A common sequence motif determines the Cajal body-specific localization of box H/ACA scaRNAs

Abstract

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., ...

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

“…Earlier experiments showed that the CAB box of U85 scaRNA is required for appropriate localization to the CB (Richard et al, 2003). Furthermore, localization of human scaRNA ACA57 to the CB was recently shown to require binding of a conserved WD40 protein to the CAB box (Tycowski et al, 2009).…”

“…Modification takes place in the nucleus after import of the U5 snRNA from the cytoplasm . CB localization of U85 scaRNA was originally demonstrated by fluorescent in situ hybridization (FISH) in mammalian and Drosophila cultured cells (Darzacq et al, 2002;Richard et al, 2003), and subsequently in various tissues of the fly (Liu et al, 2006b(Liu et al, , 2009.…”

“…Modification takes place in the nucleus after import of the U5 snRNA from the cytoplasm . CB localization of U85 scaRNA was originally demonstrated by fluorescent in situ hybridization (FISH) in mammalian and Drosophila cultured cells (Darzacq et al, 2002;Richard et al, 2003), and subsequently in various tissues of the fly (Liu et al, 2006b(Liu et al, , 2009.CBs are small nuclear bodies found in a wide variety of eukaryotic organisms, including mammals, amphibians, insects, and plants. They are most often recognized by immunostaining for the marker protein coilin Raska et al, 1991) and by their unusually high concentration of splicing snRNPs relative to the rest of the nucleus (Carmo-Fonseca et al, 1991;Matera and Ward, 1993;Spector, 1993).…”

TCAB1: Driving telomerase to Cajal bodies

“…An important insight into the mechanism of H/ACA RNA segregation between these two nuclear compartments is that scaRNAs, but not snoRNAs, contain a conserved four-base sequence called a Cajal body (CAB) box that is required for Cajal body localization. 7 It has been hypothesized that this sequence is recognized by a specific CAB box-binding protein that directs scaRNAs to and/or retains scaRNAs in Cajal bodies. Mutations in the CAB box of a scaRNA mislocalizes it to the nucleolus.…”

“…Mutations in the CAB box of a scaRNA mislocalizes it to the nucleolus. 7 Although TERC is not thought to function in spliceosomal RNA modification, TERC is a scaRNA in that it contains both H/ACA and CAB box sequences (Fig. 1B), is bound by dyskerin, and localizes to Cajal bodies.…”