Nuclear RanGTP is not required for targeting small nucleolar RNAs to the nucleolus

Abstract

The small GTPase Ran is the central regulator of macromolecular transport between the cytoplasm and the nucleus. Recent work has suggested that RanGTP also plays an important role in regulating some intra-nuclear processes. In this study, we have investigated whether RanGTP is required for the intra-nuclear transport of RNAs. Specifically, we directly analyzed the nucleolar localization of Box C/D and Box H/ACA small nucleolar RNAs (snoRNAs)in mammalian (tsBN2) cells, Saccharomyces cerevisiae and Xenopus oocytes under conditions that deplete nuclear RanGTP and prevent RNA export to the cytoplasm. Our data suggest that depletion of nuclear RanGTP does not significantly alter the nucleolar localization of U3 snoRNA in tsBN2 cells. Complementary studies in the budding yeast S. cerevisiae using conditional Ran mutants as well as mutants in Ran regulatory proteins also indicate that disruption of the Ran gradient or of Ran itself does not detectably affect the nucleolar localization of snoRNAs. Finally, microinjection into Xenopus oocytes was used to clearly demonstrate that a specific pool of snoRNAs could still be efficiently targeted to the nucleolus even when the RanGTP gradient was disrupted by microinjection of mutant Ran proteins. Taken together, our data from three phylogenetically distinct experimental systems suggest that nuclear RanGTP,which is essential for trafficking of RNAs between the nuclear and cytoplasmic compartments, is not required for nuclear retention or nucleolar localization of snoRNAs.

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5′-cap structure

“…Instead, we found that T. vaginalis snRNAs have unmodified 59 termini. The biological significance of a TMG 59-cap structure on snRNAs is not fully understood; however, roles in trafficking and snRNP assembly have been proposed (Huber et al 2002;Mouaikel et al 2002;Narayanan et al 2003;Cougot et al 2004). Whether the lack of a 59 cap influences trafficking and/or assembly of T. vaginalis snRNAs awaits further analyses.…”

“…Hypermethylation of snRNA cap structures occurs in the cytoplasm in humans and in the nucleolus in yeast. The biological significance of the distinctive 59-cap structure found in snRNAs is unclear, but it has been suggested to play roles in snRNA compartmentalization and/or transport (Huber et al 2002;Mouaikel et al 2002;Narayanan et al 2003).…”

Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

“…mca38f3; Encor Biotechnology, Alachua, FL), Nop58 For sequential RNA and DNA fluorescence in situ hybridization, HeLa cells seeded on etched CELLocate coverslips (Eppendorf, Hamburg, Germany) were injected and fixed after 2 h in 4% formaldehyde, 10% acetic acid, and PBS for 10 min. The Cy3-labeled in situ oligonucleotide probe against human U3 snoRNA was used as previously described (Narayanan et al, 2003). The etched area of the CELLocate coverslips were imaged for U3 snoRNA, DAPIstained nuclei, and phase contrast, and then the DNA in situ hybridization was performed as stated above.…”

“…The Cy3-labeled in situ oligonucleotide probe against human U3 snoRNA was used as previously described (Narayanan et al, 2003). The etched area of the CELLocate coverslips were imaged for U3 snoRNA, DAPIstained nuclei, and phase contrast, and then the DNA in situ hybridization was performed as stated above.…”

Protein delivery using engineered virus-like particles

“…Nonviral methods, including microinjection and electroporation, can also be used to deliver proteins into cells (5)(6)(7). Both antibodies (8,9) and mutant proteins (10) have been delivered into cells via microinjection. However, microinjection is limited to a relatively small number of target cells and needs specialized equipment.…”