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Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts

Nature Structural & Molecular Biology volume 30, pages 1064–1076 (2023)Cite this article

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Abstract

Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome and ensures splicing fidelity across long pre-mRNAs.

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Fig. 1: Widespread post-transcriptional splicing in human cells.
Fig. 2: Post-transcriptional splicing follows a defined order.
Fig. 3: Splicing order is consistent across cell types.
Fig. 4: Splicing order is consistent between alternative isoforms and displays later removal of AS introns.
Fig. 5: Splicing order changes upon U2-snRNA-mediated exon skipping.
Fig. 6: Perturbation of one intron disrupts excision of proximal introns.

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Data availability

Raw and processed sequencing data are available from the Gene Expression Omnibus at

accession number GSE232455. Source data are provided with this paper.

Code availability

Code for analysis of all nanopore sequencing data is available at https://github.com/churchmanlab/splicing_order.

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Acknowledgements

We thank members of the Churchman lab, W. Timp, D. Whye, and D. Wood for helpful discussions, advice, and assistance; C. Patil, H. Merens, R.S. Isaac, and N. Kramer for critical reading of the manuscript; D. Meng and Y. Jia (Tsinghua University) for raw nanopore sequencing files from Nmf291−/− mice; B. Battersby (Institute of Biotechnology, University of Helsinki) for human myoblasts; and the Biopolymers facility at Harvard Medical School and the Harvard University Bauer Core Facility for sequencing services. This work was supported by the NIH (R01-GM136794, R21-HG011682 and R01-HG010538 to L.S.C.), the Burroughs Wellcome Fund (S.R.), the Fonds de Recherche du Québec - Santé, and the Canadian Institutes of Health Research (post-doctoral fellowship awards to K.C.). This research was conducted with support from the Human Neuron Core within the Rosamund Stone Zander Translational Neuroscience Center, Boston Children’s Hospital, which is also supported by the IDDRC (NIH P50HD105351).

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Authors and Affiliations

  1. Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Karine Choquet, Autum R. Baxter-Koenigs, Brendan M. Smalec & L. Stirling Churchman

  2. Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    Sarah-Luisa Dülk & Silvi Rouskin

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Contributions

Conceptualization, K.C. and L.S.C.; Methodology, K.C. (lead), B.M.S., S.-L.D., S.R. and L.S.C.; Investigation, K.C. (lead), A.R.B.-K., S.-L.D., B.M.S.; Software/Formal Analysis, K.C. (lead) and S.-L.D.; Writing – Original Draft, K.C. and L.S.C.; Writing – Review & Editing, K.C., A.R.B.-K., S.-L.D., B.M.S., S.R. and L.S.C.; Funding Acquisition, K.C., S.R. and L.S.C.; Supervision, S.R. and L.S.C.

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Correspondence to L. Stirling Churchman.

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Extended data

Extended Data Fig. 1 Intermediate isoforms are undergoing active processing. (related to Fig. 1).

a) Distribution of RNA 3′ ends based on read splicing status in nano-COP data14 from K562 cells. 3′ end features are defined as in14. b) UpSet plot showing the position of introns present in partially spliced reads from dRNA-seq of poly(A)-selected chromatin-associated RNA. ‘Middle’ includes introns that are not the first or last intron in a transcript. Two biological replicates are displayed. c) Scatter plot of the fraction of unspliced reads per intron between two biological replicates. Pearson’s correlation coefficient is shown on the plot (p-value < 0.0001, 95% confidence interval 0.948–0.950). Only reads spanning two introns or more are included. d) Coverage tracks from dRNA-seq of poly(A)-selected chromatin-associated RNA for three genes displaying various levels of post-transcriptional splicing. Introns with fraction of unspliced reads > 0.1 are shaded in grey. e) EXOSC10 mRNA levels and f) RNA levels of two promoter upstream transcripts (PROMPT) following shRNA-mediated knockdown (KD) of EXOSC10, as measured by qRT-PCR of total RNA. Dots represent biological replicates. g) Proportion of reads mapping to each intermediate isoform for intron groups used in splicing order analyses in WT K562 cells (Fig. 2). Biological duplicates for each shRNA treatment are shown side by side. h) Proportion of intermediate isoforms and intron groups that show a significant change (FDR < 0.1 and odds ratio > 1 or < −1) in abundance upon EXOSC10 KD compared to a scrambled control. i) Distribution of median poly(A) tail lengths as a function of splicing level in K562 replicate B. All reads are classified into groups of 3-4 introns based on the number of proximal post-transcriptionally excised introns that the read covers (splicing level). The median poly(A) tail length across reads is calculated for each number of excised introns in each intron group. Splicing levels were compared using a two-sided Wilcoxon rank-sum test (p-value < 0.001 for all comparisons). In G) and I), boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers.

