Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation
- Max Planck Institute for Biophysical Chemistry, Germany
- Max Planck Institute of Molecular Physiology, Germany
- University Medical Center, Germany
Figures
Figure 1 with 3 supplements
Characterization of spliceosomes that accummulate in the presence of the small molecule inhibitor cp028.
(A) Structure of compound 028 (cp028). (B, C) Splicing was performed with 32P-MINX pre-mRNA in the presence of 10–175 µM compound 028 in HeLa nuclear extract for 60 min. RNA was analysed by …
Figure 1—figure supplement 1
Determination of IC50 and kinetics of splicing complex formation in the presence of compound 028.
(A) Quantification of splicing efficiency and determination of IC50 values. The splicing efficiency (% mRNA formed) at different concentrations was determined from three independent experiments, …
Figure 1—figure supplement 2
Identification of abundant proteins in affinity-purified A028 complexes.
(A) Proteins (larger than 25 kDa) from MS2 affinity-purified A028 complexes assembled on MINX-MS2 pre-mRNA were separated by 2D gel electrophoresis, stained with RuBPS, and the identities of single …
Figure 1—figure supplement 3
Most U4/U6.U5 tri-snRNPs remain intact in the presence of cp028.
HeLa nuclear extract plus DMSO (upper panel) or 150 µM compound 028 (lower panel) was incubated under splicing conditions (except no pre-mRNA was added) at 30° for 20 min. Extract was then (A) …
Figure 2
Identification of abundant proteins in affinity-purified B028 complexes.
(A) Proteins (larger than 25 kDa) from MS2 affinity-purified B028 complexes assembled on MINX-MS2 pre-mRNA were separated by 2D gel electrophoresis, stained with RuBPS, and the identities of single …
Figure 3 with 4 supplements
Characterization of the catalytic U2/U6 RNA network in the B028 complex.
(A) Summary of the chemical modification patterns of U6 and U2 snRNA in B028 and Bact complexes. The U2/U6 interaction network is shown forming a 3-way junction (3WJ). The different size of the dots …
Figure 3—figure supplement 1
Identification of RNA-RNA interactions in the B028 complex via psoralen crosslinking.
(A) Schematic of the PM5-10 pre-mRNA. (B) RNA composition of MS2 affinity-purified spliceosomal complexes assembled on PM5-10 pre-mRNA. RNA was recovered from the indicated complexes, separated by …
Figure 3—figure supplement 2
Structure probing of the U2 and U6 snRNAs in purified B028 complexes with kethoxal.
(A) Example of quantitation of reverse transcriptase stops after DMS, CMCT and KE probing. Primer extension using a primer complementary to the extended 3' end of U6 is shown. Bands were quantified …
Figure 3—figure supplement 3
RNA structure probing of the pre-mRNA in B028 and Bact complexes.
Primer extension analysis of the pre-mRNA from DMS and KE treated B028 and Bact complexes. Primer extension was performed with an oligonucleotide complementary to PM5-10 pre-mRNA nts 281–262.
Figure 3—figure supplement 4
U2 snRNA intramolecular helix IIc is not formed in the B028 complex.
(A) Structure of U2 stem IIc and stem–loop IIa. Nucleotides involved in stem IIc and stem loop IIa are shaded grey. Nucleotides of U2 that base pair with the branch site of the pre-mRNA are shown. (B…
Figure 4
Purified B028 complexes can be chased into catalytically-active spliceosomes.
Affinity-purified B or B028 complexes (as indicated above) formed on MINX-MS2 pre-mRNA, were incubated at 30°C for the indicated times (0–60 min) under splicing conditions in the presence of buffer …
Figure 5
Morphology of the B028 complex under the electron microscope.
(A) Overview of raw images of affinity-purified B028 complexes obtained by negative stain electron microscopy. (B) Selected class averages (columns 1–4) of the B028 complex, compared to published EM …
Figure 6 with 2 supplements
Effects of alterations in the structure of cp028 on its splicing inhibition activity.
(A–C) In vitro splicing was performed with 32P-labelled MINX pre-mRNA and HeLa nuclear extract for 60 min after addition of cp028 and various analogues of cp028 at the indicated concentrations. The …
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Figure 6—source data 1
NMR spectra of cp028 analogues synthesized in house.
Cp028 analogues synthesised in house and several intermediate compounds were characterised by nuclear magnetic resonance spectroscopy. A 1H spectrum is shown for all molecules. 13C and 19F spectra are provided for some of the analogues. The ppm values (in blue), the multiplicity and the 1H integrals (in green) are shown (see also description of the synthesis of each compound in Materials and Methods).
- https://doi.org/10.7554/eLife.23533.015
Figure 6—figure supplement 1
Structures of cp028 analogues and their effect on pre-mRNA splicing in vitro.
