The RNase P Associated with HeLa Cell Mitochondria Contains an Essential RNA Component Identical in Sequence to That of the Nuclear RNase P

Abstract

The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant (ϳ17S) very similar to that of the nuclear enzyme (nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNA Ser(UCN) precursor [ptRNA Ser(UCN) ] at the correct site. Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component. Furthermore, electrophoretic analysis of 3-end-labeled nucleic acids extracted from the peak of glycerol gradientfractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P. The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs. In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to ϳ33 to ϳ175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles. In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell. The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.The unique mode of transcription of the mammalian mitochondrial DNA in the form of giant polycistronic molecules, containing tRNA sequences regularly interspersed between the individual rRNA and mRNA sequences and in most cases butt-joined to them (39,42,43), demands the existence of a complex RNA-processing apparatus. The tRNA sequences presumably function as signals for RNA-processing enzymes, which carry out the endonucleolytic cleavages that eventually lead to the formation of the mature rRNA, mRNA, and tRNA species (43). One of these enzymatic activities would be expected to cut precisely the polycistronic transcripts on the 5Ј side of each tRNA sequence and therefore to be analogous to the RNase P, an RNA-containing enzyme first identified in Escherichia coli (3), and subsequently found ubiquitously in prokaryotic and eukaryotic organisms (4). In previous work from this laboratory, an endoribonuclease which cleaves the precursor of the E. coli suppressor tRNA Tyr at the same site as E. coli RNase P, producing the mature 5Ј end of this tRNA, has been identified. The enzyme was partially purified from HeLa cell mitochondria (and is henceforth referred to as mtRNase P) (15). An analogous enzyme ...

Protein–RNA interactions in the subunits of human nuclear RNase P

“…We show that for the eight proteins on nuclear RNase P in HeLa cells that have been cloned and sequenced, four of these proteins interact directly with H1 RNA, the RNA subunit of the enzyme+ These proteins, Rpp21, Rpp29, Rpp30, and Rpp38, both genetically and biochemically are intimately in contact with the RNA subunit+ Although we cannot be certain that these are the only proteins involved in abetting the reaction with substrates, further tests of a reconstitution assay will prove this point (see below)+ There may be other proteins that are part of the enzyme and that we have not characterized yet as part of our characterization+ Certainly, Rpp25, identified in an early study of the enzyme (Eder et al+, 1997), has yet to be tested in the manner described here+ Furthermore, both RNase MRP (see also hpop5; van Eenennaam et al+, 2000; H+ van Eenennaam, pers+ comm+) and mitochondrial RNase P (Puranam & Attardi, 2001) have proteins that may be similar to the ones we report here and that should be tested in the ways we describe here+ Our results indicate a possible path to the reconstitution reaction+ Clearly, if there is a required order of reactions to develop this assay, it might very well be the formation of a complex between H1 RNA and these four proteins (Rpp21, Rpp29, Rpp30, Rpp38) with the final proteins to be added later (Fig+ 2)+ In fact, Figure 2 does represent a speculative model of the two-and three-dimensional scheme of the organization of the subunits as determined here and elsewhere (Jiang & Altman, 2001)+ Because most work on the isolated proteins has been done with his-tagged derivatives, only the purification of large scale amounts of native enzyme complete with untagged subunits might be suitable for reconstitution+ FIGURE 1. Immunoblot using polyclonal antibodies raised in rabbits against Rpp38 and Rpp21+ The enzyme is HeLa nuclear RNase P purified through glycerol gradients as described in Materials and Methods and loaded onto a composite 9%/15% polyacrylamide SDS gel and subsequently transferred to a nitrocellulose membrane+ Lanes 1-3: Samples before incubation with streptavidin agarose beads+ Lanes 5-7: Samples after incubation with streptavidin agarose beads (not bound)+ Lane 1: Control RNase P sample, no RNA added+ Lane 2: RNase P sample crosslinked to P3 domain of H1 RNA+ Lane 3: RNase P sample crosslinked to H1 RNA+ Lane 4: Protein size marker lane+ Lane 5: Control RNase P sample+ Lane 6: RNase P sample crosslinked to P3 domain of H1 RNA+ Lane 7: RNase P sample crosslinked to H1 RNA+ The relative position and size of proteins loaded on lane 4 is indicated+ Some nonspecific, high molecular weight bands can be seen+ …”