Source data

Extended Data Fig. 2 Intron groups coverage, reproducibility and overlap with detained introns. (related to Fig. 2).

a) Features of introns that were included or not in splicing order analyses in K562 cells. Groups were compared using a two-sided Wilcoxon rank-sum test (***: p-value < 0.001). b) Expression level in K562 cells for genes (RPKM > 1) used in splicing order analyses. c) Read splicing status for the intron groups used in splicing order analyses. Two K562 biological replicates are shown. Each dot represents one intron group. d) Cumulative distribution function (CDF) of the number of reads at each splicing level for the intron groups used in splicing order analyses. e) Correlation in number of intermediate isoform reads or f) splicing order scores between K562 biological replicates prior to and after merging into replicates A and B. Each dot represents one intermediate isoform (E) or one splicing order (F). Pearson’s correlation coefficients are shown on the plots [p-value < 0.0001, intermediate isoforms: 95% confidence interval 0.88–0.90 (reps 3 vs. 4), 0.90–0.91 (reps 1 vs. 2), 0.91–0.92 (reps A vs. B), splicing order scores: 0.95–0.96 (reps 3 vs. 4), 0.95–0.96 (reps 1 vs. 2), 0.96–0.97 (reps A vs. B)]. g) Scores for splicing orders with a rank lower than 5. h) Overlap between detained introns (DIs) from31 and introns included in splicing order analyses, for introns present in genes expressed (RPKM or FPKM > 1) in K562 or HeLa cells and in at least one cell type from31. i) CDF of number of introns that are DIs or not for genes in which at least one DI was identified in31. j) Proportion of introns that were previously identified as DIs as a function of their position in the top ranked splicing order in K562 cells. Groups were compared using a two-sided Fisher’s exact test. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. For A) and C), boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. In A), points represent outliers.

Source data

Extended Data Fig. 3 Splicing order evenness and selection of coverage thresholds. (related to Fig. 2).

a) Shannon diversity index of splicing order across all analyzed intron groups, separated by the number of introns in each group (3 or 4). Shannon diversity index is compared for measured (orange) or random (grey) splicing order. Boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points: outliers. b) Splicing order plots for the intron groups with the highest evenness for groups of 3 or 4 introns, respectively. The evenness (E) is shown in red font for each intron group. c) Cumulative distribution function plot of the number of reads at each splicing level for the intron groups used in splicing order analyses as a function of evenness. Evenness was binned into quartiles separately for groups of 3 or 4 introns. d) and e) Splicing order simulations with varying levels of read coverage and evenness values for 3 (D) or 4 (E) analyzed introns. Each possible splicing order is shown in a different color and the mean splicing order score for 100 simulations is displayed with 95% confidence interval.

