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PMID: 9632784 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

In vitro genetic analysis of the RNA binding site of vigilin, a multi-KH-domain protein.

Molecular and cellular biology ·Vol. 18 ·No. 7 ·1998-07-00 ·Pages 3991-4003

Kanamori H, Dodson RE, Shapiro DJ

Abstract

The function(s) and RNA binding properties of vigilin, a ubiquitous protein with 14 KH domains, remain largely obscure. We recently showed that vigilin is the estrogen-inducible protein in polysome extracts which binds specifically to a segment of the 3' untranslated region (UTR) of estrogen-stabilized vitellogenin mRNA. In order to identify consensus mRNA sequences and structures important in binding of vigilin to RNA, before vigilin was purified, we developed a modified in vitro genetic selection protocol. We subsequently validated our selection procedure, which employed crude polysome extracts, by testing natural and in vitro-selected RNAs with purified recombinant vigilin. Most of the selected up-binding mutants exhibited hypermutation of G residues leading to a largely unstructured, single-stranded region containing multiple conserved (A)nCU and UC(A)n motifs. All eight of the selected down-binding mutants contained a mutation in the sequence (A)nCU. Deletion analysis indicated that approximately 75 nucleotides are required for maximal binding. Using this information, we predicted and subsequently identified a strong vigilin binding site near the 3' end of human dystrophin mRNA. RNA sequences from the 3' UTRs of transferrin receptor and estrogen receptor, which lack strong homology to the selected sequences, did not bind vigilin. These studies describe an aproach to identifying long RNA binding sites and describe sequence and structural requirements for interaction of vigilin with RNAs.

MeSH Terms
Animals Base Sequence Binding Sites Carrier Proteins Dystrophin/genetics Humans Molecular Sequence Data Mutagenesis Nucleic Acid Conformation RNA, Messenger/metabolism RNA-Binding Proteins/genetics,metabolism Rabbits Receptors, Estrogen/genetics Receptors, Transferrin/genetics Recombinant Fusion Proteins/genetics,metabolism Vitellogenins/genetics Xenopus
Chemicals
Carrier Proteins Dystrophin RNA, Messenger RNA-Binding Proteins Receptors, Estrogen Receptors, Transferrin Recombinant Fusion Proteins Vitellogenins high density lipoprotein binding protein
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kanamori H
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Dodson R E
Shapiro D J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.18.7.3991
Published
1998-07-00
Pages
3991-4003
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC108984
Subset
IM
Grants
NIDDK NIH HHS · DK-50080 · United States
NICHD NIH HHS · HD-16720 · United States
External Links
PubMed source DOI: 10.1128/MCB.18.7.3991 PMC Full Text View journal details
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