Cooperative structure of the heterotrimeric pre-mrna retention and splicing complex

ABSTRACT The precursor mRNA (pre-mRNA) retention and splicing (RES) complex is a spliceosomal complex that is present in yeast and humans and is important for RNA splicing and retention of

unspliced pre-mRNA. Here, we present the solution NMR structure of the RES core complex from _Saccharomyces cerevisiae_. Complex formation leads to an intricate folding of three

components—Snu17p, Bud13p and Pml1p—that stabilizes the RNA-recognition motif (RRM) fold of Snu17p and increases binding affinity in tertiary interactions between the components by more than

100-fold compared to that in binary interactions. RES interacts with pre-mRNA within the spliceosome, and through the assembly of the RES core complex RNA binding efficiency is increased.

The three-dimensional structure of the RES core complex highlights the importance of cooperative folding and binding in the functional organization of the spliceosome. Access through your

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BEING VIEWED BY OTHERS MECHANISM FOR THE INITIATION OF SPLICEOSOME DISASSEMBLY Article 26 June 2024 RNA RECOGNITION BY NPL3P REVEALS U2 SNRNA-BINDING COMPATIBLE WITH A CHAPERONE ROLE DURING

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references ACKNOWLEDGEMENTS This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center 860 (project B2 to M.Z.) and the DFG Research Unit 806 (project

A6 to M.C.W. and R.L.). We thank P. Fabrizio and K. Hartmuth for helpful discussions, T. Wandersleben for help with protein preparation and K. Giller for the preparation of expression

constructs. AUTHOR INFORMATION Author notes * Simon Trowitzsch Present address: Present address: European Molecular Biology Laboratory, Grenoble Outstation, Grenoble, France, and Unit for

Virus Host-Cell Interactions, Université Grenoble Alpes–European Molecular Biology Laboratory–National Center for Scientific Research, Grenoble, France., AUTHORS AND AFFILIATIONS *

Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Piotr Wysoczański, ShengQi Xiang, Francesca Munari, Stefan Becker & Markus

Zweckstetter * Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Cornelius Schneider, Simon Trowitzsch & Reinhard Lührmann *

Laboratory of Structural Biochemistry, Freie Universität Berlin, Berlin, Germany Markus C Wahl * German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany Markus Zweckstetter *

Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center, Göttingen, Germany Markus Zweckstetter Authors * Piotr Wysoczański View author publications

You can also search for this author inPubMed Google Scholar * Cornelius Schneider View author publications You can also search for this author inPubMed Google Scholar * ShengQi Xiang View

author publications You can also search for this author inPubMed Google Scholar * Francesca Munari View author publications You can also search for this author inPubMed Google Scholar *

Simon Trowitzsch View author publications You can also search for this author inPubMed Google Scholar * Markus C Wahl View author publications You can also search for this author inPubMed 

Google Scholar * Reinhard Lührmann View author publications You can also search for this author inPubMed Google Scholar * Stefan Becker View author publications You can also search for this

author inPubMed Google Scholar * Markus Zweckstetter View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS P.W. designed the project, conducted

protein preparation and ITC and NMR data acquisition and analysis and wrote the paper; C.S. performed immunoprecipitation of Snu17p from spliceosomal complexes; S.X. performed NMR

experiments; F.M. performed NMR data analysis; S.T. designed the project and conducted peptide arrays and protein preparation; M.C.W. designed and supervised the project and interpreted

data; R.L. designed and supervised the project; S.B. designed and supervised the project; M.Z. designed and supervised the project and wrote the paper. CORRESPONDING AUTHOR Correspondence to

Markus Zweckstetter. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 COMPARISON OF

1H-15N HSQC OF CSNU17P IN VARIOUS COMPLEXES AND SECONDARY CHEMICAL SHIFT OF FREE CPML1P AND CBUD13P. (A) 1H, 15N HSQC of cSnu17p in cPml1p–cSnu17p dimer, cBud13p–cSnu17p dimer, and cRES. (B)

1H, 15N HSQC of cSnu17p in cPml1p–cSnu17p dimer and cSnu17p monomer. (C) 1H, 15N HSQC of cSnu17p in cPml1p–cSnu17p dimer and cBud13p–cSnu17p dimer. (D) 1H, 15N HSQC of cSnu17p in

cPml1p–cSnu17p dimer and cSnu17p monomer. (E) Secondary chemical shift (Δδ) of free cBud13p. (F) Secondary chemical shift (Δδ) of free cPml1p. . SUPPLEMENTARY FIGURE 2 VALIDATION OF THE CRES

STRUCTURE WITH RESIDUAL DIPOLAR COUPLINGS (RDCS). Plots of experimental vs back-calculated RDCs before (upper panel) and after (lower panel) structure refinement with RDCs. RDCs from the