Source data

Extended Data Fig. 4 Features associated with splicing order. (related to Fig. 2).

a) Splicing index from short-read RNA-seq of rRNA-depleted nascent RNA, as a function of the position of introns in the top ranked splicing order from chromatin polyA+ dRNA-seq. b) Correlation between splicing index from A) and the number of intermediate isoform reads with a given intron excised from chromatin polyA+ dRNA-seq. Each dot represents one intron included in splicing order analyses in K562 cells. Pearson’s correlation coefficient is shown on the plot [p-value < 0.0001, 95% confidence interval 0.50–0.57 (repA chromatin), 0.50–0.56 (repB chromatin), 0.24–0.34 (repA 4sU-chromatin and repB 4sU-chromatin)]. c) Acceptor and donor bond half-lives25 as a function of the position of introns in the top ranked splicing order from chromatin polyA+ dRNA-seq. d) Splicing status of reads mapping to the intron pairs (N = 69) compared between nano-COP14 and polyA+ dRNA-seq in E. e) Splicing order for pairs of consecutive introns in nano-COP vs. polyA+ dRNA-seq. Vertical bars show the range between the two biological replicates for chromatin polyA+ dRNA-seq. Pearson’s correlation coefficient is shown on the plot (p-value < 0.0001, 95% confidence interval 0.88–0.95). f) Intron length as a function of the position of introns in the top ranked splicing order per intron group. g) Proportion of intermediate isoform reads with a given intron excised for introns with strong, moderate or weak splice sites. h) Number of intron groups used in splicing order analyses that include a first or last intron in a transcript or both. i) Number of introns that are first or last in a transcript as a function of their position in the top ranked splicing order. For A), C), F) and G), introns or groups were compared using a two-sided Wilcoxon rank-sum test. For I), groups were compared using a two-sided Fisher’s exact test. *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001. In A), C), D), F) and G, boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. For A), C) and G), points represent outliers.

Source data

Extended Data Fig. 5 Splicing order is consistent across cell types. (related to Figs. 3 and 4).

a) Western blot showing the cytoplasm, nucleoplasm and chromatin fractions obtained from cellular fractionation at each sMN differentiation timepoint. The exposition time used for each blot is noted at the bottom. More cells were used at day 9, resulting in higher abundance of the markers. b) Expression of differentiation markers during sMN differentiation, as measured by short-read RNA-seq of chromatin-associated RNA. Dots represent biological replicates. c) Principal component analysis of gene expression or exon inclusion in short-read RNA-seq across all timepoints of sMN differentiation. d) Correlation in number of intermediate isoform reads or splicing order scores between biological replicates for the intron groups used in splicing order analyses in HeLa cells and differentiating sMN at Days 9 and 14. Each dot represents one intermediate isoform (left) or one splicing order (right). For Day 14, replicates 1 and 2 were merged to achieve similar coverage as replicate 3. Pearson’s correlation coefficient is shown on the plot [p-value < 0.0001, 95% confidence interval intermediate isoform counts: 0.87–0.89 (HeLa), 0.89–0.92 (sMN Day 9), 0.86–0.88 (sMN Day 14), splicing order scores: 0.95–0.96 (HeLa), 0.96–0.97 (sMN Day 9), 0.94–0.95 (sMN Day 14)]. e) Proportion of intron groups with the same top splicing order for each pairwise comparison of cell types indicated on the x-axis. The total number of intron groups per comparison is shown on each bar. f) Evenness of splicing order for intron groups analyzed across cell types, separated by whether they share the same top splicing order across cell types or not. Boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points: outliers. g) Top: dRNA-seq coverage tracks of an example alternative cassette exon (in yellow) with differential inclusion during sMN differentiation. AS introns are shown in yellow font. Bottom: Splicing order plot for SLC37A4, shown separately for inclusion and exclusion of the alternative exon and for each timepoint that had sufficient coverage of a specific isoform.

