Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were ®rst described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize speci®cally to Cajal bodies. The small Cajal bodyspeci®c RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site-speci®c synthesis of 2¢-O-ribose-methylated nucleotides and pseudouridines in the RNA polymerase II-transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post-transcriptional modi®cation of spliceosomal snRNAs.

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

Telomerase is a ribonucleoprotein reverse transcriptase that uses its RNA component as a template for synthesis of telomeric DNA repeats at the ends of linear eukaryotic chromosomes. Here, fluorescence in situ hybridization demonstrates that in HeLa cancer cells, human telomerase RNA (hTR) accumulates in the nucleoplasmic Cajal bodies (CBs). Localization of transiently expressed hTR to CBs is supported by a short sequence motif (411-UGAG-414) that is located in the 3′-terminal box H/ACA RNA-like domain of hTR and that is structurally and functionally indistinguishable from the CB-specific localization signal of box H/ACA small CB-specific RNAs. In synchronized HeLa cells, hTR shows the most efficient accumulation in CBs during S phase, when telomeres are most likely synthesized. CBs may function in post-transcriptional maturation (e.g., cap hypermethylation of hTR), but they may also play a role in the assembly and/or function of telomerase holoenzyme.

Box H/ACA RNAs represent an abundant, evolutionarily conserved class of small noncoding RNAs. All H/ACA RNAs associate with a common set of proteins, and they function as ribonucleoprotein (RNP) enzymes mainly in the site-specific pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Some H/ACA RNPs function in the nucleolytic processing of precursor rRNA (pre-rRNA) and synthesis of telomeric DNA. Thus, H/ACA RNPs are essential for three fundamental cellular processes: protein synthesis, mRNA splicing, and maintenance of genome integrity. Recently, great progress has been made toward understanding of the biogenesis, intracellular trafficking, structure, and function of H/ACA RNPs.

Biogenesis of functional spliceosomal small nuclear RNAs (snRNAs) includes the post-transcriptional covalent modi®cation of numerous internal nucleotides. We have recently demonstrated that synthesis of 2¢-O-methylated nucleotides and pseudouridines in the RNA polymerase II-synthesized Sm snRNAs is directed by sequence-speci®c guide RNAs. Here, we provide evidence supporting the notion that modi®ca-tion of Sm snRNAs occurs in nucleoplasmic Cajal bodies (CBs), where modi®cation guide RNAs accumulate. We show that short fragments of Sm snRNAs are correctly and ef®ciently modi®ed when targeted to CBs, but not when these same fragments are targeted to the nucleolus. We also demonstrate that internal modi®cation of the U2 snRNA occurs exclusively after nuclear import of the newly assembled Sm snRNP from the cytoplasm. Finally, we show that p80 coilin, the CB marker protein, is not required for snRNA modi®cation. In coilin knockout cells, Sm snRNAs and their modi®cation guide RNAs colocalize in residual CBs, which do not stockpile ®brillarin and fail to recruit the U3 small nucleolar RNA.

To better understand intranuclear-targeting mechanisms, we have studied the transport of U3 snoRNA in human cells. Surprisingly, we found that PHAX, the snRNA export adaptor, is highly enriched in complexes containing m7G-capped U3 precursors. In contrast, the export receptor CRM1 is predominantly bound to TMG-capped U3 species. In agreement, PHAX does not export m7G-capped U3 precursors because their caps become hypermethylated in the nucleus. Inactivation of PHAX and CRM1 shows that U3 first requires PHAX to reach Cajal bodies, and then CRM1 to be routed from there to nucleoli. Furthermore, PHAX also binds the precursors of U8 and U13 box C/D snoRNAs and telomerase RNA. PHAX was previously shown to discriminate between small versus large RNAs during export. Our data indicate that the role of PHAX in determining the identity of small RNAs extends to nonexported species, and this appears critical to promote their transport within the nucleus.

