RNA import elements for transport into mitochondria

The invention relates to small RNAs encoded within the nucleus of mammalian cells that specifically import to the mitochondria. The RNAs bind to several nucleolar peptides and thus provide potential carriers for import of biological molecules, including metabolites and proteins, into the mitochondrial compartment. Mitochondrial dysfunction in several maternally inherited human diseases may be correctable employing linkage of mitochondrial import signal to mitochondrial tRNA sequences expressed from nuclear trans-genes without requirement for direct genetic transformation of mitochondria.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to the field of molecular biology 
and to its application in the utilization of nucleic acid transcripts 
which import biological molecules from the cell cytoplasm into 
mitochondria. The invention also concerns vectors incorporating DNA 
encoding the transcripts and various peptide/RNA mitochondrial import 
complexes. The nucleic acid segments copy RNA molecules which can 
transport additional RNA sequences, peptides or DNA molecules into the 
mitochondrial matrix. Also disclosed are methods of gene therapy directed 
at mitochondrial gene defects. 
2. Description of Related Art 
Mitochondria have been recognized as having a role in certain diseases and 
may be directly related to some aspects of the ageing process. An 
increased understanding of the function of mitochondrial genes in relation 
to the nuclear genes has therefore been the subject of much interest and 
research. 
Current views of mitochondria hold that mitochondria import most small 
molecules and proteins from the cytoplasm. There is some evidence that RNA 
may also be imported, although the mechanism is not clear (Vestweber and 
Schatz, 1989). 
The mammalian mitochondrial genome is different from nuclear genes in both 
structure and function. The human mitochondrial genome is small and 
economically packaged, lacking the large intervening sequences of 
non-coding DNA (introns) that constitute most of the nuclear genome. 
Mitochondrial amino acids are determined by a different genetic code than 
those in nuclear genes. Numerous other differences exist between nuclear 
and mitochondrial genes; for example, the monocistronic transcription from 
individual promoters and the extensive post transcriptional cleavage and 
ligation steps. Characteristic of mRNA synthesis directed by nuclear genes 
mitochondrial DNA is transcribed as a single polycistronic message 
subsequently cleaved to produce individual transfer, ribosomal and 
messenger RNA transcripts. 
Mitochondrial biogenesis requires the participation of two distinct genetic 
compartments: the nuclear genome that contributes the vast majority of 
mitochondrial proteins and the mitochondrial genome that contributes 13 
protein subunits to inter membrane enzymes of the respiratory chain 
(Anderson et al., 1981; Bibb et al., 1981). With the exception of two 
ribosomal RNA subunits and a complete set of tRNA species, the gene 
products necessary for replication transcription and translation of 
mitochondrial genes in cells of higher eukaryotes are derived entirely 
from the nucleus (Kruse et al., 1989; Parisi and Clayton, 1991; Attardi 
and Schatz, 1988). The set of nuclear genes required for replication and 
expression of the mitochondrial genome appears to include not only protein 
coding genes but loci that encode small RNA transcripts. Nuclear-encoded 
tRNAs have been observed in mitochondria from lower eukaryotes (Mottran et 
al., 1991; Nagley, 1989) and mitochondrial RNaseP in mammalian cells may 
include a nuclear encoded RNA subunit (Doersen et al., 1985). Even more 
definitive evidence for the participation of small RNA transcripts of 
nuclear origin in essential mitochondrial functions lies in the discovery 
of a mammalian RNase MRP that is presumed to generate primers for 
mitochondrial DNA replication (Chang and Clayton, 1987a; 1987b). This 
enzyme requires an RNA subunit (MRP-RNA) encoded by a single copy nuclear 
gene that is highly conserved among the mammalian species (Chang and 
Clayton, 1989; Yuan et al., 1989; Gold et al., 1989). 
Most of the total cellular pool of MRP-RNA is localized within nucleoli 
(Reimer et al., 1988) but a small fraction of MRP-RNA partitions to 
mitochondria (Chang and Clayton, 1987b). The nuclear and cytoplasmic 
enzymes associated with MRP RNA exhibit some distinctions in RNase 
activity (Karwan et al., 1991). The mitochondrial RNase MRP cleaves a 
mitochondrial RNA substrate (transcribed in vitro) at a unique site 
between conserved sequence blocks (CSB) II and III. Mutations in CSB II 
and III severely inhibit the cleavage (Bennett and Clayton, 1990). In 
contrast, nuclear RNase MRP cleaves the same RNA substrate in multiple 
sites. Variations in the enzymatic properties of mitochondrial and nuclear 
RNases that contain identical MRP-RNA subunits are attributable to 
differences in apoprotein components (Karwan et al., 1991). 