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Extended Data Fig. 6 Splicing order is consistent between alternative isoforms and displays later removal of AS introns. (related to Fig. 4).

a) Number of intron groups as a function of the order in which AS introns are removed in the top ranked splicing order per intron group. Each x-axis label compares the splicing order in inclusion vs. exclusion isoforms (top) or Day 9 vs. Day 14 (bottom). b) Correlation in splicing order scores for AS introns between exclusion and inclusion isoforms (top) or between Days 9 and 14 (bottom). Each dot represents one possible splicing order for one intron group. Pearson’s correlation coefficient is shown on the plot [p-value < 0.001, 95% confidence interval 0.82–0.95 (Day 9), 0.62–0.88 (Day 14), 0.72–0.91(exclusion), 0.48–0.87 (inclusion)]. c) Evenness for inclusion and exclusion isoforms. Isoforms were compared using a two-sided Wilcoxon rank-sum test (p-value > 0.05). d) Splicing index from short-read RNA-seq of chromatin-associated RNA for all intron groups that contain a differentially included exon between Days 9 and 14 (right) or for only those that had sufficient coverage to be analyzed by dRNA-seq (left). ‘Inclusion intron 1’, ‘inclusion intron 2’ and ‘exclusion intron’ refer to the three possible AS introns that flank or overlap an alternative exon. ‘Upstream’ and ‘downstream’ intron refer to the next proximal introns upstream or downstream of the AS introns. e) Splicing order for four mutually exclusive exons (MXE) AS events. Splicing order plots are shown for each isoform and time point that has sufficient coverage. The percent spliced in (PSI) for isoform #2 is shown for each timepoint. In C) and D), boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. In C), points represent intron groups. In D), points represent outliers.

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Extended Data Fig. 7 Splicing order changes upon U2-snRNA-mediated exon skipping. (related to Fig. 5).

a) NET-seq pause index upon control or U2 snRNA KD. b) Percent of excised introns as a function of the distance transcribed past the 3′ SS for nano-COP reads from control and U2 snRNA KD. Two biological replicates per condition are displayed. c) Splicing order in nano-COP data from control and U2 snRNA KD for pairs of consecutive introns. Dots represent biological replicates. d) Total RNA-seq splicing index for introns that are sensitive to U2 snRNA KD compared to those that are stable. Three biological replicates (‘rep’) per condition are shown. e) Summed splicing order score for introns that are sensitive or stable upon U2 snRNA knockdown as a function of their position in splicing orders in WT HeLa cells. f) Number of reads mapping to each intron group as a function of the number of excised introns and their status upon U2 snRNA KD. g) Proportion of genes with and without intron retention in short-read RNA-seq from upon U2 snRNA KD, as a function of the exon skipping level difference between control and U2 snRNA KD in the same genes. h) Number of genes showing co-occurrence of skipped exons (SE) and retained introns (RI) in dRNA-seq, as depicted in Fig. 5f. i) Summed splicing order score for RIs upon U2 snRNA knockdown as a function of their position in splicing orders in WT HeLa cells. Intron groups are composed of one intron involved in RI and two introns involved in SE. j) Number of genes showing co-occurrence of SE(s) and RI(s) in cDNA-PCR nanopore sequencing data from WT and NMF291−/− (U2 snRNA mutant) mice53. K) Example of a SE and RI co-occurrence in Puf60 in NMF29−/− mice. ***: p-value < 0.001 in the one-sided binomial test assessing co-occurrence of SE and RI. In A), D), E) and I), boxplots elements are shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. In E) and I), groups were compared using a two-sided Wilcoxon rank-sum test; *: p-value < 0.05; ***: p-value < 0.001.

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Extended Data Fig. 8 Splicing order changes in IFRD2 upon U2 snRNA KD. (related to Fig. 5).

