(Received for publication, February 14, 1995; and in revised form, August 17, 1995)
From the
Three novel genes encoding small RNAs homologous to human and mouse RNase P RNA have been isolated from a mouse genomic library. As assessed by Northern blot analysis and nuclease protection assays, transcripts derived from one or more of these genes are expressed in murine cells and tissues. The RNA products of these RNase P RNA-homologous genes are smaller in size (238-248 nucleotides) than the 305-nucleotide transcript previously identified. These smaller transcripts are uniformly less abundant than the larger RNase P RNA, but their expression varies severalfold among different mouse tissues. Similar short homologues of RNase P RNA also are expressed in rat, rabbit, and human cells. We conclude that higher vertebrates express multiple isoforms of RNase P RNA.
RNase P is a site-specific endoribonuclease that cleaves tRNA
precursor molecules to generate the 5` termini of mature tRNAs in both
prokaryotic and eukaryotic cells(1, 2, 3) .
The holoenzyme is a ribonucleoprotein, the RNA subunit of which, termed
RNase P RNA (RPR), ()exhibits considerable variability in
size among different species, ranging from 140 to 490 nucleotides in
length(4, 5, 6) . The RNA moiety alone, as
isolated from Escherichia coli or Bacillus subtilis,
is capable of catalyzing the site-specific cleavage reaction in
vitro in the absence of its apoprotein(7, 8) .
Eukaryotic RNase P, in contrast, requires assembly of the holoenzyme
for activity(9, 10, 11, 12) .
In yeasts, RNase P is compartmentalized; both the apoprotein and RNA components of mitochondrial RNase P are distinct and arise from different genes than those encoding components of the nuclear enzyme (4, 13, 14, 15, 16) . In Saccharomyces cerevisiae, the protein subunit of mitochondrial RNase P is derived from a nuclear gene(15, 16) , whereas the mitochondrial RPR is encoded within the mitochondrial genome(4) . In mammalian cells, RNase P activity is present in both nuclear and mitochondrial fractions(11, 17) , but only a single form of RPR has been identified(18, 19) , and no RPR-homologous sequences can be identified within the more compact mammalian mitochondrial genome.
The present study was designed to test the hypothesis that mammals express multiple isoforms of RPRs. We screened a mouse genomic library using a PCR-amplified mouse RPR sequence as the probe. Three new genes encoding homologues of RNase P RNA (RPRH) were isolated and sequenced. All of these encode RNA molecules with a high degree of sequence identity to the human and mouse RPR genes published previously(18, 19) but are smaller in size. Transcripts derived from one or more of these novel genes are expressed in murine cells, and multiple sizes of RPR-related transcripts are expressed in other mammalian species.
Figure 2: Nucleotide sequences of three RPRH genes, as compared with the previously published mouse RPR gene. Putative TATA boxes and transcription termination signals are underlined. Asterisks indicate the 5` and 3` termini of the RPRH4 transcript, as mapped by S1 nuclease protection assays. Nucleotide positions in the RPRH genes that differ from the published mouse RPR sequence are shown in lowercase, and nucleotides within the 5` and 3` flanking regions at which complete divergence begins are italicized.
Figure 1: Mouse genomic DNA blot. 10 µg of mouse DNA were digested by each of five restriction enzymes, as indicated, and hybridized with the mouse RPR cDNA probe. kb, kilobase pairs.
Eight phage clones were isolated by screening a
mouse genomic library. Restriction mapping and Southern blot
hybridization analysis indicated that these
clones contained
murine DNA inserts that could be grouped into four nonoverlapping
genomic sequences. Three of these clones included BamHI
fragments of 0.95, 2.3, and 5.2 kilobase pairs, respectively (data not
shown), identical in size to genomic BamHI fragments seen in
the original Southern blots (Fig. 1). Approximately 1 kilobase
pair from each of these three clones (termed RPRH2, RPRH3, and RPRH4)
were sequenced in both orientations. A portion of the sequence data is
shown in Fig. 2.