C-terminal α-helix (117-126) backbone amides are indicated in red, those of cPml1p in yellow and cBud13p in blue. SUPPLEMENTARY FIGURE 3 1H-15N CHEMICAL-SHIFT PERTURBATION (CSP) OF

CSNU17P–CBUD13P OR CSNU17P–CPML1P WHEN TITRATED WITH CPML1P OR CBUD13P, RESPECTIVELY. (A) Plot of CSP imposed on cSnu17p–cBud13p dimer when titrated with cPml1p. (B) The aforementioned plot

mapped onto the structure of cRES with spheres colored as described above; T49 is indicated (C) Residues that are common between this CSP experiment (when cSnu17p-cBud13 dimer is titrated

with cPml1p) and when cRES is titrated with RNA (CUUCAUCUUUUUG) are labeled. (D) Plot of CSP imposed on cSnu17p–cPml1p dimer when titrated with cBud13p. (E–F) The aforementioned plot mapped

onto the structure of cRES with spheres colored as described above; T49 is indicated. Only 224 to 238 residues of cBud13p are shown for clarity. Significant CSPs were grouped and color-coded

into three categories according to: medium (light pink) if 2σ>CSP>1σ, strong (pink) if 3σ>CSP>2σ, very strong (red) if CSP>3σ, were σ is the standard deviation of the mean.

SUPPLEMENTARY FIGURE 4 ANALYSIS OF CSNU17P MONOMER, CSNU17P–CPML1P DIMER, CSNU17P–CBUD13P DIMER AND CRES DYNAMICS. (A) Plot of residue specific hydrogen-deuterium exchange half-life H-D1/2

for cRES (black), cSnu17p–cPml1p dimer (yellow), cSnu17p–cBud13p dimer (blue), cSnu17p monomer (red). (B) R2 (black) and R1ρ (grey) plots (left panel) for the aforementioned complexes. Rex

estimates derived from R2 and R1ρ difference (right panel). Positions marked with an asterisk correspond to the five most broadened peaks in C). (C) Normalized intensity vs 1H full width at

half maximum (FWHM) of 1H, 15N HSQC peaks for the aforementioned complexes and cSnu17p monomer. Intensity values are offset between each group by 1. Please note that both cRES and

cSnu17p–cBud13p dimer experience a decrease in the overall tumbling due to apparent increase in size related to peptide binding and/or the presence of C-terminal α-helix. This effect is

small in cSnu17p–cPml1p dimer since cPml1p is shorter and C-terminal α-helix is folded. (D) S2 order parameter derived from the chemical shift for the aforementioned complexes. SUPPLEMENTARY

FIGURE 5 RES-RNA INTERACTION. (A) 1H, 15N chemical shift perturbation (CSP) imposed on cSnu17p in cSnu17p–cPml1p dimer and in cSnu17p–cBud13p dimer upon titration with CUUCAUCUUUUUG RNA.

CSP mapped on the structure of cRES (left of each graph). Significant CSPs were grouped and color-coded into three categories according to: medium (light pink) if 2σ>CSP>1σ, strong

(pink) if 3σ>CSP>2σ, very strong (red) if CSP >3σ, were σ is the standard deviation of the mean for the CUUCAUCUUUUUG to cRES titration. Only 224 to 238 residues of cBud13p are

shown for clarity. (B) CSP binding curves derived from a representative set of residues experiencing high CSP and residue-averaged dissociation constants (_K_d) of CUUCAUUCUUUUUG and all

four members of the cRES assembly pathway. (C) Uncropped western blots from main text Fig. 5a. From left to right, Ponceau-stained membrane, peroxidase-anti-peroxidase detection of RES-TAP

and autoradiography. SUPPLEMENTARY FIGURE 6 1H-15N NORMALIZED CHEMICAL-SHIFT PERTURBATION (CSP) IMPOSED ON CSNU17P IN CRES UPON TITRATION WITH VARIOUS RNAS. (A) NCSP mapped on the structure

of cRES (left of each graph). Significant NCSPs were grouped and color-coded into three categories according to: medium (light pink) if 2σ>NCSP>1σ, strong (pink) if 3σ>NCSP>2σ,

very strong (red) if NCS >3σ, were σ is the standard deviation of the mean. Only 224 to 238 residues of cBud13p are shown for clarity. The given RNA sequence is indicated above each

graph. (B) CSP binding curves derived from a representative set of residues experiencing high CSP upon ACGAAUUAGA titration and average binding affinity (below). * value derived from CSP of

two residues. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–6 (PDF 2505 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Wysoczański, P., Schneider, C., Xiang, S. _et al._ Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex. _Nat Struct Mol Biol_ 21, 911–918 (2014).

https://doi.org/10.1038/nsmb.2889 Download citation * Received: 14 February 2014 * Accepted: 15 August 2014 * Published: 14 September 2014 * Issue Date: October 2014 * DOI:

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