human cells whose distal polyA isoforms are retained in chromatin/nuclear matrix and whose proximal polyA isoforms are released into the cytoplasm. Global metabolic PAS-seq and Nanopore long-read RNA-sequencing provide further evidence that the strong distal polyA sites are processed first and the resulting transcripts are subsequently anchored in chromatin/nuclear matrix to serve as precursors for further processing at proximal polyA sites. Inserting an autocleavable ribozyme between the proximal and distal polyA sites, coupled with a Cleave-seq approach that we describe here, confirms that the distal polyA isoform is indeed the precursor to the proximal polyA isoform. Therefore, unlike alternative splicing, APA sites are recognized independently, and in many cases, in a sequential manner. This provides a versatile strategy to regulate gene expression in mammalian cells.

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Fig. 1: Isoforms with extended 3′ UTR are restricted in the NM.
Fig. 2: Characterization of the proteins associated with NM polyA+ RNA.
Fig. 3: Distinct sequence features between pPAS and dPAS pairs.
Fig. 4: Isoforms with extended 3′ UTR are intermediates for shorter ones.
Fig. 5: Identification of the 3′ cleavage fragments by Cleave-seq.
Fig. 6: Progressive polyadenylation prevents the leakage of unspliced introns.
Fig. 7: Progressive polyadenylation model for polyadenylation.

Data availability

All deep sequencing data from this study have been deposited in the Gene Expression Omnibus under series accession number GSE165742. Source data are provided with this paper. Other data are available upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This study was supported by grants from the China NSFC projects (grant nos. 31922039, 31670827 and 31871316) and the National Key R&D Program of China (grant no. 2017YFA0504400) to Y.Z., the China NSFC projects (grant no. 31800689) to P.T., and Hubei Provincial Natural Science Foundation of China (grant nos. 2020CFA057 to Y.Z. and 2020CFA017 to Y.Z. and Y.X.). Part of computation in this work was done on the supercomputing system in the Supercomputing Center of Wuhan University.

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Authors and Affiliations

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Contributions

P.T. and Y.Z. conceived the study. P.T., G.L., L.H., W.R. and C.Z. performed the experiments. Y.Y., M.W., X.G. and Y.Z. performed the analysis of the sequencing data. X.Z., D.L. and Y.X. contributed to critical experimental information. P.T. and Y.Z. wrote the manuscript with input from Y.Y. X.-D.F. participated in the project from 2016 to 2018 as a visiting professor and then contributed to manuscript packaging and revision in 2021. All authors discussed the results and approved the manuscript.

Corresponding authors

Correspondence to Xiang-Dong Fu or Yu Zhou.

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The authors declare no competing interests.

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Nature Structural and Molecular Biology thanks Nicholas Proudfoot and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Anke Sparmann and Beth Moorefield were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Nascent RNAs are tightly associated with the nuclear matrix.

a, Relative RNA amount extracted from different cellular fractions. Data are presented as mean values ±SD (n = 3). b, Western blotting of the extracts from different fraction with the antibody N20 targeting the largest subunit of RNA polymerase II. Data were from n = 1 independent experiment. c, Histogram of coSI (completed Splicing Index) values of exons. d, Boxplots of coSI values in bins of exons by their distances to the annotated polyA site. e, UCSC genome browser tracks showing the rRNA depleted RNA-seq signals in the nuclear matrix across the genes ADK and GPHN, respectively. The lower and upper hinges of the boxplots correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. The centre line represents the median value.

Source data

Extended Data Fig. 2 The distribution of long 3′UTR isoform in three cellular compartments.

a, UCSC genome browser tracks showing the genes with a higher percentage of longer 3′UTR isoforms (dotted box) in both NM and NP, compared with CY, from polyA+ RNA-seq. NM: nuclear matrix, NP: nucleoplasm, CY: cytoplasm. b, Gene examples with higher percentage of long 3′UTR isoforms mainly observed in NM, and similar lever in NP and CY. c, Illustration of Percentage of dPAS Usage Index (PDUI) computation. d, Distribution of the PDUI in three cellular compartments. PDUI values are from the 654 genes identified in Fig. 1 f.

Extended Data Fig. 3 Characterization of nuclear matrix polyA- RNA.

a, Percentages of exonic and intronic reads in CY, NP, NM polyA+ and NM polyARNA-seq data. b,c, UCSC genome browser view of NM polyA+ and polyA RNA signals in the region from TSS (transcription start site) to 10 kb downstream TES (transcription end site) for FUBP1 (b) and hnRNPA2B1 (c). d, Meta profiles of NM polyA+ and polyA RNA signals in the ±250 nt region flanking the pPAS (left) and dPAS (right). The mean read densities were normalized and shown in arbitrary units (a.u.).

Extended Data Fig. 4 The distal polyA sites are stronger than the proximal polyA sites.

a, Reporter constructed to test the strengths of polyA sites. Fluc and Rluc are Firefly luciferase and Renilla luciferase, respectively. The polyA site (PAS) to be tested is the region ±200 nt flanking the cleavage site. IRES, internal ribosomal entry site. b, Relative luciferase activity of different polyA sites represented by mean ± SD (n = 5). The p-values are based on a two-tailed unpaired t test: ***p < 0.001. c, Constructs with the paired proximal and distal PASs in two different orders. The primers used for RT-PCR were indicated. d, RT-PCR results of two different constructs (C1 and C2) are shown for PASs from 4 genes. Data were from n = 1 independent experiment.

