Human U4/U6 snRNP recycling factor p110: mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling.
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.
Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Jan
J
Medenbach
Silke
S
Schreiner
Sunbin
S
Liu
Reinhard
R
Lührmann
Albrecht
A
Bindereif
D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D000951:Antigens, Neoplasm; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; C416530:SART3 protein, human