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UV‐crosslinking of E1 small nucleolar RNA to proteins in frog oocytes
Author(s) -
Smith James L.,
Walton Andrew H.,
Eliceiri George L.
Publication year - 2005
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.20223
Subject(s) - rna , small nucleolar rna , rnase p , small nuclear rna , non coding rna , microbiology and biotechnology , nuclease protection assay , biology , rna dependent rna polymerase , rna editing , ribosomal rna , chemistry , biochemistry , gene
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 [α‐ 32 P]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. © 2004 Wiley‐Liss, Inc.