Source data

Extended Data Fig. 5 Quality control for the SLAM PAS-seq.

a, Rates of nucleotide substitutions with or without DRB treatment. b, Relative T-C nucleotide conversion of mRNA and mitochondria RNA w/o DRB treatment. Data are presented as mean values ± SD (n = 2). c, Correlation coefficients of expression values in CPM between pairs of samples. total: total reads; new: nascent reads with T-C conversion. d, Difference of d/p ratios between the nascent RNA and steady state RNA. NM, NM&NP, NP, and others are different groups of genes with longer 3′UTR isoform enriched in the NM only, both NM and NP, NP only (groups as Fig. 1 g), non-enriched in neither NM nor NP (gray points in Fig. 1e,f), respectively. NP: nucleoplasm; NM: nuclear matrix. The p-values are based on a two-tailed unpaired t test. e, Scatter plot showing the d/p ratios of total RNA-seq with or without 4sU treatment in cell culture. f, Gene counts with different numbers of APA pairs using the most distal PAS as reference. g, Summary of genes classified by the d/p ratio of the nascent over steady state RNA of their APA pairs. h, Box plot showing the d/p ratios of total RNA is altered slightly upon transcription inhibition with DRB treatment. The dotted lines represent the value ±1. The lower and upper hinges of the boxplots correspond to the first and third quartiles (the 25th and 75th percentiles). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. The centre line represents the median value.

Source data

Extended Data Fig. 6 Sequence features around the proximal and distal polyA sites.

a, Enrichment score (log2[frequency in PAS / frequency in random sequence]) for AAUAAA, UGUA, and GU/U-rich motifs around the cleavage sites (±100 nt) of all polyA sites. b, Nucleotide frequency around (±100 nt) pPAS and dPAS in 4 different groups as in Fig. 3c. c, Distribution of U-rich 6-mer motifs (described in the Methods) in the region −100–0 nt upstream of the pPAS (blue) and dPAS (yellow) from 4 different groups.

Extended Data Fig. 7 Isoforms with extended 3′UTR do not produce proteins.

a, Illustration of alternative UTR (aUTR) and its insertion in the 3′UTR of GFP reporter gene. b, The effect of aUTR from CPSF6 and FUBP1 on the expression reporter detected by WB. Another flag expression plasmid was used as transfection control. Data were from n = 2 independent experiments. c, Two models for the processing of the proximal polyA sites. Model 1: the proximal and distal polyA sites are processed independently from each other (left); Model 2: the processing of distal polyA site is prior to the proximal one and the dPAS isoform acts as the intermediate for the proximal one (right). d, Validation of the inserted ribozyme by Sanger sequencing.

Source data

Extended Data Fig. 8 Little impact of ribozyme insertion downstream of the distal PAS on gene expression.

a, Diagram of the reporters with ribozyme insertion downstream of dPAS in the FUBP1-based or hnRNPA2B1-based reporter. b,c, The effects of ribozyme insertion downstream of dPAS in FUBP1 (b) or hnRNPA2B1 (c) on the exogenous reporter at the protein (left) and RNA (right) levels. The GFP plasmid served as control for transfection. Data were from n = 3 independent experiments and are presented as mean values± SD for the two genes. d, Diagram of ribozyme insertion upstream (up) or downstream (down) of dPAS in endogenous hnRNPA2B1. e, PCR validation of ribozyme insertion downstream of dPAS in hnRNPA2B1. Data were from n = 1 independent experiment. f, Effects of ribozyme insertion downstream of dPAS in endogenous hnRNPA2B1 at the protein (left) and RNA (right) levels. Bar graph represents mean ± SD (n = 3). The p-values are based on a two-tailed unpaired t test: *p < 0.05, n.s. represents non-significant.

Source data

Extended Data Fig. 9 Features of the 5′ end signals of Cleave-seq.

a, UCSC genome browser view of the 5′ end signals of polyA+ RNA from Cleave-seq on DGCR8 gene. The zoom-in view of the 20 nt region flanking the Drosha cleavage site (left dotted) is shown in the dotted box on the right (* marks the 5′ end base). b, Upset plot of the identified cleavage sites from 4 Cleave-seq libraries. Only the peaks found in at least 2 samples were included. For the 654 genes with longer 3′UTR enriched in NM, the number of peaks located in the downstream 10 nt window of their pPAS and dPAS are shown in the upper right box. c, Violin plot depicting the ratio of the number of Cleave-seq reads in the 10 nt window downstream pPAS over that of dPAS (n = 160). The dotted grey line represents 10-fold ratio. The red line represents the median. The lower and upper hinges of the box plots correspond to the first and third quartiles (the 25th and 75th percentiles). The p-value was determined using the two-sided Wilcoxon test.

Extended Data Fig. 10 Regulation of splicing through progressive polyadenylation.

a,b, Splicing analysis of the rescue reporters by RT-PCR for construct C5 (a) and C6 (b) in Fig. 6b. Data were from n = 1 independent experiment. c, Illustration of the formula to calculate the PIR score. d, Proposed model for progressive polyadenylation mediated splicing regulation.

Source data

Supplementary information

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Supplementary Table 1

Supplementary Table 1 Nuclear matrix polyA+ RNA interacting proteins. Supplementary Table 2 Primers used in this study.

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Source data for NM RNA interactome mass spectrometry.

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Tang, P., Yang, Y., Li, G. et al. Alternative polyadenylation by sequential activation of distal and proximal PolyA sites. Nat Struct Mol Biol 29, 21–31 (2022). https://doi.org/10.1038/s41594-021-00709-z

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