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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)
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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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Publikationstyp Artikel: Journalartikel
Dokumenttyp Meeting abstract
ISSN (print) / ISBN 0006-3495
e-ISSN 1542-0086
Zeitschrift Biophysical Journal
Quellenangaben Band: 106, Heft: 2, Seiten: 465A Artikelnummer: , Supplement: ,
Verlag Elsevier
Verlagsort Cambridge
Begutachtungsstatus Peer reviewed