“…Unlike prokaryotic RNase P, which consists of one RNA and one protein subunits, eukaryotic nuclear RNase P (Hela nuclear RNase P) consists of many proteins associated with an essential RNA subunit+ This latter RNA subunit, H1 RNA, has not been shown to be catalytic in vitro (Altman & Kirsebom, 1999)+ No HeLa cell protein subunits have been identified as essential participants in catalytic function in a reconstitution assay for RNase P (Nichols et al+, 1988;True & Celander, 1998;Altman & Kirsebom, 1999), as has been done with the protein subunit from the enzyme of Escherichia coli (Kole & Altman, 1979;Guerrier-Takada et al+, 1983)+ However, these subunits may have a direct role in the catalytic activity of the enzyme+ Of course, some of the same protein subunits are also participants in the complex of eukaryotic RNase MRP (Lee et al+, 1996;Pluk et al+, 1999;van Eenennaam et al+, 2000) and possibly in mammalian mitochondrial RNase P (Puranam & Attardi, 2001), but no function of the subunits has yet been shown for these other organisms+ A critical tool in the analysis of the complex subunit architecture of the human nuclear RNase P holoenzyme and in the function of individual subunits is the ability to reconstitute active enzymes from these subunits+ Assembly of the holoenzyme may proceed through a well-defined series of steps that involve addition of particular subunits to form and then to add to a core structure+ On the presumption that H1 RNA, the RNA subunit of the enzyme, would be a component of the core structure, we have employed both a genetic (three-hybrid; Sengupta et al+, 1996) and a direct biochemical method (UV crosslinking) to determine which protein subunits may also be part of that core structure+ The results of these analyses, in conjunction with those of proteinprotein subunit interactions, have enabled us to construct a rough three-dimensional picture of the holoenzyme and to design a reconstitution assay+…”

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

“…Among them one can find systems with relatively low specificity for RNA transported (for example, tRNA import in trypanosomatids) (Schneider and Marechal-Drouard 2000), systems with several cytosolic RNA species specifically imported (like tRNA import into plant mitochondria) (Kumar et al 1996), as well as very specific, selective systems, usually with but few RNAs imported (the best-studied case-tRNA Lys CUU import in yeast) (Tarassov et al 1995;Entelis et al 1998;Kazakova et al 1999), one out of three imported yeast tRNA species (Rinehart et al 2005). The pathway of directing the 5S rRNA into human mitochondria is obviously an example of highly selective targeting systems, since this is a single RNA species described to be efficiently targeted into the human organelle, while data on possible import of two other RNAs-RNA components of RNase P and MRP-are still contradictory and their import efficiency seems very low (Kiss and Filipowicz 1992;Li et al 1994;Puranam and Attardi 2001). Although the selectivity of the 5S rRNA import has been demonstrated, no explanation for this specificity existed.…”

Human ribonuclease P: Subunits, function, and intranuclear localization

“…Extensive purification analysis of RNase P from HeLa cells has revealed that the nuclear form of this tRNA processing holoenzyme is composed of at least 10 protein subunits associated with a single RNA species, H1 RNA (Table 1; Eder et al+, 1997;)+ These protein subunits are designated Rpp14, Rpp20, Rpp21, Rpp25, Rpp29, Rpp30, Rpp38, Rpp40, hPop5, and hPop1 (Lygerou et al+, 1996b;Eder et al+, 1997;Jarrous et al+, 1998Jarrous et al+, , 1999avan Eenennaam et al+, 1999van Eenennaam et al+, , 2001)+ The tight association of these proteins with highly purified nuclear RNase P obtained by different purification schemes implies that they constitute the core structure of the holoenzyme )+ A recent study has shown that the mitochondrial form of HeLa RNase P possesses an RNA that is identical to the H1 RNA (Puranam & Attardi, 2001)+ This enzyme has a sedimentation coefficient of ;17S in glycerol gradients, compared to ;15S of the nuclear counterpart, and exhibits properties typical of a ribonucleoprotein enzyme (Puranam & Attardi, 2001)+ This finding contradicts earlier biochemical analyses that support the concept that human mitochondrial RNase P is not a ribonucleoprotein complex (Rossmanith et al+, 1995;Rossmanith & Karwan, 1998)+ These opposing findings provoke further debate on the biochemical nature of mitochondrial and other organellar RNase P enzymes (Frank & Pace, 1998;Schon, 1999;Altman et al+, 2000;Gegenheimer, 2000;Salavati et al+, 2001), and therefore more biochemical and genetic means should be applied to resolve this issue+ Nuclear RNase P purified from Saccharomyces cerevisiae has nine distinct protein subunits and genetic studies established that these subunits are essential for yeast viability and enzyme activity in tRNA processing (see Xiao et al+, 2001)+ A multi-subunit ribonucleoprotein has also been described for nuclear RNase P purified from Aspergillus nidulans (Han et al+, 1998)+ In addition to the protein subunits described above (Table 1), several other proteins transiently interact with RNase P in yeast and human cells+ As judged by twohybrid genetic screens in yeast, the subunit Rpp20 interacts with the heat shock protein Hsp27 and the subunit Rpp14 contacts several proteins, including the LIM domain protein 1 (LIMD1) and HSPC232 ; the latter is an SR-rich protein that exhibits partial similarity to splicing factors SC-35 and SRp46+ The interaction of Rpp20 with Hsp27 was verified by biochemical analysis )+ In S. cerevisiae, a complex of seven Sm-like proteins (Lsm2-8) is associated with the precursor RNA subunit, Rpr1, of nuclear RNase ...…”