Characterization of U6 snRNA–protein interactions

• Published in: RNA (Cambridge) Vol. 5; no. 11; pp. 1470 - 1481
• Main Authors: VIDAL, VALERIE P.I., VERDONE, LOREDANA, MAYES, ANDREW E., BEGGS, JEAN D.
• Format: Journal Article
• Language: English
• Published: United States Cambridge University Press 01.11.1999
• Subjects: Base Sequence; Conserved Sequence; Cross-Linking Reagents; Escherichia coli - genetics; Lsm proteins; Oligodeoxyribonucleotides - chemistry; Prp8 protein; Ribonucleoprotein, U4-U6 Small Nuclear - chemistry; Ribonucleoprotein, U4-U6 Small Nuclear - metabolism; RNA, Bacterial - chemistry; RNA, Bacterial - radiation effects; RNA, Small Nuclear - chemistry; RNA, Small Nuclear - metabolism; RNA, Transfer - chemistry; RNA, Transfer - radiation effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Sm proteins; snRNA U4; snRNA U5; snRNA U6; Spliceosomes - metabolism; Ultraviolet Rays; cross-linking; snRNP; yeast; Lsm; pre-mRNA splicing
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1017/S1355838299991355

Through a combination of in vitro snRNP reconstitution, photocross-linking and immunoprecipitation techniques, we have investigated the interaction of proteins with the spliceosomal U6 snRNA in U6 snRNPs, U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs. Of the seven Lsm (Sm-like) proteins that associate specifically with this spliceosomal snRNA, three were shown to contact the RNA directly, and to maintain contact as the U6 RNA is incorporated into tri-snRNPs. In tri-snRNPs, the U5 snRNP protein Prp8 contacts position 54 of U6, which is in the conserved region that contributes to the formation of the catalytic core of the spliceosome. Other tri-snRNP-specific contacts were also detected, indicating the dynamic nature of protein interactions with this important snRNA. The uridine-rich extreme 3′ end of U6 RNA was shown to be essential but not sufficient for the association of the Lsm proteins. Interestingly, the Lsm proteins associate efficiently with the 3′ half of U6, which contains the 3′ stem-loop and uridine-rich 3′ end, suggesting that the Lsm and Sm proteins may recognize similar features in RNAs.