1. Volume 165, Issue 2, Pages 372-381 (April 2016)
Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II 
Fernando Carrillo Oesterreich, Lydia Herzel, Korinna Straube, Katja Hujer, Jonathon Howard, Karla M. Neugebauer 
Cell 
Volume 165, Issue 2, Pages (April 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Single-Molecule Intron Tracking Measures Co-transcriptional Splicing Kinetics
(A) Single-molecule intron tracking (SMIT) workflow. Nascent RNA is purified from chromatin (intron, red; exons, black). Nascent RNA molecules of interest are selected by PCR with a forward primer in exon 1. Pol II position (3′ end) and splicing state are determined by paired-end sequencing. Single-molecule observations are corrected for PCR length bias, grouped by Pol II position and the fraction spliced is calculated. Saturation values (cyan), defining a statistically determined maximum value, allow calculation of ½ max Pol II positions (50% of saturation, magenta). For details see the Supplemental Information.
(B) SMIT profiles for three representative endogenous genes. Fraction spliced (mean ± SD) is plotted versus distance from 3′ splice sites (SSs). Co-transcriptional splicing saturation value (cyan dotted line), half-saturation Pol II position (magenta dotted line), and polyA site (gray dotted line, if in range) are indicated.
See Figures S1, S2, and S3 for characterization of SMIT and additional SMIT profiles.
See also Tables S1, S2, and S3.
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4. Figure 2
Long Read Sequencing Detects Splicing of Nascent RNA when Pol II Is <100 Nucleotides Downstream of 3′SSs
(A) Long read sequencing of nascent RNA workflow. Nascent RNA molecules are 3′ end ligated and reverse transcribed. A PCR targeting all cDNAs generates double-stranded cDNAs for long read sequencing.
(B) Long read sequencing observations of S. cerevisiae nascent RNA molecules for six representative genes. Presence (thick line) or absence (thin line) of intron sequence and 3′ end position indicate splicing state and Pol II position, respectively.
(C) Long read sequencing observations of S. pombe nascent RNA molecules is represented as described in (B).
See also Tables S2 and S3.
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5. Figure 3
Spliced Nascent RNAs Are First Observed When Pol II Is Just Downstream of 3′SSs
(A) Pol II position (distance from 3′SS) at which 10%, 50%, or 90% of splicing saturation is reached (n = 87 endogenous genes). Gene-specific values and summary statistics are shown.
(B) Cumulative distribution of Pol II positions of 158 spliced long read observations from 83 endogenous genes (black). A model assuming uniform sampling is indicated (red). Splicing onset inferred by the model (x-intercept: 36 ± 11 nt, mean ± 95% confidence interval) and measured by SMIT (gray line ± 95% confidence interval) is indicated. See also Figure S4.
(C) Pol II density over terminal exons of all 87 genes analyzed by SMIT. Densities were derived at nucleotide resolution by 3′ end sequencing of nascent RNA, grouped in 20 nt bins and encoded by color (Log10 scale, see color map). Rows represent individual terminal exons (extended by 100 nt up- and downstream) sorted by length and aligned to the 3′SS. Distance from the 3′SS is indicated and gray line shows average Pol II position of splicing onset (10% of saturation value).
See also Table S2.
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6. Figure 4 5′SS Sequence Determines Co-transcriptional Splicing Kinetics
(A) SMIT (black) and MS2 ChIP (gray) profiles (mean ± SD) for integrated HZ18 reporter with canonical 5′SS. Signals are plotted versus distance from 3′SSs.
(B) Long read sequencing observations from nascent RNA of integrated HZ18 reporter with consensus 5′SS. Gene architecture and representative single nascent RNA observations are shown. 5′SS sequence is indicated above the graph.
(C and D) SMIT (black), MS2 ChIP (gray) profiles (mean ± SD), as well as long read sequencing observations deduced from HZ18 reporter gene with mutated 5′SS splice-site are shown as described above.
See also Figure S5 and Tables S2 and S3.
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7. Figure 5
Pol II Elongation Rate Is a Determinant of Co-transcriptional Splicing Kinetics
(A) SMIT profiles (mean ± SD) for two representative endogenous genes with WT Pol II (gray) and mutated Pol II with increased elongations rates (fast Pol II, black). Fraction spliced is plotted versus distance from 3′SSs. Half-saturation Pol II positions are indicated.
(B) Pol II positions (distance from 3′SS) at which 10%, 50%, or 90% of splicing saturation is reached (n = 24 endogenous genes). Data points represent gene-specific values and boxplots summary statistics.
See also Figure S6 and Tables S2, S3, and S4.
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8. Figure 6 The Onset of Splicing Coincides with Intron Exit from Pol II
(A) Splicing catalysis starts when WT and Fast Pol II reach ∼26 and 27 nt downstream of 3′SSs (green), respectively. Splicing saturation is reached within ∼130 and 353 nt (blue), respectively. The spliceosome and mutant Pol II are separated by a longer stretch of nascent RNA at splicing completion.
(B) Schematic diagram showing that 15 nt of nascent RNA is protected by Pol II, and 9 nt is bound by the spliceosome (gray shades). At splicing onset 26–27 nt downstream of 3′SSs, the spliceosome and Pol II are separated by only 2–3 nt nascent RNA. Introns (red) and exons (black) are depicted.