Post-transcriptional synthesis of 2¢-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modi®cation in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and speci®cally targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-speci®c localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5¢ and 3¢ hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism. Keywords: box C/D RNA/box H/ACA RNA/Cajal body/ RNA localization/scaRNA IntroductionIn eukaryotes, most aspects of the nuclear biogenesis of cellular RNAs can be linked to distinct subnuclear domains, also called nuclear organelles (Lamond and Earnshaw, 1998;Misteli and Spector, 1998;Lewis and Tollervey, 2000). The structural and functional compartmentalization of the nucleus requires speci®c targeting of many RNAs to their correct destination. Therefore, intranuclear RNA transport plays a fundamental role in eukaryotic gene expression. However, in contrast to the extensively studied nucleo-cytoplasmic RNA transport, little is known about the principles governing RNA localization within the nucleus. In this study, we investigate the mechanism directing speci®c localization of small Cajal body (CB)-speci®c RNAs (scaRNAs) to the nucleoplasmic CBs.CBs are evolutionarily conserved nucleoplasmic organelles that contain many protein and ribonucleoprotein (RNP) factors involved in mRNA and rRNA biogenesis (Bohmann et al., 1995;Matera, 1999;Gall, 2000;Dundr and Misteli, 2001;Ogg and Lamond, 2002). The list of proteins and RNPs detected in CBs includes RNA polymerase I, II and III subunits, basal transcription factors, small nuclear RNPs (snRNPs) mediating mRNA splicing and small nucleolar RNPs (snoRNPs) required for rRNA modi®cation and nucleolytic processing. CBs, however, do not contain nascent mRNAs and rRNAs, indicating that neither synthesis nor processing of these RNAs occur in this organelle. Instead, the available data are most consistent with the idea that the major function of CBs is in maturation, assembly and/or traf®cking of snRNPs, snoRNPs and transcription complexes which later function in mRNA and rRNA biogenesis in other nuclear compartments (Gall et al., 1999;Sleeman and Lamond, 1999;Gall, 2001;Carmo-Fonseca, 2002;Ogg and Lamond, 2002;Verheggen et al., ...

Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10 -40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance. INTRODUCTIONTelomeres are specific nucleoprotein complexes protecting the termini of eukaryotic linear chromosomes from degradation, end-to-end fusion and undesired recombination (for recent reviews, see Wong and Collins, 2003;Ferreira et al., 2004;Smogorzewska and de Lange, 2004;Blasco, 2005). In humans, the DNA component of telomere is made up of ϳ5-15 kb of double-stranded 5Ј-TTAGGG-3Ј repeats that terminates in a 3Ј overhang of ϳ50 -300 bases (Makarov et al., 1997;Wright et al., 1997). By invading into proximal double-stranded telomeric sequences, the single-stranded overhang supports formation of a large duplex loop, termed the T-loop (Griffith et al., 1999). Because of the unidirectionality of conventional DNA polymerases, the ends of telomeres cannot be fully duplicated and human telomeres lose ϳ50 -200 base pairs during each cell division cycle and eventually, critically short telomeres induce cell cycle arrest called proliferative senescence. Therefore, by imposing a limit on the replicative life span of somatic cells, telomere erosion represents an innate mechanism for tumor suppression (Smogorzewska and de Lange, 2004).The replicative telomere erosion can be balanced by the telomerase reverse transcriptase that adds telomeric DNA repeats to the 3Ј overhang of telomeres (reviewed in Collins and Mitchell, 2002;Cong et al., 2002). In humans, telomerase activity is not detectable in most somatic cells, but germ line and other highly proliferative cells as well as the majority of tumor-derived cell lines possess highly active telomerase, indicating that maintenance of telomere length is necessary for indefinite proliferation of human cells (Kim et al., 1994;Greider, 1996;Shay and Bacchetti, 1997). Telomerase is a ribonucleoprotein (RNP) enzyme that is minimally composed of the telomerase RNA (TR) that spec...