Fig 2: CDK7 inhibition with SY-351 causes widespread defects in splicing. (A) Differentially expressed mRNAs following SY351 treatment (DESeq2). Replicate RNA-seq data sets were generated from rRNA-depleted total RNA from HL60 cells treated with DMSO (CTRL) or SY-351 (50 nM in DMSO) for 5 h. (B) GSEA analysis. (C) SY-351 favors exon inclusion over exon skipping, splicing over retention of introns, and alters 5′ and 3′ splice site selection equally, without preference for upstream or downstream sites. Alternative splicing events significantly affected by SY-351 were identified in replicate RNA-seq data sets using MAJIQ (Vaquero-Garcia et al. 2016). (D) IGV genome browser Sashimi plot of a segment of the LTV1 gene. Normalized read numbers for DMSO control and SY-351 treated samples are shown on the Y-axis. Splice junction read numbers for sense strand transcripts are shown in blue and red. Note reduced exon inclusion (green arrow) and retention of both flanking introns (black arrows) in SY-351. (E) Immunofluorescence microscopy of endogenous SF3B1 (green) and Hoechst (blue) in DMSO and SY-351 conditions. (F) Quantitation of SF3B1 puncta per nucleus in DMSO and SY-351 conditions, P = 0.001 (n = 3 biological replicates).

Fig 3: Cytoplasmic forces accelerate molecular-scale kinetics in nuclear condensates and regulate mRNA processing.a Scheme of droplet surface fluctuations as a function of cytoplasmic stirring (left); representative nuclear SRSF2-GFP droplet surface fluctuations in orange (above) and surface fluctuation intensity heatmaps of comparably-sized droplets (below) in SN oocytes with Control, amplified, or disrupted cytoplasmic forces; droplet radii, 2 to 2.7 μm; droplet number, Control = 25; Nocodazole = 12; FMN2−/− = 21; FMN2−/− + Nocodazole = 15. b FRAP sequences of SRSF2-GFP droplets in Control, FMN2−/−, and FMN2−/− + FMN2 SN oocytes. c Fluorescence recovery curves (mean ± s.e.m.) with simple exponential fits of SRSF2-GFP droplets in Control, FMN2−/−, and FMN2−/− + FMN2 SN oocytes; insets, apparent diffusion coefficients (Dapp); droplet number, Control = 28, FMN2−/− = 13, FMN2−/− + FMN2 = 11. d Representative co-immunostainings of nuclear speckles and phosphorylated SF3b155 (pT313) in SN Control, FMN2−/−, and FMN2−/− + FMN2 oocytes with magnifications of single droplets. e Quantifications of droplet-specific pT313-SF3b155 intensities in growing Control and FMN2−/− oocytes, and in FMN2−/− + FMN2 SN oocytes; Droplet number, Control NSN = 34, Trans = 37, SN = 38, FMN2−/− NSN = 31, Trans = 33, SN = 56, FMN2−/− + FMN2 SN = 26. f, g Differential mRNA exon usage versus mean abundance or differential mRNA isoform usage versus isoform switch Padj in SN FMN2−/− oocytes relative to Control SN FMN2+/− oocytes; Colored dots are over/underrepresented exons or isoforms in FMN2−/− oocytes with a BH-adjusted (exons) or FDR-adjusted (isoforms) P value Padj < 0.05. h, i About 10 × 10 dot plots showing percentages of mRNA alternative splicing patterns detected in FMN2−/− (n = 3565 patterns) and percentages of predicted consequences of mRNA isoform switches in FMN2−/− (n = 2178 consequences). j Jaccard measures of overlaps between sets of genomic sites, through computation of the ratio of their intersections to their union; differentially used exon sites and alternative splice sites of transcripts of FMN2−/− oocytes were tested bidirectionally for spatial correlation with SRSF1, SRSF2, MBNL3, YY1 RNA-binding sites, and against in silico controls corresponding to the 50 first (Prom50) or last (Term50) nucleotides of all RefSeqNCBI transcripts. Violin plots with median ± quartiles (c, e); P values derived from two-tailed Mann–Whitney U-tests (c, e) and Kruskal–Wallis tests (e), ns not significant, P > 0.0678, **P < 0.0099, ***P < 0.0006, ****P < 0.0001; scale bars, 5 μm. Source data are provided as a Source Data file.

Fig 4: CDK7 kinase activity is negatively regulated by TFIIH. (A) SYPRO-stained gel of human TFIIH. (B) TFIIH activates CDK7 kinase toward the CTD, but reduces or prevents CDK7 phosphorylation of DSIF, SF3B1, U2AF2, and TFIIF. Within the CAK, CDK7 is activated toward these substrates. (C) SYPRO-stained gel of human CAK complex (MAT1 is GST-tagged). (D) Quantitation of CDK7 kinase results across biological replicates (n = 3) with autorad signal normalized to CAK phosphorylated DSIF; P-values as shown.

Fig 5: Biological pathway, molecular functions, and network analysis based on intron retention(A) For biological pathway analysis, we took the top 25% of transcripts with high intron retention in CLL as compared with normal B cells (p < 0.05, FDR = 0.1). The gene symbols for corresponding transcripts were used as input and subjected for Reactome Pathway. The p value was adjusted using a Bonferroni correction. The x axis shows the % of the genes enriched and y axis shows the description of the pathways. (B) Molecular function analysis was done for the top 25% of transcripts with high intron retention CLL/NBC Network analysis by selecting transcripts with high intron retention in CLL as compared with normal B cells using WebGestalt. The height of the bar represents the % of genes observed in the category. (C) Subnetwork centered on SF3B1 and its first neighbors. The SF3B1 centered subnetwork was based on Reactome FI network database and cytoscape V3.4 was used to identify the first neighbors of SF3B1. Nodes and links represent genes and functional interactions, respectively. The color of each node scale with log2 intron retention ratio in CLL cells vs. NBCs as indicated in the scale at the bottom.