Journal: bioRxiv

Article Title: Efficient co-transcriptional splicing enforces rapid microexon definition and inclusion by SRRM4

doi: 10.64898/2026.05.02.722430

Figure Lengend Snippet: (A) Examples of long reads aligned to Calr, Eif4g1, Plxna3, and Nop56 , colored by splicing status (All spliced: blue, Partially spliced: green, All unspliced: tan). Percent of reads aligning to each gene in each category is depicted in bar plot to the right of each plot. Number of reads (all replicates combined) is indicated in each plot. Spliced-out introns are indicated by thin lines. (B) Histogram of mean co-transcriptional splicing efficiency values (CoSE) calculated for each intron. (C) Scatterplot of SPI vs. CoSE calculated for each intron using short and long reads respectively. Pearson’s correlation coefficient was used to assess correlation and is displayed in plot. Color indicates intron long-read coverage for individual introns. Short read and long-read samples were prepared from the same biological replicates of nascent RNA.

Article Snippet: The PacBio SMRTbell prep kit was used for final library preparation according to the manufacturer (Pacific Biosciences) and sequenced on a PacBio Sequel II Long-Read Sequencer (Table S1).

Techniques:

Journal: bioRxiv

Article Title: Efficient co-transcriptional splicing enforces rapid microexon definition and inclusion by SRRM4

doi: 10.64898/2026.05.02.722430

Figure Lengend Snippet: (A) Schematic depicting how splicing status and Pol II position are derived from long-read sequencing of nascent RNA. (B) Histogram of distance in nucleotides between Pol II and last upstream splice junction (e.g. the distance Pol II transcribed into the gene body before splicing can take place). (C) Scatterplot of mean distance to upstream splice junction in nucleotides vs. CoSE for each intron. Long-read coverage for each intron is indicated by color. Pearson’s correlation coefficient was used to assess correlation and is displayed in plot. (D) Zoomed-in Examples of long reads aligned to Map4k4, and Kif1b. Exon of interest is colored according to length and dependence on SRRM4 for inclusion (Independent microexon: green, SRRM4-dependent microexon: red). Spliced-out introns are indicated by thin lines. (E) Mean distance Pol II transcribes before the intron upstream of an exon of interest (Ei) is excised, where Ei represents constitutive long exons (>27 nt) (n=38,191), alternative long exons (n=10,377), SRRM4-dependent long exons (n=7), SRRM4-independent microexons (≤27 nt) (n=148), and SRRM4-dependent microexons (n=4). Statistical significance was determined using a Mann-Whitney U test, *p<0.05. (F) Mean distance Pol II transcribes before the intron downstream of an exon of interest (Ei) is excised, where Ei represents constitutive long exons (>27 nt) (n=19.595), alternative long exons (n=4,553), SRRM4-dependent long exons (n=8), SRRM4-independent microexons (≤27 nt) (n=111), and SRRM4-dependent microexons (n=10). Statistical significance was determined using a Mann-Whitney U test, *p<0.05.

Article Snippet: The PacBio SMRTbell prep kit was used for final library preparation according to the manufacturer (Pacific Biosciences) and sequenced on a PacBio Sequel II Long-Read Sequencer (Table S1).

Techniques: Derivative Assay, Sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Efficient co-transcriptional splicing enforces rapid microexon definition and inclusion by SRRM4

doi: 10.64898/2026.05.02.722430

Figure Lengend Snippet: (A) Schematic of targeted PCR amplification strategy for generating gene-specific long-read sequencing libraries. (B) Histogram of read lengths for three biological replicates (all replicates combined) of PacBio long-read sequencing datasets prepared from N2a nascent RNA and amplified with gene-specfic forward primers in first exons. (C) Read coverage plots from long-read sequencing libraries prepared by targeted PCR amplification of Ank2 , Kif1b , Mon2 , and Clasp2 . Gene diagram is displayed at the top of each plot. Long-read coverage of for libraries prepared using global amplification (G), amplification with a forward primer in the first exon of each gene of interest (FE), and amplification with a forward primer in the exon upstream of the microexon of interest (UE) are displayed in the middle with zoom-ins indicated by each window. Short-read (SR) coverage from global nascent RNA-seq dataset is displayed in green at the bottom of each plot for comparison. (D) Zoomed-in examples of long reads aligned to Ank2, Kif1b, Mon2, and Clasp2. Constituitive exons are colored in blue. Microexons are colored in red. Spliced-out introns are indicated by thin lines (top). Mean distance between Pol II position and upstream splice junctions for introns in corresponding reads plots (bottom). Introns with no data are boxed in red. Median distance to upstream splice junctions for the entire gene is indicated by dashed gray line.

Article Snippet: The PacBio SMRTbell prep kit was used for final library preparation according to the manufacturer (Pacific Biosciences) and sequenced on a PacBio Sequel II Long-Read Sequencer (Table S1).

Techniques: Amplification, Sequencing, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Efficient co-transcriptional splicing enforces rapid microexon definition and inclusion by SRRM4

doi: 10.64898/2026.05.02.722430

Figure Lengend Snippet: (A) Maximum entropy scores for 3’ splice sites (left) and 5’ splice sites (right) for exons with significant coverage in long-read sequencing dataset. (B) Maximum entropy scores for 5’ splice sites vs. distance between Pol II and upstream splice junctions for introns upstream of exons of interest (left) or downstream of exons of interest (right). (C) Logo plots of 5′ splice sites of independent long exons, independent microexons, SRRM4-dependent long exons, and SRRM4-dependent microexons generated using exons found in global long-read sequencing dataset. Junction positions are indicated by horizontal lines. (D) Clasp2 -based minigene constructs. Teal regions indicate mutations expected to enhance strength of motif. Red regions indicate mutations expected to hamper strength of motif. Bolded nucleotides indicate changes to consensus splice sites or polypyrimidine tracts. Blue boxes represent exons, while gray boxes represent introns. Salmon box indicates 24-nucleotide microexon. Schematic of minigene transfection and RT-PCR is shown to the left of minigene constructs. (E) RT-PCR gel for Clasp2-based minigene constructs. Upper bands represent microexon-included isoform while lower bands represent microexon-skipped isoform. (F) RT-PCR gel for Vav2(B)-based minigene constructs. Upper bands represent microexon-included isoform while lower bands represent microexon-skipped isoform.

Article Snippet: The PacBio SMRTbell prep kit was used for final library preparation according to the manufacturer (Pacific Biosciences) and sequenced on a PacBio Sequel II Long-Read Sequencer (Table S1).

Techniques: Sequencing, Generated, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction