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Molecular and Cellular Biology, October 2001, p. 7010-7019, Vol. 21, No. 20
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.20.7010-7019.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

MSY2 and MSY4 Bind a Conserved Sequence in the 3' Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

Flaviano Giorgini, Holly G. Davies,dagger and Robert E. Braun*

Department of Genetics, University of Washington, Seattle, Washington

Received 14 May 2001/Returned for modification 26 June 2001/Accepted 16 July 2001

Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5'-UCCAUCA-3', present in the 3' untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU (subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

* Corresponding author. Mailing address: Department of Genetics, Box 357360, 1959 NE Pacific St., University of Washington, Seattle, WA 98195. Phone: (206) 543-1818. Fax: (206) 543-0754. E-mail: braun{at}u.washington.edu.

dagger Present address: National Institutes of Health, National Institute of Diabetes and Digestive Kidney Disorders, Laboratory of Cellular and Developmental Biology, Bethesda, MD 20892-8028.

Molecular and Cellular Biology, October 2001, p. 7010-7019, Vol. 21, No. 20
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.20.7010-7019.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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