UV-crosslinking of E1 small nucleolar RNA to proteins in frog oocytes.
E1/U17 small nucleolar RNA (snoRNA) is a box H/ACA snoRNA. To detect protein bands that UV-crosslink to E1 RNA primarily at uridines, frog oocytes were injected with [alpha-32P]UTP-labeled E1 RNA and incubated, isolated nuclei were UV irradiated, and nuclear contents were digested with RNase A. Wild-type E1 RNA specifically UV-crosslinked to several protein bands. To identify E1 RNA sites involved in these interactions, we tested 21 E1 RNA mutants, each consisting of substitutions in a conserved sequence or structure. UV-crosslinking of different protein bands to E1 RNA depended on one of the following sets of conserved E1 RNA segments: two 5' end RNA sites; five 5' half RNA sites; two 3' half RNA sites; or 14 sites located throughout E1 RNA. Of these conserved E1 RNA sites, UV-crosslinking apparently depended on sequences at 11 sites, and structures at 2 sites. Gel electrophoresis with and without RNA competition detected protein bands that are not common to all of the box H/ACA snoRNAs.
Smith
James L
JL
Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104-1028, USA.
Walton
Andrew H
AH
Eliceiri
George L
GL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D002474:Cell-Free System; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000737:chemistry; D010777:Photochemistry; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays; D014529:Uridine / Q000378:metabolism; D014982:Xenopus laevis
D011506:Proteins; D020537:RNA, Small Nucleolar; D014529:Uridine