Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: Introns and exons are schematized as blue lines and rectangles, respectively, with A indicating the branchpoint. For bulk analysis ( B ) trace-labeled 5i3 was incubated with nuclear extracts, aliquots were analyzed on denaturing gel (15%) and second step splicing products quantified in graph. Second step splicing efficiency (±s.d.) was calculated as the amount of ligated exon product relative to the amount of 5i3 starting material at time zero. For single-molecule analysis ( C ), dyes tethered to the surface (red, green stars) were visualized using total internal reflection fluorescence microscopy using alternating red and green laser excitation (arrows); dye-labeled molecules in solution are not detectable. Fraction (±s.e.) of labeled introns remaining was calculated as the fraction of the N molecules retaining the exon dye fluorescence (green star) through the entire experiment duration which retained intron dye fluorescence (red star) at a particular time. Labeling of spliceosomal subcomplexes is shown in – .

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Labeling, Incubation, Fluorescence, Microscopy

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: Fluorescence and Coomassie images of SDS-PAGE gel lanes show specific dye labeling of tagged spliceosomal components in nuclear extracts. Indicated migration position of unreacted dye was determined using control untagged nuclear extract spiked with the green-excited dye-benzylguanine adduct.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Fluorescence, SDS Page, Labeling, Migration, Control

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: ( A ) Schematic of a CoSMoS experiment in which green dye-labeled U1 is observed binding to red dye-labeled, surface-tethered RNAs. Introns and exons are schematized as blue and magenta lines and rectangles, respectively, with A indicating the branchpoint. Dyes (stars) linked to tethered RNAs were visualized using total internal reflection fluorescence microscopy using alternating red and green laser excitation (arrows); dye-labeled molecules in solution are not detectable. ( B ) Schematic of 5i3 and 3e5 RNAs, with features indicated as in ( A ). See for RNA sequences. ( C ) Protocol: 5i3 (blue) and 3e5 (magenta) RNAs were sequentially deposited and located (squares) under red laser excitation. Non-overlapping control ‘no RNA’ locations (gray) were selected. Then, extract was introduced and spliceosomal subcomplex (e.g., U1 ) binding to individual RNA molecules was visualized under green laser excitation. Images (grayscale) are a small portion (2.6 μm x 2.6 μm) of the microscope field of view recorded at each stage of the process. See for complete field of view. ( D ) Time series images (1 s per frame; 1.3 μm x 1.3 μm) of U1 fluorescence from example surface locations containing a single 5i3 RNA (top), a single 3e5 RNA (middle) or no detected RNA (bottom). Images with fluorescence spots (highlighted) indicate U1 binding. See for additional traces and detected events. ( E ) Rastergrams aggregating U1, U2, and U5 binding time courses from random samples of 50 individual 5i3 and 3e5 RNA molecules over 2,400 s. Each row in these plots contains data from a single RNA molecule; color indicates presence and white indicates absence of bound spliceosomal subcomplex. In each panel, RNA molecules are sorted by the time of first subcomplex binding (latest to earliest); the percentage (±s.e.) of N observed RNA molecules that exhibited subcomplex binding during the experiment is indicated. Rastergrams for ‘no RNA’ control locations are shown in . ( F ) Cumulative distributions of U1, U2, and U5 dwell times on N observed 5i3 and 3e5 RNAs or control ‘no RNA’ locations. Data show the mean frequency per RNA molecule (or per ‘no RNA’ location) of subcomplex binding events with durations greater than or equal to the indicated dwell time. All frequencies on RNAs are substantially higher than the non-specific binding seen at ‘no RNA’ locations (note logarithmic scale). ( G ) Total frequencies (±s.e.) per RNA molecule of RNA-specific subcomplex binding. These RNA-specific binding frequencies correspond to the RNA minus the no RNA vertical axis intercepts of the curves in ( F ); they represent the total rate of subcomplex-RNA binding throughout the 2,400 s experiment averaged over all observed RNA molecules. ( H ) Frequencies (±s.e.) per RNA molecule of the subsets of RNA-specific subcomplex binding events shorter or longer than 50 s. ( I ) Specific occupancy (±s.e.), corresponding to the fraction of RNA molecules bound by the indicated fluorescent subcomplex averaged over the duration of the experiment. Numbers of RNA molecules observed in ( G–I ) are the same as those reported in ( F ). The specific occupancy values are calculated as described (see Materials and methods) to correct for the small amount of binding observed at ‘no RNA’ locations. Source data for : SourceDataFigure2.zip. 10.7554/eLife.37751.012 Figure 2—Source data 1. Data from the single-molecule experiments.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Labeling, Binding Assay, Fluorescence, Microscopy, Control, RNA Binding Assay

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: Data are from the experiments shown in . The stated fraction (±s.e.) of the no RNA locations that exhibited subcomplex binding was calculated from the total sample of N no RNA locations reported in .