Figure 3: Northern blot of mouse RNAs. Upper panel, 10 µg of total RNA were loaded in each lane and hybridized with a 230-nt RPRH4 DNA probe. The sizes (nucleotide bases) of three major RNA bands are indicated. Lower panel, the same blot hybridized to a probe complementary to U1 RNA and used as a loading control.
To verify that the 238- and 248-nt RNAs are indeed transcribed from RPRH gene(s) and not generated by partial degradation of the 305 nt of RNase P RNA, we performed S1 nuclease protection mapping using two single-stranded antisense DNA probes prepared from the RPRH4 gene (Fig. 4). The 5` end protection probe, extending 210 nucleotides upstream from the EcoRI site, detected two transcription start sites (Fig. 4A). One of the start sites is located at 127/126 nt upstream from the EcoRI site, a position that corresponds to the 5` end of the 238-nt RNA transcript predicted from the RPRH4 gene. The other start site is located 135-138 nt from the EcoRI site, corresponding to the 5` end of a 248-nt RNA transcript predicted from RPRH4 gene. The 3` end protection probe, extending 347 nucleotides downstream from the EcoRI site, detected a RNA transcript ending 112 nt downstream from the EcoRI site (Fig. 4B), consistent with the predicted 3` end of RPRH4 transcript.
Figure 4:
S1 nuclease protection mapping of the
RPRH4 transcript. A, mapping of the 5` end. Lane 1,
probe only; lane 2, no RNA added but with 400 units of S1
nuclease; lanes 3-5, each reaction containing 30 µg
of mouse liver RNA, 10 cpm of the 5` end protection probe
and increasing amounts of S1 nuclease, as indicated. The sizes
(nucleotide bases) of the protected fragments are indicated. B, mapping of the 3` end. Lane 1, probe only; lane 2, no RNA added but with 400 units of S1 nuclease; lanes 3-6, each reaction contained 40 µg of mouse
liver RNA, 10
cpm of the 3` end protection probe, and
increasing amounts of S1 nuclease, as indicated. C, schematic
presentation showing the S1 nuclease protection probes and the 5` and
3` ends of RPRH4 transcripts protected from S1 nuclease digestion. The
sizes of the probes and the predicted 5` and 3` ends are indicated as
nucleotide bases. Cleavages sites at the termini or at sites of
sequences mismatched between the 305-nt RPR transcript and the probes
based on the RPRH4 sequence are indicated (open
triangles).
These fragments of probes based on the RPRH4 gene, when bound to their complementary RNA sequences from murine cells, were resistant to high concentrations of S1 nuclease, providing evidence that the 238- and 248-nt RNA species are not degradation products of the 305-nt RPR transcript but bona fide products of the RPRH4 gene. The 3` end analysis further supports this conclusion in that the predicted hybridization product formed between the RPRH4 probe, and the 305-nt RPR transcript (115 nt) is present and abundant at low concentrations of S1 nuclease (Fig. 4B) but disappears at the higher concentrations of S1 nuclease necessary to digest single base mismatches with the probe. Spatial relationships between these probes and transcripts derived from the RPR and RPRH4 genes are summarized in Fig. 4C.
Figure 5: Southern blot of genomic DNAs isolated from rat or rabbit kidney or human WI-38 cells. The rat (A) and rabbit (B) DNAs were hybridized with the mouse RPR cDNA probe. The human (C) DNA was hybridized with the human RPR cDNA probe. kb, kilobase pairs.
Northern blot hybridization using the mouse and human RPR sequence to probe RNAs isolated from rat, rabbit, and human cells also revealed at least two major RPR isoforms in each of these mammalian species: a larger RPR that corresponds to the 305 nt form in the mouse, and a smaller and less abundant form that corresponds to the RPRH transcripts we have identified in murine cells (Fig. 6). When human RNA and genomic DNA were probed with the mouse, instead of human, RPR sequence, only the RNase P RNA but not the RPR homologues was detected (data not shown), suggesting that the human RPRH is more diverged from the RNase P RNA than those in mouse, rat, and rabbit. In summary, these data indicate that most, if not all, mammalian species contain and express multiple genes encoding isoforms of RNase P RNA.