(A) Compound 028. Analogues of cp028 in which the p-fluorophenyl group was replaced that (B) have little or no effect on splicing inhibition activity, (C) enhance slightly inhibition activity or (D) …
Figure 6—figure supplement 2
Effect of cp028 analogues on in vitro splicing.
(A, B) In vitro splicing was performed with 32P-labelled MINX pre-mRNA and HeLa nuclear extract for 60 min after addition of cp028 and 50 μM of various analogues of cp028 as indicated above each …
Scheme 1
General Synthesis of cp028 (R = 4fluoro) and analogues.
https://doi.org/10.7554/eLife.23533.018
Chemical structure 1
1-(2-Ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (C).
https://doi.org/10.7554/eLife.23533.019
Chemical structure 2
5-(4-Fluorophenyl)furan-2-carbaldehyde (D1).
https://doi.org/10.7554/eLife.23533.020
Scheme 2
Aldehydes used for the synthesis of the cp028 analogues.
https://doi.org/10.7554/eLife.23533.021
Chemical structure 3
(E/Z)−1-(2-Ethylphenyl)−5-((5-(4-fluorophenyl)furan-2-yl)methylene)pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of E/Z isomers) (cp028).
https://doi.org/10.7554/eLife.23533.022
Chemical structure 4
(+/-)1-(2-Ethylphenyl)−5-((5-(4-fluorophenyl)furan-2-yl)methyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (racemic mixture) (343139).
https://doi.org/10.7554/eLife.23533.023
Chemical structure 5
(E/Z)−5-((5-(4-Acetylphenyl)furan-2-yl)methylene)−1-(2-ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of E/Z isomers) (343870).
https://doi.org/10.7554/eLife.23533.024
Chemical structure 6
(E/Z)−5-((5-(4-(Dimethylamino)phenyl)furan-2-yl)methylene)−1-(2-ethylphenyl) pyrimidine-2,4,6 (1H,3H,5H)-trione (1.5:1 mixture of E/Z isomers) (343869).
https://doi.org/10.7554/eLife.23533.025
Chemical structure 7
(E/Z)−5-((5-(3,4-dimethoxyphenyl)furan-2-yl)methylene)−1-(2-ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of E/Z isomers) (343877).
https://doi.org/10.7554/eLife.23533.026
Chemical structure 8
(E/Z)−5-((5-(3,5-Dichlorophenyl)furan-2-yl)methylene)−1-(2-ethylphenyl) pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of E/Z isomers) (343873).
https://doi.org/10.7554/eLife.23533.027
Chemical structure 9
(E/Z)−1-(2-Ethylphenyl)−5-((5-(3-fluorophenyl)furan-2-yl)methylene)pyrimidine-2,4,6 (1H,3H,5H)-trione (1.2:1 mixture of E/Z isomers) (343867).
https://doi.org/10.7554/eLife.23533.028
Chemical structure 10
(E/Z)−5-((5-(3-Chlorophenyl)furan-2-yl)methylene)−1-(2-ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of E/Z isomers) (343871).
https://doi.org/10.7554/eLife.23533.029
Chemical structure 11
(E/Z)−5-(4-Chlorobenzylidene)−1-(2-ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of E/Z isomers) (1:1 mixture of E/Z isomers) (343878).
https://doi.org/10.7554/eLife.23533.030
Chemical structure 12
(E/Z)−5-(4-Bromobenzylidene)−1-(2-ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (1.7:1 mixture of isomers E/Z) (343879).
https://doi.org/10.7554/eLife.23533.031
Chemical structure 13
(E/Z)−5-((5-(3,4-Dichlorophenyl)furan-2-yl)methylene)−1-(2-ethylphenyl) pyrimidine-2,4,6 (1H,3H,5H)-trione (1.4:1 mixture of isomers E/Z) (343872).
https://doi.org/10.7554/eLife.23533.032
Chemical structure 14
(E/Z)−5-((5-([1,1'-Biphenyl]−4-yl)furan-2-yl)methylene)−1-(2-ethylphenyl)pyrimidine-2,4,6 (1H,3H,5H)-trione (1:1 mixture of isomers E/Z) (343868).
https://doi.org/10.7554/eLife.23533.033
Chemical structure 15
5-((5-(4-fluorophenyl)furan-2-yl)methylene)pyrimidine-2,4,6 (1H,3H,5H)-trione.
https://doi.org/10.7554/eLife.23533.034Additional files
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Supplementary file 1
Protein composition of A028 and B028 complexes as determined by mass spectrometry.
Proteins identified by LC-MS/MS in human spliceosomal B and Bact complexes, as well as complexes stalled in the presence of compound 028 (B028). Total spectral counts of sequenced peptides are shown. Peptides and proteins were identified by searching fragment spectra against the NCBI database (taxonomy human) using Mascot as search engine and were annotated with Scaffold software. Proteins are grouped according to function or association. Common contaminants, such as ribosomal proteins, are not shown.
- https://doi.org/10.7554/eLife.23533.035