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: ( A ) Rastergrams showing U2 binding data on 50 randomly selected individual 5i3 and 5iX pre-mRNA molecules and 'no RNA' control locations over the course of 2,400 s, sorted by the time of the first binding event to each RNA. The percentage (±s.e.) of N observed RNA molecules that exhibited subcomplex binding during the experiment is indicated. ( B ) Cumulative dwell time distributions of U2 binding events on wild-type 5i3 and mutated 5iX cross-intron RNAs, and at randomly selected locations with no RNA. ( C ) Frequencies (±s.e.) of RNA-specific U2 binding to 5i3 and 5iX RNAs calculated as in . ( D–F ) Same as ( A–C ), except for the 3e5 and Xe5 RNAs. Data in all three panels are from the same experiment. 10.7554/eLife.37751.023 Figure 3—figure supplement 1—source data 1. Data from the single-molecule experiments.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay, Control

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: Data are from the experiment in . The percentage (±s.e.) of N observed RNA molecules (see ) that exhibited subcomplex binding during the experiment is indicated.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: ( A ) Rastergrams showing U1 binding data on 50 randomly selected individual 5i3 and Xi3 pre-mRNA molecules and control no RNA locations from the same experiment, sorted by the time of the first binding event at each RNA or no RNA location. The percentage (±s.e.) of N observed RNA molecules that exhibited subcomplex binding during the experiment is indicated. ( B ) Cumulative dwell time distributions of U1 binding events on wild-type 5i3 and mutated Xi3 cross-intron RNAs. ( C ) Frequency (±s.e.) of RNA-specific U1 binding to 5i3 and Xi3 RNAs calculated as in . Data in panels A, B, and C are all from the same experiment. ( D–F ) Same as ( A–C ), except for the 3e5 and 3eX RNAs. Data in all three panels are from the same experiment. 10.7554/eLife.37751.024 Figure 3—figure supplement 3—source data 2. Data from the single-molecule experiments.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay, Control

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: The percentage (±s.e.) of N observed RNA molecules (see ) that exhibited subcomplex binding during the experiment is indicated. Data for the left and right halves of the figure are taken from the same experiments as the left and right halves of .

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: ( A ) Rastergrams showing U5 binding to 50 randomly selected individual 5i3 and Xi3 pre-mRNA molecules and to 'no RNA' control locations over the course of 2,400 s, sorted by the time of the first binding event to each RNA. The percentage (±s.e.) of N observed RNA molecules that exhibited subcomplex binding during the experiment is indicated. ( B ) Cumulative dwell time distributions of U2 binding events on wild-type 5i3 and mutated Xi3 cross-intron RNAs, and at randomly selected locations with no RNA. ( C ) Frequencies (±s.e.) of RNA-specific U5 binding to 5i3 and Xi3 RNAs calculated as in . 10.7554/eLife.37751.025 Figure 3—figure supplement 5—source data 3. Data from the single-molecule experiments.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay, Control

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: ( A ) Bulk splicing assay (as in ) shows specific inhibition of 5i3 pre-mRNA splicing upon addition of anti-U1 AMO at 10 µM final concentration as previously reported . A non-complementary control AMO of the same length showed minimal inhibition . ( B ) Single molecule observations of U2 binding to RNAs in extract preincubated with 10 µM control AMO. Rastergrams show U2 binding to 50 randomly selected individual 5i3 and 3e5 pre-mRNA molecules and control no RNA locations from the same experiment, sorted by the time of the first binding event at each RNA or no RNA location. The percentage (±s.e.) of N observed RNA molecules that exhibited subcomplex binding during the experiment is indicated. ( C ) Cumulative dwell time distributions of all U2 binding events from the experiment excerpted in ( B ). ( D,E ) Same as ( B,C ), except that the extract was preincubated with 10 µM anti-U1 AMO. ( F ) Frequency (±s.e.) of RNA-specific U2 binding in the presence of control and anti-U1 AMO to 5i3 (left) and 3e5 (right) calculated from data in ( B–E ) as in . 10.7554/eLife.37751.026 Figure 3—figure supplement 6—source data 4. Data from the single-molecule experiments.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Splicing Assay, Inhibition, Concentration Assay, Control, Binding Assay

Journal: eLife

Article Title: Synergistic assembly of human pre-spliceosomes across introns and exons

doi: 10.7554/eLife.37751

Figure Lengend Snippet: The percentage (±s.e.) of N (see ) observed RNA molecules or no RNA locations that exhibited subcomplex binding during the experiment is indicated.

Article Snippet: Plasmids containing spliceosomal subcomplex protein-fSNAP fusions ( ) were generated by PCR amplification from HEK293 cDNA for U1-70K and U2B'', and from Kazusa DNA Research Institute cDNA clone ORK00375 ( ) for Snu114 using the specified primers ( ) and cloning the products into pcDNA5-TetO-fSNAPc using the KpnI and NotI restriction sites.

Techniques: Binding Assay