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9. Figure S1 Single-Molecule Intron Tracking, Related to Figure 1
(A) 3′ end linker ligation of nascent RNA is very efficient. Unligated and ligated nascent RNAs were analyzed with 10% TBE-Urea PAGE. Separation of nascent RNAs shorter than 1000 nt is observed and the nascent RNA smear is shifted toward higher molecular weights upon ligation.
(B) Single-molecule intron tracking (SMIT) PCR for 9 genes with forward primers in the first exon and reverse 3′ end linker primer produce gene-specific cDNA smear. The SMIT PCR products were analyzed by agarose gel electrophoresis. No product is detected in samples without 3′ end ligation.
(C) High numbers of nascent RNA observations per Pol II position. Histogram of Illumina sequencing read counts (3′ ends) per position from 87 SMIT genes (mean 287 3′ end counts/Pol II position). The dotted line marks the minimal read count per position required for analysis.
(D) High numbers of nascent RNA observations per gene. Histogram of total Illumina sequencing read counts (3′ ends) per SMIT genes (mean 2 × 105 3′ end counts/gene).
(E) Distribution of gene-specific number of Pol II positions covered by SMIT. A Pol II position is reported if it is characterized by at least 10 distinct reads (spliced or unspliced). The average gene has 329 nt of these Pol II positions.
(F) The distance of the last Pol II position (at least 10 reads) to the 3′SS is plotted against annotated terminal exon length. A linear model with intercept 0 and slope 1 indicates the position for which the last Pol II position aligns exactly with the polyA site. For most (short) genes, the last Pol II position is clearly downstream of this point.
(G) The insert length bias was characterized by determining the insert length distribution for intronless genes and plotted as a cumulative probability distribution (black). This distribution can be well described by an exponential model (red), which is used to correct for the insert length bias in all further analyses.
(H) Correlation of read count (spliced and unspliced) per Pol II position was determined for each gene between all replicates. The Pearson correlation coefficient between pairs of replicates shows high correlation between replicates with the same library preparation protocol (short RNAs < 250 nt and all sizes of nascent RNAs). Hierarchical clustering was done using the individual Pearson correlation coefficients between samples.
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10. Figure S2 SMIT Profiles for Endogenous Genes, Related to Figure 1
Fraction spliced was plotted versus distance to 3′SS. Co-transcriptional splicing saturation value (cyan dotted line), half-saturation Pol II position (magenta dotted line) and polyA-site (gray dotted line) are indicated.
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11. Figure S3
Maximal Co-transcriptional Splicing Values Correlate Well with Previous Estimates, Related to Figure 1
(A) Frequency distribution of co-transcriptional splicing saturation values for 87 endogenous genes.
(B) Gene specific co-transcriptional splicing values measured by SMIT versus corresponding values determined by microarray analysis of nascent RNA (Carrillo Oesterreich et al., 2010). Correlation coefficient (cor) and mean squared error (MSE) are given.
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12. Figure S4
Long Read Sequencing of Nascent RNA of Endogenous Genes, Related to Figure 3
(A) Frequency distribution of long read nascent RNA sequencing observations per gene.
(B) Empirical cumulative frequency distribution of Pol II position relative to the 3′SS for all 421 spliced nascent RNA molecules from 116 endogenous genes. Minimal terminal exon length (230 nt) is indicated (dashed line). The full data range (black) and probabilistic model (red) assuming uniform distribution between 0 nt and 230 nt are shown.
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13. Figure S5
Splicing of Integrated HZ18 Reporter Gene, Related to Figure 4
(A) Analysis of RNA transcribed from the integrated HZ18 reporter gene by PCR. Bands representing spliced and unspliced are indicated. Nascent RNA (n) and mRNA (m) from integrated reporter constructs with canonical (wt) and mutated (mut, GUAcGU) 5′SS were analyzed.
(B and C) Co-transcriptional spliceosome assembly on integrated HZ18 with (B) canonical and (C) mutated 5′SS. Nascent RNA association of U1-snRNP (red), U2-snRNP (green), U5-snRNP (blue) and NTC (magenta) was measured by ChIP and plotted versus distance to the 3′SS. Pol II positions are indicated where 50% spliced (SMIT, black dashed line) and 50% anti-MS2 ChIP signal (gray dashed line) are reached. Spliceosome components associate with both reporter constructs in a step-wise fashion. In agreement with previous characterizations, U1-snRNP associates early, followed by U2-snRNP and is then replaced by U5-snRNP and NTC. In agreement with delayed co-transcriptional splicing as measured by SMIT, replacement of U1-snRNP by U5-snRNP-NTC is significantly delayed in the construct with impaired 5′SS. In both reporter genes, the increase of SMIT signal correlates well with increase of U5-snRNP-NTC ChIP signal. In contrast, the anti-MS2 ChIP signal increases in the construct upon decrease of U5-snRNP-NTC ChIP signal. Interestingly, levels of U5-snRNP-NTC remain high in the reporter with mutated 5′SS. Thus, co-transcriptional splicing kinetics measured by SMIT agree with spliceosome assembly and offer a high resolution measure of splicing kinetics.
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14. Figure S6 SMIT on WT and Fast Pol II, Related to Figure 5
SMIT profiles for two endogenous genes with WT Pol II (gray) and mutated Pol II (E1103G) with increased elongations rates (black). Fraction spliced is plotted versus nt distance from 3′SSs. Co-transcriptional splicing saturation values and ½ max values are indicated.
Cell  , DOI: ( /j.cell )
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