Figure 6: Northern blot of RNAs isolated from mouse, rat, or rabbit kidney or human HeLa cells. The mouse, rat, and rabbit RNAs were hybridized with the mouse RPR cDNA probe. The human RNA was hybridized with the human RPR cDNA probe.
Genes encoding an RNase P RNA were cloned previously from human and mouse and encode transcripts ranging from 305 nt in mice to 341 nt in humans(18, 19) . We have cloned three new murine genes homologous to the published RPR sequence but predicted to generate shorter RNA transcripts. Each of these RPRH genes includes a segment almost identical (88-92%) to the 5` region of the previously described mouse RPR sequence but truncated by the presence of a transcriptional termination signal at a position corresponding approximately to nt 240 of the 305-nt RPR.
Two lines of evidence
rule out the possibility that these genes are derived from a cloning
artifact. First, BamHI fragments of clones that contain
RPRH genes are matched in size to BamHI fragments detected in
a mouse genomic Southern blot. Second, two of the novel RPRH gene
sequences (RPRH2 and RPRH4) were present in two or more independent
clones containing overlapping mouse genomic DNA fragments and an
identical RPRH sequence. Other data demonstrate that the RPRH sequences
that we have identified are not pseudogenes. Transcripts corresponding
in size to the products predicted from RPRH genes are present in murine
cells, and nuclease protection experiments confirm that the shorter
forms of RPR-homologous RNA observed in Northern blots are not
degradation products of the 305-nt RPR. Short forms of RPR-homologous
RNA also are present in rat, rabbit, and human cells.
The structure and function of RNase P RNAs have been studied by computer modeling(28) , chemical cleavage(29) , nuclease protection (30) , and mutational or phylogenetic analyses(31, 32, 33, 34) . Although the size and sequence of RNase P RNAs have diverged considerably during evolution, a similar three-dimensional structure appears to be conserved(1, 2, 3, 19, 28, 32, 33) . The 305-nt mouse RPR is predicted to have the core structure common to other RNase P RNAs, which includes three major rings formed by internal base pairing(19) . The tertiary structure is established through base pairing between the rings(19) . Because of their shorter length, mouse RPRH transcripts may form only two of the three rings of the common RNase P RNA core structure.
The functional properties of RPRH transcripts in mammalian cells have not yet been determined. Truncated forms of human RPRs retain enzymatic activity in reconstitution assays in vitro(35) , suggesting that RPRH transcripts potentially serve as functional components of holoenzyme complexes. An interesting alternative possibility is that these naturally occurring truncated forms of RNase P RNA may be capable of binding to the apoprotein constituents of RNase P ribonucleoprotein complexes but are enzymatically inactive due to the disruption of the RNA tertiary structure. In this way, RPRH gene products could function as negative regulators of RNase P. It should be possible, in future studies, to assess the functional characteristics of the RPRH gene products that we have identified, as well as their protein subunit and subcellular distribution. Because yeast expresses different forms of RNase P RNA in mitochondria and the nucleus(4, 13, 14) , perhaps one or more of the RPRH transcripts will have a similarly compartmentalized function.
Comparison of the primary sequences of the previously defined mouse RPR gene and the new RPRH genes also suggests certain interesting possibilities concerning their evolutionary origins. An analysis using a Jotun Hein alignment method (DNASTAR), as shown in Table 1, indicates that all three RPRH genes are clearly related to each other but diverge considerably from the RPR gene downstream of their 3` transcriptional termination signals. Within the group of RPRH genes, RPRH2 has diverged from the RPRH3 and RPRH4 genes both within the transcribed regions and within flanking sequences, whereas the latter two genes are almost identical throughout the entire 1-kilobase pair region we sequenced. These comparisons suggest that the present diversity within this gene family has arisen from sequential gene duplication events.
In summary, three novel genes encoding sequences homologous to RNase P RNA have been isolated from a mouse genomic library. Transcripts derived from one or more of these RPRH genes are expressed in murine cells, and multiple sizes of RPR-related transcripts are present in other mammalian species as well. We conclude that higher vertebrates express multiple isoforms of RNase P RNA.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U31003 [GenBank](RPRH2), U31227 [GenBank](RPRH3), and U31228 [GenBank](RPRH4).