The mitochondrial inner membrane is impermeable to charged molecules so 
that transport of metabolites and proteins is accomplished by specialized 
carriers for small molecules (Aquila et al., 1987) and by an elaborate 
import apparatus for proteins (Sollner et al., 1991; Manning-Krieg et al., 
1991). The partitioning of MRP-RNA to the mitochondrial compartment after 
transcription within the nucleus suggests the existence of a pathway by 
which RNA transcripts exit the nucleus and are imported across both the 
outer and inner mitochondrial membranes to the site of holoenzyme assembly 
within the mitochondrial matrix. However, such an import pathway has not 
been elucidated, nor has there been identification of sequence-specific 
targeting that might provide a signal for mitochondrial import. 
The variable proportion of mutant mitochondrial genomes per cell results in 
cells with a range of bioenergetic capacities. Moreover, the expression of 
the whole genome is essential for the maintenance of mitochondrial 
bioenergetic function. Despite this knowledge of mitochondrial gene 
structure and of the biochemical steps involved in mitochondrial gene 
expression, relatively little is known about processes that regulate the 
expression of mammalian mitochondrial genes. 
Mitochondrial function has been the subject of numerous studies, both in 
energy regulation and as a source of DNA mutations that may contribute to 
aging and degenerative diseases. Age dependent increases in deleted 
mitochondrial DNA, for example, have been found in the human heart 
(Hattori et al., 1991). Certain diseases such as Parkinson's disease, 
appear to be closely related to aging. Deletions in aging heart tissue are 
similar to those found in some Parkinson's patients and it has been 
speculated that some factors that accelerate mitochondrial DNA mutations 
may contribute to both Parkinson's disease and cardiomyopathy. 
Other diseases and conditions may also be associated with defects in 
mitochondrial DNA. These include Kearns-Sayre syndrome and retinitis 
pigmentosa, ataxia, seizures, dementia and proximal muscle weakness 
(Grossman, 1990). A single base change in human mitochondrial DNA has been 
correlated with the appearance of Leber Hereditary Optic Neuropathy 
(LHON). LHON is a form of central optic nerve death resulting in blindness 
in affected individuals at a relatively early age, typically in their 
early twenties. 
The identification of a region of RNA that may serve as a mitochondrial 
targeting signal has potential clinical significance. Several maternally 
inherited human diseases are associated with deletions and point mutations 
in the mitochondrial genome (Holt et al., 1988; Wallace et al., 1988; 
Shoffner et al., 1990; Goto et al., 1990). For example, myoclonic epilepsy 
and ragged-red fiber disease (MERRF) and mitochondrial myopathy, 
encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) are 
attributable to single base substitutions in tRNA.sup.Lys and 
tRNA.sup.Leu, respectively (Shoffner et al., 1990; Goto et al., 1990). The 
tRNA.sup.Lys mutation causes a general reduction in mitochondrial protein 
synthesis (Chomyn et al., 1991). 
Prospects for gene therapy directed at mitochondrial gene defects are 
limited currently by the absence of methods for efficient introduction of 
foreign genetic material into mitochondria (discussed by Lander and 
Lodish, 1990). Mitochondrial dysfunction in cells of MERRF and MELAS 
patients may be correctable by linkage of a mitochondrial import signal to 
mitochondrial tRNA sequences expressed from nuclear trans-genes, without a 
requirement for direct genetic transformation of mitochondria. 
SUMMARY OF THE INVENTION 
The present invention seeks to address the problems and needs inherent in 
the prior art by elucidating RNA transcripts with unique mitochondrial 
transport functions. Identification of the novel RNA transcripts provides 
routes to novel therapies directed toward mitochondrial gene defects and 
dysfunction. The invention also includes novel peptide/nucleic acid 
compositions, RNA transport elements and methods of introducing genetic 
material into the mitochondrion. 
The inventors have discovered MRP-RNA transcripts that import to the 
mitochondria. Specific regions of a MRP-RNA gene have been identified that 
are essential for import of small RNA transcripts into the mitochondria 
but at the same time are dispensable for transcription, nuclear 
partitioning or stability of the transcript in the cytoplasmic 
compartment. The inventors have shown that specific sequence elements are 
required to direct RNA to a mitochondrial import pathway. 
It has been found that transcriptional control elements sufficient to 
direct MRP gene transcription reside within the proximal 700 bp of 5' 
flanking DNA. This upstream region of mouse MRP-RNA gene includes an array 
of regulatory motifs similar to those of the U6 and 7SK RNA genes. 
