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.
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.
Granneman
Sander
S
161 Department of Biochemistry, University of Nijmegen, P. O. Box 9101, The Netherlands.
Pruijn
Ger J M
GJ
Horstman
Wendy
W
van Venrooij
Walther J
WJ
Luhrmann
Reinhard
R
Watkins
Nicholas J
NJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020537:RNA, Small Nucleolar / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid
D020537:RNA, Small Nucleolar; C117378:RNA, U3 small nucleolar; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins