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Voith von Voithenberg, L.* ; Sanchez-Rico, C. ; Warner, L. ; Zhang, Y. ; Sattler, M. ; Lamb, D.C.*

Conformational dynamics during spliceosome assembly investigated by single-pair FRET.

Biophys. J. 106, 465A (2014)
Open Access Green as soon as Postprint is submitted to ZB.
The ribonucleoprotein (RNP) machinery of the spliceosome is composed of several subunits, which assemble stepwise during the process of splicing. U2 auxiliary factor (U2AF), a heterodimer comprised of a large (65 kDa) and a small (35 kDa) subunit, is involved in the early recognition of the intron and stabilization during splicing reactions. U2AF65 specifically recognizes the polypyrimidine tract in pre-mRNA introns and additionally contacts further splicing factors, such as mBBP/SF1 and SAP155 [1,2]. U2AF65 contains three RNA recognition motifs (RRM), where RRM1 and RRM2 mediate RNA binding while RRM3 is a U2AF homology motif (UHM) that mediates the interaction with U2AF35 [3]. A nuclear magnetic resonance spectroscopy study of recombinant human U2AF65 (RRM1-RRM2) showed a closed conformational state for the splicing factor in absence of RNA, while an open conformation is induced upon binding to polypyrimidine stretches [4]. Here, conformational subpopulations of the protein were investigated using single-pair FRET in solution with multiparameter fluorescence detection and pulsed interleaved excitation [5]. Information on FRET efficiency, stoichiometry, and lifetime revealed different conformational states dependent on substrate recognition and provided clues for dynamic motions of the splicing factor. These were further analyzed on a total internal reflection microscope using molecules immobilized in lipid vesicles. Changes in FRET efficiency over time showed a highly flexible U2AF65 protein with stabilization of specific conformational states upon RNA binding. Single-pair FRET measurements provide detailed insights into the mechanistic action of polypyrimidine tract recognition.
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Publication type Article: Journal article
Document type Meeting abstract
Corresponding Author
ISSN (print) / ISBN 0006-3495
e-ISSN 1542-0086
Journal Biophysical Journal
Quellenangaben Volume: 106, Issue: 2, Pages: 465A Article Number: , Supplement: ,
Publisher Elsevier
Publishing Place Cambridge
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Structural Biology (STB)
Institute of Virology (VIRO)