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  ATPγS stalls splicing after B complex formation but prior to spliceosome activation.

Agafonov, D. E., van Santen, M., Kastner, B., Dube, P., Will, C. L., Urlaub, H., et al. (2016). ATPγS stalls splicing after B complex formation but prior to spliceosome activation. RNA, 22(9), 1329-1337. doi:10.1261/rna.057810.116.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-3371-B Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-9FE2-5
Genre: Journal Article

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 Creators:
Agafonov, D. E.1, Author              
van Santen, M.1, Author              
Kastner, B.1, Author              
Dube, P.2, Author              
Will, C. L.1, Author              
Urlaub, H.3, Author              
Lührmann, R.1, Author              
Affiliations:
1Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society, ou_578576              
2Research Group of 3D Electron Cryo-Microscopy, MPI for biophysical chemistry, Max Planck Society, ou_578577              
3Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              

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Free keywords: ATPγS; Prp19/CDC5L complex; pre-mRNA splicing; small molecule; spliceosome
 Abstract: The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (B(ATPγS)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATPγS) complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B(ATPγS) complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to B(act) complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B(act) spliceosomal complex.

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Language(s): eng - English
 Dates: 2016-07-132016-09
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1261/rna.057810.116
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Title: RNA
Source Genre: Journal
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Pages: - Volume / Issue: 22 (9) Sequence Number: - Start / End Page: 1329 - 1337 Identifier: -