Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells.

Abstract

Abstract. The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These resuits demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA. cessing) was initially identified as an endoribonu-,Ik ~ clease that produces, in vitro, RNA primers for the initiation of mitochondrial DNA replication (Chang and Clayton, 1987a). When RNase MRP was subsequently discovered to be a ribonucleoprotein enzyme and its RNA (MRP RNA; see Fig. 1) component was sequenced (Chang and Clayton, 1987b), it turned out to be identical to a previously defined nucleolar RNA, termed 7-2 RNA (Reddy et al., 1981;Gold et al., 1989;Yuan et al., 1989). MRP RNA (7-2 RNA) is essential for RNase MRP enzymatic activity in vitro and is encoded by a nuclear gene (Chang and Clayton, 1987b;Yuan et al., 1989; Tooper and Clayton, 1990) that is transcribed by RNA polymerase III (Chang and Clayton, 1989). MRP RNA has been detected by in situ hybridization in both mitochondria and nucleoli of mouse cardiac myocytes (Li et al., 1994). The great majority of MRP RNA (7-2 RNA) in the cell fractionates with nucleoli (Reddy et al., 1981 Chu et al., 1994;Lygerou et al., 1994). In HeLa cells, nuclear MRP RNA is associated with large nucleolar structures sedimenting at N80 S (Kiss et al., 1992). MRP RNA-containing RNP particles can be immunoprecipitated by human To (Reddy et al., 1983), Th (Hashimoto and Steitz, 1983), and Wa (Reimer et al., 1988) sera (To, Th, and Wa representing patient codes for autoimmune sera that contain antibodies directed against the MRP RNA-containing RNP particle). The protein recognized by anti-To and anti-Wa antibodies is a ~40,000-mol wt nucleolar protein (Reimer et al., 1988). The 40,000-mol wt To autoantigen-binding domain was subsequently localized to nucleotides 21-64 of human MRP RNA (Yuan et al., 1991).In the present investigation, we have examined the subcellular localization of fluorescently tagged MRP RNA after microinjection into the nucleus of living cells. This method, which we term fluorescent RNA cytochemistry, is integrated with high re...

Worldwide mutation spectrum in cartilage-hair hypoplasia: ancient founder origin of the major70A→G mutation of the untranslated RMRP

“…12,28 Mutations in RMRP seem to avoid nucleotides 23 -62 which have been reported to be a nucleolar localisation signal region. 27 We found a compound heterozygous mutation at nucleotide 57 in one patient, but due to the sequence content of this duplication it does not change the sequence of the nucleolar localisation signal. On the other hand, the mitochondrial localisation signal region has been reported to reside between nucleotides 118 -175 in the mouse 29 which corresponds to nucleotides 118 -167 in the human RMRP.…”

“…In addition, we have characterised herein three insertions and duplications in the 5' end of the transcript, which form a third group of mutations in RMRP. These mutations are gathered around the nucleolar localisation signal region 27 and the regions reported to be important for binding the different proteins of the RNase MRP complex. 13 Only heterozygosity of insertion and further duplication mutations in the promoter region or 'null' alleles of RMRP were detected, further suggesting that expression of the RNase MRP RNA is indeed essential for life.…”

Distinct Effects of Topoisomerase I and RNA Polymerase I Inhibitors Suggest a Dual Mechanism of Nucleolar/Nucleoplasmic Partitioning of Topoisomerase I

“…This behavior of topo I closely resembles that of RNA polymerase I itself (21,25). Thus, topo I does not make an exception to the general rule that molecules are targeted to the nucleolus due to their interaction with one of the three nucleolar building blocks that are structured around rDNA and/or its transcripts (35)(36)(37). Even during mitosis, where rDNA is not transcribed, topo I remains associated with the corresponding chromosomal structures that harbor rDNA and RNA polymerase I (5,38,39).…”

An essential protein-binding domain of nuclear RNase P RNA

“…P3 domain comparisons from RNase P and RNase MRP RNAs+ A: Secondary structure of S. cerevisiae RNase P RNA based on phylogenetic and biochemical analysis (Tranguch et al+, 1994)+ The location of the P3 RNA domain is shown in bold+ The location and sequence of the inserted hybridization tag (Bertrand et al+, 1998) between positions 133 and 139 is shown in italics+ B: P3 RNA domains from RNase P and MRP are shown from five eukaryotes, with intraspecies nucleotide conservations in bold+ S. cerevisiae and S. pombe RNase P sequence is from Tranguch & Engelke (1993) and Krupp et al+ (1986); human, mouse, and X. laevis RNase P is from Pitulle et al+ (1998); S. pombe RNase MRP is from Paluh and Clayton (1995); S. cerevisiae, human, mouse, and X. laevis RNase MRP is from Schmitt et al+ (1993)+ Engelke, 1993;Paluh & Clayton, 1995;Pitulle et al+, 1998)+ The P3 domains from Saccharomyces cerevisiae RNases P and MRP have been shown to be functionally equivalent, because swapping domains between enzymes was permitted (Lindahl et al+, 2000)+ Minimization experiments using domain deletions from yeast RNase P RNA have shown the P3 domain to be essential for generating a functional enzyme (PaganRamos et al+, 1994)+ Recent studies have further identified the P3 domain, in both human RNase P and RNase MRP, as required for binding the To antigen, a 40-kDa protein immunoprecipitated by human autoimmune sera (Reddy et al+, 1983;Liu et al+, 1994;Eder et al+, 1997)+ Microinjection experiments using synthetic RNase MRP RNA result in nucleolar localization of the RNA, consistent with the role of RNase MRP in processing prerRNA (Jacobson et al+, 1995)+ Microinjected RNase P RNA alone localized to the nucleolus only transiently (Jacobson et al+, 1997)+ Transient nucleolar localization of RNase P RNA was dependent on the P3 domain, suggesting a potential role for P3 in nucleolar localization+ It is currently not clear whether RNase P is also a nucleolar enzyme, and to what extent this is consistent among species+ Yeast nuclear RNase P appears largely nucleolar (Bertrand et al+, 1998), and in situ studies of mammalian RNase P have indicated either transient nucleolar association (Jacobson et al+, 1997) or limited steady-state localization to a "perinuclear compartment" (PNC) located on the nucleolar periphery (Matera et al+, 1995;Lee et al+, 1996)+ It has not been clear from these data whether the role of the P3 domain in localization is to direct holoenzyme assembly, which in turn causes localization, or whether the RNA interacts directly with some...…”