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A first exon termination checkpoint preferentially suppresses extragenic transcription

Nature Structural & Molecular Biology volume 28, pages 337–346 (2021)Cite this article

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Abstract

Interactions between the splicing machinery and RNA polymerase II increase protein-coding gene transcription. Similarly, exons and splicing signals of enhancer-generated long noncoding RNAs (elncRNAs) augment enhancer activity. However, elncRNAs are inefficiently spliced, suggesting that, compared with protein-coding genes, they contain qualitatively different exons with a limited ability to drive splicing. We show here that the inefficiently spliced first exons of elncRNAs as well as promoter-antisense long noncoding RNAs (pa-lncRNAs) in human and mouse cells trigger a transcription termination checkpoint that requires WDR82, an RNA polymerase II–binding protein, and its RNA-binding partner of previously unknown function, ZC3H4. We propose that the first exons of elncRNAs and pa-lncRNAs are an intrinsic component of a regulatory mechanism that, on the one hand, maximizes the activity of these cis-regulatory elements by recruiting the splicing machinery and, on the other, contains elements that suppress pervasive extragenic transcription.

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Fig. 1: Effects of ZC3H4 depletion on extragenic transcription.
Fig. 2: Recruitment of the WDR82–ZC3H4 complex to genomic sites with high RNA Pol II occupancy in mouse macrophages.
Fig. 3: Control of lncRNA production by WDR82–ZC3H4.
Fig. 4: lncRNAs suppressed by WDR82–ZC3H4 contain inefficiently spliced exons.
Fig. 5: A first exon transcription termination checkpoint.

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Data availability

The complete list of datasets used in this study is reported in Supplementary Table 10. Datasets generated in this study are available in the Gene Expression Omnibus (GEO) database under the accession number GSE133109. Source data are provided with this paper.

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Acknowledgements

We thank B. Amati (IEO) and S. Monticelli (IRB, Bellinzona) for critical comments on the manuscript. This work was supported by the European Research Council (Advanced ERC grant no. 692789 to G.N).

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Author notes

  1. These authors contributed equally: Liv M. I. Austenaa, Viviana Piccolo, Marta Russo.

Authors and Affiliations

  1. European Institute of Oncology (IEO) IRCCS, Milan, Italy

    Liv M. I. Austenaa, Viviana Piccolo, Marta Russo, Elena Prosperini, Sara Polletti, Danilo Polizzese, Serena Ghisletti, Giuseppe R. Diaferia & Gioacchino Natoli

  2. Department of Surgery and Cancer, Imperial College London, London, UK

    Iros Barozzi

  3. Humanitas University (Hunimed), Milan, Italy

    Gioacchino Natoli

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  1. Liv M. I. Austenaa
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Contributions

L.M.I.A., V.P., M.R. and G.N. conceptualized the study. L.M.I.A. and M.R. generated all data with contributions from E.P., D.P., S.G. and G.R.D. V.P. analyzed all data with contributions from I.B. S.P. generated and processed sequencing libraries. G.N. wrote the manuscript with contributions from all authors. G.N. supervised the study. G.N. was responsible for funding acquisition.

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Correspondence to Liv M. I. Austenaa or Gioacchino Natoli.

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Peer review information Nature Structural & Molecular Biology thanks Christopher Glass and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. 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.

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Extended data

Extended Data Fig. 1 Extragenic transcription in cells depleted of WDR82 or other transcription terminators.

a, The effects of the depletion of known termination factors on extragenic transcription was measured by 4sU labeling and sequencing in mouse bone marrow-derived macrophages. We considered the n=2,870 extragenic regions whose transcription was increased in macrophages depleted of WDR82 at 45′ after LPS stimulation and measured their 4sU labeling in macrophages depleted of the indicated proteins. Each transcript was assigned to the nearest annotated enhancer, Transcription Start Site (TSS) or Transcription End Site (TES). The log2-transformed fold change (sh vs. scramble) for each depletion experiment is shown. Statistical significance was assessed using the two-tailed Wilcoxon signed rank test and a p-value ≤ 0.01 was considered significant. p-values for transcripts assigned to Enhancers: Exosc3 p-value= 2.2e-208, Ars2 p-value= 2.9e-206, Ints11 p-value= 1.4e-217, CFIm25 p-value= 4.4e-189, Xrn2 p-value= 9.2e-239. p-values for transcripts assigned to TSS: Exosc3 p-value= 4.6e-69, Ars2 p-value= 3.7e-73, Ints11 p-value= 1.6e-72, CFIm25 p-value= 7.1e-72, Xrn2 p-value= 7.8e-101. p-values for transcripts assigned to TES: Exosc3 p-value= 1.5e-14, Ars2 p-value= 5.8e-10, Ints11 p-value= 6.7e-26, CFIm25 p-value= 0.149775, Xrn2 p-value= 1.0e-81. *** = p-value <0.01. ns: not statistically significant. Inside the boxplot, the median value for each fold change is shown with a horizontal black line. Boxes show values between the first and the third quartile. The lower and upper whisker show the smallest and the highest value, respectively. Outliers are not shown. The notches correspond to ~95% confidence interval for the median. b, Comparison of the effects of the depletion of WDR82 and INTS11 on transcription termination at snRNA genes. c, A representative genomic region on mouse chromosome 11 containing multiple snRNA genes. d, Snapshots of genomic regions showing the effects of the depletion of WDR82 and other termination factors on extragenic transcription.

