Nuclear Retention Elements of U3 Small Nucleolar RNA

Abstract

The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C′ and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5′ cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.

ACKNOWLEDGMENTS

We kindly thank Reinhard Lührmann (m^2,2,7G cap), Elsebet Lund (m⁷G cap), Joseph Gall (p80 coilin monoclonal antibody H1), and Michael Pollard and Eng Tan (fibrillarin monoclonal antibody 72B9) for providing antibodies used in this study. We are grateful to Claiborne V. C. Glover III and members of our laboratory for critical reading of the manuscript and to James Griffith, Thomas Eades, and Ellie Kalwerisky for assistance in generating many of the mutant templates used in this work.

This work was supported in part by a Basil O’Conner Starter Scholar Research Award from the March of Dimes Birth Defects Foundation and by a grant from the National Institutes of Health (GM54682) to M.P.T.