Terminal Minihelix, a Novel RNA Motif That Directs Polymerase III Transcripts to the Cell Cytoplasm: TERMINAL MINIHELIX AND RNA EXPORT

Carole Gwizdek,Edouard Bertrand,Catherine Dargemont,Jean-Claude Lefebvre,Jean-Marie Blanchard,Robert H Singer,Alain Doglio

doi:10.1074/jbc.m100493200

Abstract

Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport. We have characterized the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic RNA transcribed by RNA polymerase III. Using a large panel of VA1 mutants in both transfected COS cells and injected Xenopus oocytes, we showed that the terminal stem of VA1 is necessary and sufficient for its export. Surprisingly, we found that the nucleotide sequence within the terminal stem is not important. Rather, the salient features of this motif are its length and its relative position within the RNA. Such stems thus define a novel and degenerate cytoplasmic localization motif that we termed the minihelix. This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway.

Highlights

Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport
We focused on polymerase III transcripts,1 and we have analyzed in detail the adenovirus VA1 RNA (VA1)
Export of Stem17 was inhibited by an excess of VA1 competitor (Fig. 4, lane 6 versus lane 3), and, reciprocally, export of VA1 was blocked by an excess of Stem17 (Fig. 4, lane 12 versus lane 3). These competitions were specific because export of U1⌬Sm was not affected. These results demonstrated that artificial RNAs displaying a terminal stem with structural features similar to the one of VA1 were localized in the cytoplasm of both mammalian cells and Xenopus oocytes

Summary

TERMINAL MINIHELIX AND RNA EXPORT*

Such stems define a novel and degenerate cytoplasmic localization motif that we termed the minihelix This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway. Our study led us to identify a new cis-acting RNA export motif that we termed the minihelix motif This motif is encountered in a large family of small viral and cellular transcripts, which all are transcribed by pol III

EXPERIMENTAL PROCEDURES

Nuclear export in Xenopus oocytesc

RESULTS

DISCUSSION