a) Working model to explain splicing order changes upon U2 snRNA KD. b) RT-PCR products from amplification of exons 2 to 5 and 9 to 12 of IFRD2, showing increased retention of introns 2 to 4 and 9 to 11 upon U2 snRNA KD compared to control. The identity of the spliced and unspliced products is shown on the right of the gel. Two biological replicates per knockdown condition are displayed. c) Scatter plots showing the correlation in frequency of intermediate isoform reads per splicing level for cDNA-PCR sequencing of IFRD2 exons 4–10 from chromatin-associated RNA with 12, 16 or 20 PCR cycles. Pearson’s R correlation coefficients are shown on each plot (p-value < 0.0001, 95% confidence interval: control KD, 12 cycles: 0.91–0.97; control KD, 16 cycles: 0.95–0.99; U2 snRNA KD, 12 cycles: 0.91–0.98; U2 snRNA KD, 16 cycles: 0.97–0.99). The data obtained from 20 cycles was used for subsequent analyses. d) Representation of reads mapping to introns 4 to 9 of IFRD2 upon cDNA-PCR sequencing of chromatin-associated RNA from control or U2 snRNA KD. Each line represents one read and each column represents one intron. For each number of excised introns, 100 reads were randomly subsampled from the total dataset. A black rectangle highlights the splicing order reversal upon U2 snRNA KD. e) Splicing order plots for IFRD2 in the absence (top) or presence (bottom) of SEs. On the y-axis, introns that are removed together to result in SE are shown separated by an underscore. The top 10 splicing orders per category (with or without SE) are shown. Experiments shown in C), D) and E) were performed on biological duplicates and data is displayed for one representative replicate per condition.

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Extended Data Fig. 9 Perturbation of one intron disrupts excision of proximal introns. (related to Fig. 6).

a) RT-PCR following treatment with a positive control ASO resulting in the use of an alternative 5′SS in BCL2L1 (red asterisk). b) RT-PCR following treatment with ASOs targeting the 3′SS of introns 4, 5 and 9 of IFRD2. Asterisks indicate the alternative splicing events in which exons 5 to 8 are skipped, without (orange) or with (red) intron 9 retention. c) Proportion of reads with different alternative splicing events observed in cDNA-PCR nanopore sequencing of total RNA following treatment with ASOs targeting IFRD2. RI: retained intron, SE: skipped exon(s), int: intron, ex: exon. d) Deletion tiling of IFRD2 using CRISPR-Cas9 editing. The resulting deletions are shown as black horizontal lines delimited by vertical lines aligned to the sgRNA(s) used (blue triangles). Left: Percent of reads with the expected deletion in cDNA-PCR nanopore sequencing of chromatin-associated RNA. Deletions made with only one sgRNA and non-targeting controls were not assessed (N/A). Right: Percent of reads with exon skipping in each deletion. ‘Undetermined splicing pattern’ refers to reads in which a splicing event does not map to annotated intron-exon junctions and results from detection of the deletion and/or from the use of a cryptic splice site as a result of the deletion. The reads shown on the left and right plots are not mutually exclusive, where reads containing the deletion (left) can also be classified as ‘undetermined splicing pattern’ (right) if the deletion overlaps with exon(s). e) Top: Chromatograms from Sanger sequencing of pools of cells edited with a non-targeting sgRNA and one targeting the end of exon 5. The bottom bar plot shows the predicted type of indel introduced. The x-axis indicates the position relative to the expected cut site. f) RT-PCR of introns 2 to 4 of IFRD2 for the deletions shown in D) that are not displayed in Fig. 6f. g) Expected outcomes for intron 4 excision based on the intron and exon definition models of splice site recognition58,59. The observation of intron 4 retention (Fig. 6) is consistent with exon definition.

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Extended Data Fig. 10 DMS-MaPseq analysis of IFRD2 intron 9. (related to Fig. 6).

Top: DMS-MaPseq reactivity for IFRD2 intron 9 (grey shade) +/− 10 nucleotides in the in vitro transcribed RNAs shown above. Individual dots represent two biological replicates. Bottom: Heatmap showing the log2 fold change of DMS reactivity in U2_SE or del_int4-8 relative to WT. Positions corresponding to G’s and T’s, which do not react with DMS, are shown in light grey. Positions where the largest fold change is observed in both mutated constructs are shown in orange font. Predicted branch points45,60 are shown as brown rectangles and the two predicted SRSF1 binding sites (ESEFinder46) with the highest scores are shown as beige rectangles.

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Choquet, K., Baxter-Koenigs, A.R., Dülk, SL. et al. Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts. Nat Struct Mol Biol 30, 1064–1076 (2023). https://doi.org/10.1038/s41594-023-01035-2

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