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Molecular and Cellular Biology, September 2004, p. 7392-7401, Vol. 24, No. 17
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.17.7392-7401.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

Jan Medenbach,1 Silke Schreiner,1 Sunbin Liu,2 Reinhard Lührmann,2 and Albrecht Bindereif1*

Institut für Biochemie, Justus-Liebig-Universität, Giessen,1 Abteilung Zelluläre Biochemie, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany2

Received 27 February 2004/ Returned for modification 29 March 2004/ Accepted 1 June 2004

After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

* Corresponding author. Mailing address: Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. Phone: 49-641-99 35 420. Fax: 49-641-99 35 419. E-mail: albrecht.bindereif{at}chemie.bio.uni-giessen.de.

Molecular and Cellular Biology, September 2004, p. 7392-7401, Vol. 24, No. 17
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.17.7392-7401.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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