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J. Biol. Chem., Vol. 277, Issue 50, 48490-48500, December 13, 2002
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From the The 15.5K protein directly binds to the 5'
stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA
box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C
motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box
B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of
15.5K to the box B/C motif. The association of hU3-55K with the U3BC
RNA was found to be also dependent on a conserved RNA structure that
flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our
data support a new structural model of the box B/C region of the U3
snoRNA in which the box B/C motif is base-paired to form a structure
highly similar to that of both the U4 5' stem-loop and the box C/D motif.
The hU3-55K Protein Requires 15.5K Binding to the Box B/C Motif
as Well as Flanking RNA Elements for Its Association with the U3 Small
Nucleolar RNA in Vitro*
,,,, 161 Department of Biochemistry, University
of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
and § Max-Planck-Institute of Biophysical Chemistry, Am
Fassberg 11, D-37077 Göttingen, Germany
*
This work was supported by Deutsche Forschungsgemeinschaft
Grants Lu294/12-1 and Fonds der Chemischen Industrie (to R. L.), the
Council for Chemical Sciences of the Netherlands Organization for
Scientific Research (NWO-CW) Grant 97-033 (to W.J.vV.), and by an EMBO
Short Term Fellowship ASTF 9781 (to S. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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