ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.
Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.
Alvi
R K
RK
School of Biological Sciences, University of Manchester, United Kingdom.
Lund
M
M
Okeefe
R T
RT
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D005091:Exons; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D012342:RNA, Small Nuclear; C405485:U5 small nuclear RNA; D000255:Adenosine Triphosphate