Extended Data Fig. 2 Interaction of WDR82 with the zinc finger protein ZC3H4.

a,b, Immunoprecipitations were carried out either with an anti-Flag antibody on extracts of HEK-293 cells transduced with a Flag-mouse ZC3H4 expression vector (A) or with an anti-ZC3H4 rabbit polyclonal antibody on extracts from Raw264.7 mouse macrophages (B). Different parts of the western blot membrane were hybridized with the indicated antibodies. Data are representative of n=4 independent experiments. The position of molecular weight markers (kDa) is shown on the right. Uncropped images are available online as Source Data. c, Upper panel: Schematic representation of (A) the full length human ZC3H4 protein and (B to E) its deletion mutants used in transfection and co-immunoprecipitation experiments. The ZC3H4 domains annotated in UniProt are shown. Bottom panel: lysates from HEK-293 cells, either untransfected (-) or transduced with the indicated Flag-ZC3H4 expression vectors (A-E) were used in co-immunoprecipitation experiments with an anti-Flag antibody. Inputs (left) and immunoprecipitates (right) were immunoblotted and probed with an anti-FLAG (top) or an anti-WDR82 (bottom) antibody as indicated. The position of molecular weight markers (kDa) is shown on the right. Uncropped images are available online as Source Data. d, The Flag-tagged ZC3H4 C-terminal fragment (804–1303) was expressed in HeLa cells. Lysates were immunoprecipitated with an anti-ZC3H4 antibody directed against aa. 677–765 and blotted with anti-Flag or anti-WDR82 antibody. Inputs are shown on the left and molecular weight markers (kDa) on the right. Uncropped images are available online as Source Data.

Source data

Extended Data Fig. 3 Effects of WDR82 and ZC3H4 co-depletion on extragenic transcription in HeLa cells.

a, The effects of WDR82, ZC3H4 or their combined depletion by siRNA transfection were measured on selected extragenic transcripts, as indicated. In co-depletion experiments, a double amount of siRNA was used, as indicated. The bar plots show the mean ± SD of n=4 biological replicates. The data were normalized on the housekeeping gene CDC25b. Light grey columns: 30pmol siRNA, dark grey columns, 60pmol siRNA. b, Depletion efficiency of WDR82 (left) and ZC3H4 mRNA (right) in individual and combined depletions. The bar plots show the mean ± SD of n=4 independent experiments.

Extended Data Fig. 4 Distribution of WDR82, ZC3H4 and RNA Pol II ChIP-seq peaks.

a, Classification of WDR82 and ZC3H4 ChIP-seq peaks based on their genomic location. TSS: Transcription Start Site; TES: Transcription End Site. Data are from n=2 independent experiments. b, Transcribed protein-coding genes (n=10,917) were divided into quartiles of increasing RNA Pol II occupancy. The heatmaps show WDR82, ZC3H4 and RNA Pol II ChIP-seq signals at genes of the 1st and 4th quartiles.

Extended Data Fig. 5 Analysis of spliced and unspliced lncRNAs suppressed by WDR82.

Extragenic transcripts upregulated upon WDR82 depletion in mouse macrophages (n=2,870; top) or in HeLa cells (n=1,509; bottom) were first divided into spliced (left) and unspliced RNA species (right). Then within each of these two groups they were further divided based on their overlap with lncRNAs in the NONCODE v5 database of non-coding RNAs, classified into single exon and multi-exonic ncRNAs.

