Nuclear domains of the RNA subunit of RNase P

Marty R. Jacobson 2 Long-Guang Cao 2 3 Krishan Taneja 1 Robert H. Singer 0 1 Yu-li Wang 2 Thoru Pederson 2 0 Present address: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine , Bronx, NY 10461-1975 , USA 1 Department of Cell Biology, University of Massachusetts Medical Center , Worcester, MA 01655 , USA 2 Cell Biology Group, Worcester Foundation for Biomedical Research , Shrewsbury, MA 01545 , USA 3 Present address: Department of Molecular Genetics and Microbiology, University of Florida , PO Box 100266, Gainesville, FL 32610 , USA The ribonucleoprotein enzyme RNase P catalyzes the 5 processing of pre-transfer RNA, and has also recently been implicated in pre-ribosomal RNA processing. In the present investigation, in situ hybridization revealed that RNase P RNA is present throughout the nucleus of mammalian cells. However, rhodamine-labeled human RNase P RNA microinjected into the nucleus of rat kidney (NRK) epithelial cells or human (HeLa) cells initially localized in nucleoli, and subsequently became more evenly distributed throughout the nucleus, similar to the steadystate distribution of endogenous RNase P RNA. Parallel microinjection and immunocytochemical experiments revealed that initially nucleus-microinjected RNase P RNA localized specifically in the dense fibrillar component of the nucleolus, the site of pre-rRNA processing. A mutant RNase P RNA lacking the To antigen binding domain (nucleotides 25-75) did not localize in nucleoli after nuclear SUMMARY Ribonuclease P (RNase P) is a phyletically ubiquitous ribonucleoprotein enzyme responsible for the endonucleolytic cleavage of transfer RNA (tRNA) precursor molecules, generating the 5 termini of mature tRNAs (Altman et al., 1988; Baer et al., 1989; Pace and Smith, 1990; Altman, 1990; Darr et al., 1992), a process which takes place predominantly in the nucleus of eukaryotic cells (Melton et al., 1980). Although it has not yet been purified as an intact enzyme, mammalian RNase P contains a ~340 nt RNA molecule and its buoyant density suggests that it may be greater than 50% protein (Bartkiewcz et al., 1989). The RNA subunits of several mammalian RNase P enzymes have been identified and cloned (Altman et al., 1993), and human RNase P RNA was shown to be transcribed from a single copy gene by RNA polymerase III (Baer et al., 1990; Hannon et al., 1991). In contrast to the RNA subunit of prokaryotic RNase P, mammalian RNase P RNA is not catalytically active in vitro in the absence of the RNase P protein component(s) (for a review see Darr et al., 1992). A nucleolar ribonucleoprotein enzyme closely related to RNase P is RNase MRP, an endoribonuclease that participates in pre-ribosomal RNA processing (Schmitt and Clayton, 1993; microinjection. In contrast, a truncated RNase P RNA containing the To binding domain but lacking nucleotides 89341 became rapidly localized in nucleoli following nuclear microinjection. However, unlike the full-length RNase P RNA, this 3 truncated RNA remained stably associated with the nucleoli and did not translocate to the nucleoplasm. These results suggest a nucleolar phase in the maturation, ribonucleoprotein assembly or function of RNase P RNA, mediated at least in part by the nucleolar To antigen. These and other recent findings raise the intriguing possibility of a bifunctional role of RNase P in the nucleus: catalyzing pre-ribosomal RNA processing in the nucleolus and pre-transfer RNA processing in the nucleoplasm. Chu et al., 1994; Lygerou et al., 1994). Although the RNA components of human RNase P and RNase MRP have only limited sequence homology (Gold et al., 1989), they can be folded into similar theoretical caged pseudoknot structures (Foster and Altman, 1990; Schmitt et al., 1993). Human RNase P and RNase MRP share at least two protein components, the 40 kDa To (or Th) antigen (Reddy et al., 1983; Liu et al., 1994) and the ~100 kDa Pop1 protein (Lygerou et al., 1996). Both enzymes cleave substrate RNAs to generate 5 phosphates and 3 hydroxyl termini in a divalent cation-dependent reaction, and both are capable of cleaving the same substrate RNA in vitro (Potuschak et al., 1993). These findings and other considerations suggest a close evolutionary relatedness of these two ribonucleoprotein enzymes (Morrissey and Tollervey, 1995; Lygerou et al., 1996). Candidate protein components of mammalian RNase P have recently been described (Eder et al., 1997). In bacteria, which have only the RNase P enzyme, a transfer RNA element is located in the pre-ribosomal RNA spacer region between the 16 S and 23 S rRNA sequences which can act as a substrate for RNase P. Significantly, this region corresponds to ITS1 in eukaryotes, which contains the RNase MRP cleavage site A3 (reviewed by Morrissey and Tollervey, 1995). Recently, Chamberlain et al. (1996) have shown that mutation of conserved positions of the Saccharomyces cerevisiae RNase P RNA subunit (T315D T307) negatively affects pre-ribosomal RNA processing. In addition to this recent finding, there have been several clues that RNase P, in addition to its role in pretransfer RNA processing, might also play a role in eukaryotic pre-ribosomal RNA processing (reviewed by Clayton, 1994). Little is known about the nuclear sites of RNase P ribonucleoprotein assembly or function. In the present investigation we have employed in situ hybridization and fluorescent RNA cytochemistry techniques to examine the intranuclear localization of RNase P RNA. Our results suggest that the RNA subunit of RNase P has a nucleolar association in its RNA processing, ribonucleoprotein assembly or function, in accord with recent findings suggesting an involvement of RNase P in preribosomal RNA processing, but that RNase P RNA also exists throughout the nucleoplasm as well, where it presumably is involved in pre-transfer RNA processing. The concept emerges of a bifunctional role of RNase P in the nucleus. MATERIALS AND METHODS The methods used were essentially as described previously (Wang et al., 1991; Jacobson et al., 1995) with only minor modifications. Human RNase P RNA was transcribed from DraI digested plasmid pGEM1/H1 (Bartkiewicz et al., 1989) using T7 RNA polymerase (Gibco/BRL, Bethesda, MD). A RNase P To domain RNA, consisting of only nucleotides 1-88 of human RNase P RNA was transcribed from BfaI digested plasmid pGEM1/H1 using T7 RNA polymerase. A mutant RNase P RNA lacking the To antigen binding domain was transcribed using T3 RNA polymerase (Gibco/BRL) from Ecl136II digested plasmid pMH1-1. Plasmid pMH1-1 was constructed by deleting the KpnI-XbaI fragment (vector multiple cloning site sequences) from a mouse RNase P RNA clone originally constructed by Kathleen Collins and Carol Greider (Cold Spring Harbor Laboratory) and kindly given to us by Sidney Altmans laboratory (Yale University). This clone consists of nucleotides 61-297 of the mouse RNase P RNA sequence (Altman et al., 1993; corresponding to nucleotides 71-329 (...truncated)