Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C', box D, and the 3' terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.

Originally identified as an essential component of the herpes simplex virus immediate early (IE) gene enhancer complex, the transcriptional coactivator host cell factor-1 (HCF-1) has been implicated in a broad range of cellular regulatory circuits. The protein mediates activation through multiple interactions with transcriptional activators, coactivators, and chromatin remodeling complexes. However, the mechanisms involved in HCF-1-dependent transcriptional stimulation were undefined. By using a minimal HCF-1-dependent promoter and a model activator, the varicella zoster IE62 protein, it was determined that HCF-1 was not required for the assembly of the RNAPII basal complex, which depended solely on IE62 in conjunction with the cellular factor Sp1. In contrast, HCF-1 was required for recruitment of the histone methyltransferases Set1 and MLL1 (mixed-lineage leukemia 1), leading to histone H3K4 trimethylation and transcriptional activation. Similarly, in a varicella zoster virus lytic infection, HCF-1, Set1, and MLL1 were recruited to the viral genomic IE promoter, suggesting an essential role for HCF-1 in chromatin modification and remodeling during initiation of lytic infection. The results indicate that one biological rationale for the incorporation of the viral IE activators in the viral particle is to recruit HCF-1/histone methyltransferase complexes and promote assembly of the viral IE gene promoters into transcriptionally active chromatin. These studies also contribute to the model whereby the induced nuclear transport of HCF-1 in sensory neurons may be critical to the reactivation of latent herpesviruses by promoting the activation of chromatin modifications.chromatin ͉ histone methyltransferase ͉ chromatin modifications ͉ Sp1 ͉ transcription T he cellular transcriptional coactivator host cell factor-1 (HCF-1) was originally isolated as a component of the herpes simplex virus (HSV) immediate early (IE) gene enhanceosome complex containing the cellular POU domain protein Oct-1 and the viral transactivator VP16 (1-6). The protein has been most thoroughly studied in this context where it mediates the VP16 transcriptional activation of the viral IE genes (7,8). In an analogous manner, HCF-1 also mediates the induction of the related varicella zoster virus (VZV) IE genes by the viral transactivators ORF10 and IE62 (8). The transcription of these ␣-herpesvirus genes is regulated by multiple mechanisms and factors via complex combinatorial enhancer-promoter domains. Strikingly, HCF-1 has been shown to be essential for IE gene expression, suggesting that it mediates a common rate-limiting step. Most intriguingly, both HSV and VZV establish latency in the neurons of sensory ganglia. In these cells, HCF-1 is uniquely sequestered in the cytoplasm of sensory neurons and is rapidly transported to the nucleus upon stimulation that results in viral reactivation (9). Therefore, whereas the protein is essential for viral lytic replication, it may also be a key component in the ␣-herpesvirus latency reactivation cycle.Sinc...

The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3Ј hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5Ј or 3Ј hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.

U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5′ trimethylated guanosine cap, 13 pseudouridines, and 10 2′-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.