Extended Data Fig. 6 Analysis of splice efficiency and splice site sequences of lncRNAs suppressed by ZC3H4-WDR82 in HeLa cells.

a, Splicing efficiency at WDR82-suppressed lncRNA junctions (n=3,717) (top panel) and at a randomly selected set of premRNA junctions (n=4,000) (bottom panel) in HeLa cells. A window of ±− 10 nucleotides centered on the 5′ splice sites was used to measure read counts in polyA RNA-seq data. b, log2-transformed ratio of polyA RNA-seq reads in a 20 nt window centered on the 5′ splice sites of WDR82-suppressed lncRNA or of randomly selected set of mRNAs with at least one splice junction. c, Analysis of 5′ (left) and 3′ (right) splice site strength (measured as MaxEnt scores) at WDR82-suppressed lncRNAs. Statistical significance was assessed using the two-tailed Wilcoxon rank sum test in correspondence of both the 5′ (p-value=1.2e-21) and the 3′ (p-value=2.6e-06) splice sites. ***=p-value <0.01. Nucleotide frequencies at splice sites are shown as sequence logos. Donor and acceptor splice sites are indicated as black triangles. d, Effects of the depletion of WDR82-ZC3H4 on transcription of protein coding genes in HeLa cells. Expressed protein-coding genes (n=8,804) were divided into deciles based on their sensitivity to the depletion of WDR82 in 4sU-seq data, with the 10th decile including the most upregulated genes. Log2-transformed RNA fold changes (polyA and 4sU RNA-seq data) and log2-transformed reads ratio across the first exon-intron junction, as annotated in GENCODE, are shown for the 10th, 5th and 1st deciles. Statistical significance was assessed using the two-tailed Wilcoxon rank sum test (pvalue = 2.0e-21). ***=p-value<0.01. Data were from n=3 independent experiments. In the boxplots in panels B, C, D the median value for each group is shown with a horizontal black line. Boxes show values between the first and the third quartile. The lower and upper whisker show the smallest and the highest value, respectively. Outliers are not shown. The notches correspond to ~95% confidence interval for the median.

Extended Data Fig. 7 Relationship between gene transcript expression and splicing efficiency.

a, Genes were ranked into deciles of decreasing expression based on 4sU-seq data in macrophages (left) and HeLa cells (right). In both panels, expression of the lncRNAs upregulated in WDR82-depleted cells is shown in the red boxes on the right. Data are from n=3 independent experiments. b, Splicing efficiency of the 1st exon of the ranked genes was measured by dividing the sequencing reads in the 10nt upstream by those in the 10nt downstream of the 5′ splice junction in polyA RNA-seq data. Left: macrophages (n=6,280 junctions); right: HeLa (n=8,804 junctions). Data are from n=3 independent experiments. Boxes show values between the first and the third quartile. The lower and upper whisker show the smallest and the highest value, respectively. Outliers are not shown. The notches correspond to ~95% confidence interval for the median.

Extended Data Fig. 8 Exonic splice enhancer (ESE) sequences in exons of WDR82-suppressed lncRNAs and in mRNAs.

a, Number of ESE per exon in lncRNAs and in mRNAs suppressed by WDR82 in macrophages. Data are from n=3 independent experiments. b, Distance between ESEs and 5′ splice sites in lncRNAs and in mRNAs suppressed by WDR82. Data are from n=3 independent experiments. c, Number of ESEs per exon recognized by individual SRSF proteins in lncRNAs and in mRNAs suppressed by WDR82.

Extended Data Fig. 9 Characterization of splicing efficiency and splice site quality of extragenic transcripts not affected by WDR82 depletion.

a, log2-transformed ratio of polyA RNA-seq reads upstream and downstream of the 5′ splice sites of WDR82-suppressed and WDR82-insensitive lncRNAs in HeLa cells. Statistical significance was assessed using the two-tailed Wilcoxon rank sum test (p-value= 1.8e-175 for the controls and p-value=1.3e-199 in WDR82-depleted cells). ***p-value < 0.01. Data are from n=3 independent experiments. b, Analysis of 5′ (left) and 3′ (right) splice site strength at WDR82-suppressed and WDR82-insensitive lncRNAs in HeLa cells. MaxEnt scores for both donor and acceptor splice sites were measured. Statistical significance was assessed using the two-tailed Wilcoxon rank sum test in correspondence of both the 5′ (p-value= 1.3e–20) and the 3′ (p-value= 3e-04) splice sites. *** p-value < 0.01. Data are from n=3 independent experiments. Boxes show values between the first and the third quartile. The lower and upper whisker show the smallest and the highest value, respectively. Outliers are not shown. The notches correspond to ~95% confidence interval for the median.

Extended Data Fig. 10 First exon deletions in protein coding genes.

