Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila

Abstract

To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. Eighty-seven percent of the introns assayed manifest >50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly or slowly, with ∼3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources. Importantly, introns with low cotranscriptional splicing efficiencies are present in the same primary transcript with efficiently spliced introns, indicating that splicing is intron-specific. The analysis also indicates that cotranscriptional splicing is less efficient for first introns, longer introns, and introns annotated as alternative. Finally, S2 cells expressing the slow RpII215(C4) mutant show substantially less intron retention than wild-type S2 cells.

Figure 1.

Most introns in Drosophila S2 cells are efficiently spliced cotranscriptionally. (A) An image of the sequencing reads for a typical gene, gp210, in the Affymetrix Integrated Genome Browser. pA RNA is in gray, NUN RNA is in black, and gene structure is in black. Note breaks in sequencing reads coinciding with intron position. (B) Quantitation of intron retention for the introns of gp210. Intron retention = reads per base pair in introns/reads per base pair in all exons. (C) A histogram of the percent of all introns of abundantly transcribed genes, grouped by intron retention. pA RNA is in gray, and NUN RNA is in black.

Figure 2.

Select introns are dramatically retained in the nascent transcript, while others in the same gene are efficiently spliced. (A) A gene, CG12030, that has an intron with high retention (arrow) in the NUN RNA sample (black) and no matching intron signal in the pA (gray) control sample. Gene structure is in black. (B) Quantification of intron retention for CG12030. (C) A Venn diagram demonstrating that poorly spliced introns are found in ∼43% of analyzed genes and coexist with introns undergoing efficient cotranscriptional splicing in 36% of cases.

Figure 3.

A similar degree of efficient cotranscriptional splicing is present in heterogeneous fly head tissue as well as S2 cells. (A) A histogram of the percent of all introns of abundantly transcribed genes, grouped by intron retention. S2 cell NUN RNA is in blue, and fly head NUN RNA is in purple. Retention = reads per base pair in introns/reads per base pair in all exons. (B) A scatter plot of intron retention values for individual introns in both S2 cells and fly heads. Although there are small variances in splicing of individual introns between tissues, there is a high correlation (Spearman's ρ = 0.641, P < 0.01). (C) An example gene, Ppn, whose first intron (red arrow) has dramatic differences in cotranscriptional splicing between the fly head (purple) and S2 cell (blue) NUN RNA fractions. (D) Quantification of intron retention for the introns of Ppn.

Figure 4.

Intron retention is dependent on intron length and position and whether the intron is annotated as alternative or constitutive. (A,B) Longer introns show greater retention in both S2 cells (A) and fly head (B) populations (P < 0.001, Kruskal-Wallis test, all pairwise comparisons). (C,D) First introns show greater retention than all other introns in both S2 cells (C) and fly heads (D) (P < 0.001, Mann-Whitney U-test). (E,F) Alternatively annotated introns show a higher 3′ SS ratio than constitutive introns in both S2 cell (E) and fly head (F) populations (P < 0.001, Mann-Whitney U-test).

Figure 5.

The slow-elongating RpII215^C4 mutant increases cotranscriptional splicing efficiency in S2 cells. (A) A histogram of the percent of all introns of abundantly transcribed genes, grouped by intron retention. S2 cell wild-type NUN RNA is in blue, and RpII215^C4 NUN RNA is in green. Retention = reads per base pair in introns/reads per base pair in all exons. (B) A scatter plot of intron retention values for individual introns in both wild-type and RpII215^C4 S2 cells. There is a high correlation (Spearman's ρ = 0.683, P < 0.01) between the splicing of individual introns, and the slope of the regression line is flatter toward the wild type, indicating greater retention in that population. (C) A pie chart illustrating the percent of introns analyzed from both data sets that showed an increase or decrease in intron retention from wild-type to RpII215^C4 S2 cells. (D) A visualization of CG12030 that has a high degree of retention in its first intron (red arrow) in the wild-type S2 cell NUN RNA sample (blue) and a dramatic decrease in the RpII215^C4 (green) S2 cell sample. Gene structure is in black. (E) Quantification of intron retention for the introns of CG12030.