a, Schematic representation of the deletion of the first exons of protein coding genes. sgRNAs were designed to remove a genomic sequence that included the first exon from 30–50 nt downstream of the TSS to the intronic sequences just downstream of the 5′ splice site. b, Expression of the indicated gene mRNAs was measured by qRT-PCR in bulk populations of wild type or first exon-deleted HeLa cells after transduction of the indicated siRNAs. Primers used were specific for spliced mRNAs and were designed on downstream exons (Methods). The plot shows the mean ± s.d. of n=3 independent experiments. * P < 0.05; **P < 0.01, by two tailed t-test. The data were normalized on the housekeeping gene NRSN2. P-values for COG2: WT vs. DEL siCtl = 1.75E-07; DEL siCtl vs. siWdr82 = 0.78 (n.s.); DEL siCtl vs. siWdr82 = 0.80 (n.s.). P-values for FAM174a: WT vs. DEL siCtl = 0.0005; DEL siCtl vs. siWdr82 = 0.85 (n.s.); DEL siCtl vs. siWdr82 = 0.15 (n.s.). P-values for RRP15: WT vs. DEL siCtl = 0.013; DEL siCtl vs. siWdr82 = 0.70 (n.s.); DEL siCtl vs. siWdr82 = 0.65 (n.s.). c, First exon deletion efficiency at the three genes tested was analyzed by genomic PCR. The quantification of the wild type allele gel band in wt cells and cells in which the first exon was deleted using sgRNAs+Cas9 is shown on the right. Uncropped images are available online as source data.

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Supplementary Figs. 1–5.

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Supplementary Table 1

Extragenic transcription changes in primary mouse bone-marrow-derived macrophages depleted of WDR82 or other transcription termination factors. 4sU–seq datasets were generated in mouse bone- marrow-derived macrophages depleted of the indicated factors by retroviral delivery of shRNAs. Two different shRNAs per target were used as indicated. Data for each depleted factor are shown in different sheets. Data are from n = 4 independent experiments for WDR82 and n = 2 independent experiments for the other factors. False discovery rate (FDR) was calculated based on the Benjamini–Hochberg correction.

Supplementary Table 2

Deregulation of extragenic transcription in primary mouse bone-marrow-derived macrophages depleted of ZC3H4. 4sU–seq datasets were generated in macrophages transduced with retroviral vectors expressing ZC3H4-targeting shRNAs. Data are from n = 3 independent experiments. False discovery rate (FDR) was calculated based on the Benjamini–Hochberg correction.

Supplementary Table 3

Extragenic transcription changes in HeLa cells upon disruption of the WDR82–ZC3H4 complex. Tabs 1 and 2: HeLa cells were transfected with siRNAs targeting WDR82 or ZC3H4, and 4sU–seq datasets were generated. Data are from n = 3 independent experiments. Tab 3: HeLa cells were transfected with an expression vector encoding ZC3H4(804–1303) and, 48 h later, 4sU–seq datasets were generated. Data are from n = 3 independent experiments. False discovery rate (FDR) was calculated based on the Benjamini–Hochberg correction.

Supplementary Table 4

WDR82, ZC3H4 and RNA Pol II ChIP–seq datasets from mouse macrophages. False discovery rate (FDR) was calculated based on the Benjamini–Hochberg correction.

Supplementary Table 5

Overlap of annotated lncRNAs in the NONCODE v.5 database with extragenic transcripts upregulated in mouse macrophages upon WDR82 or ZC3H34 depletion. Extragenic regions showing increased transcription in 4sU–seq datasets from macrophages depleted of WDR82 or ZC3H4 were overlapped with annotated lncRNAs from the NONCODE v.5 database. The identity of the annotated lncRNA corresponding to each region upregulated in WDR82- or ZC3H4-depleted cells is shown.

Supplementary Table 6

Noncoding RNA junctions overlapping with extragenic transcripts upregulated in macrophages upon depletion of WDR82. polyA-RNA-seq datasets were used to identify splice junctions in the extragenic regions detected as upregulated in 4sU–seq data from WDR82-depleted macrophages. Exonic splice enhancers are also indicated. Data are from n = 3 independent experiments.

Supplementary Table 7

Overlap of annotated lncRNAs in the NONCODE v.5 database with extragenic transcripts upregulated in HeLa cells upon WDR82 or ZC3H34 depletion. Extragenic regions showing increased transcription in 4sU–seq datasets from HeLa cells depleted of WDR82 or ZC3H4 were overlapped with annotated lncRNAs from the NONCODE v.5 database. The identity of the annotated lncRNA corresponding to each region upregulated in WDR82- or ZC3H4-depleted cells is shown.

Supplementary Table 8

Noncoding RNA junctions overlapping with extragenic transcripts upregulated in HeLa cells upon depletion of WDR82. PolyA-RNA-seq data were used to identify splice junctions in the extragenic regions detected as upregulated in 4sU–seq data obtained in WDR82-depleted HeLa cells.

Supplementary Table 9

Sequences of the primers used in this study.

Supplementary Table 10

List of the datasets generated in this study.

Source data

Source Data Extended Data Fig. 2

Uncropped western blots.

Source Data Extended Data Fig. 10

Uncropped agarose gels.

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Austenaa, L.M.I., Piccolo, V., Russo, M. et al. A first exon termination checkpoint preferentially suppresses extragenic transcription. Nat Struct Mol Biol 28, 337–346 (2021). https://doi.org/10.1038/s41594-021-00572-y

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