Deletion of either the putative distal or proximal sequence elements from 
the MRP-RNA promoter eliminates transcription. This places the MRP-RNA 
gene with U6 and 7SK RNA as a member of the set of POLIII transcribed 
genes controlled by upstream sequences without a requirement for internal 
elements governing transcription. 
In particular, the inventors have isolated an RNA transcript which 
functions as a mitochondrial import carrier. The transcript is encoded 
within the mid-portion of mammalian MRP-RNA gene between coding region 
nt100 and nt200 (SEQ ID NO: 1, mouse; SEQ ID NO: 8, human); more 
particularly, between coding region nt118 and 175 (SEQ ID NO: 9). Part of 
this region, namely the region between nt144 and nt156 (SEQ ID NO: 3) 
includes an evolutionary conserved sequence within a flexible base pairing 
region and a stable stem loop. The stem loop is preserved in all forms of 
MRP-RNA that import efficiently into mitochondria. These structural 
features of the RNA segment between nt118 and nt175 (SEQ ID NO: 9) of 
MP-RNA may provide a recognition signal for proteins that service carriers 
in the pathway between the nucleus and the mitochondrial matrix. 
Therefore, mitochondrial carriers are likely not limited to transcripts 
defined by the complete region between nt118 and nt175 (SEQ ID NO: 9), but 
may include shorter regions. Such regions are expected to include the 
highly conserved sequence between nt144 to nt156, as well as some 
structural features enabling the formation of a stable stem loop. Thus RNA 
transcripts comprising relatively short regions of at least 10 base pairs 
or longer regions of 20, 30, 40 base pairs up to 100 base pairs or longer 
which include this conserved sequence may be expected to function as 
carriers between the nucleus and the mitochondria. 
A further aspect of the invention is the discovery that particular 
mitochondrial import RNA transcripts are stabilized by binding with 
nucleolar proteins. The inventors have discovered that reduced stability 
occurs in some mutant RNA transcripts. Such unstable transcripts lack the 
region of MRP-RNA containing the binding site for To/Th antigen. It is 
known that To/Th antigen has high affinity for MRP-RNA and apparently 
confers stability upon binding with the RNA. Impaired mitochondrial 
partitioning of certain MRPF mutants may therefore be attributable to 
failure of the transcript to be recognized by a protein carrier of a 
mitochondrial import apparatus and lability may be due to defective 
binding of a protein that stabilizes the transcript within the nucleolar 
compartment. Binding sites for protein stabilization sequences could be 
engineered into import RNA sequences. 
In further embodiments, the present invention contemplates compositions 
comprising nucleic acid segments, i.e., DNA segments or RNA segments, 
which have a sequence in accordance with or complementary to SEQ ID NO: 1 
or 8 or to segments of SEQ ID NO: 1 or 8 which represents part of MRP-RNA 
gene. Certain nucleic acid segments comprising the sequence between coding 
region nt100 and nt200 are useful as DNA probes of the MRP-RNA gene. 
Probes selected from segments of the MRP-RNA gene between nt100 and nt200 
are useful for identifying specific regions of that coding region. One 
may, for example, wish to identify shorter or longer regions of targeting 
RNA transcripts in order to modify import properties. One generally wishes 
to select probes at least 10 base pairs in length up to the length of the 
mitochondrial import RNA transport region encoded within the mid-portion 
of the mammalian MRP-RNA gene which is 100 base pairs in length. 
In addition to utility as probes for isolating and identifying regions of 
RNA import transcripts, DNA segments may be employed as primers to amplify 
particular regions of interest of the MRP-RNA gene. In preferred 
embodiments, one may employ different oligonucleotide primers for such 
amplifications. Deletion mutants, for example, may be generated by PCR 
primer-guided synthesis. DNA sequences complementary to isolated RNA 
transcripts are readily generated based on techniques known to those in 
the art. Typically employed primers are from 10 to 30 base pairs in 
length, although most preferable lengths are 15-25 base pairs. 
Further aspects of the invention contemplate stabilized complexes between 
the disclosed RNA transcripts and cellular peptides. Such peptides will 
bind to the RNA to confer stability. Cellular peptides may include 
nucleoplasmins, chaperonins, heat shock protein 70, signal recognition 
particles, .alpha.-lytic factor prosequence, ubiquitinated ribosomal 
proteins, trigger factor, sec B protein, Pap D protein or other unrelated 
classes of proteins that act as molecular chaperones (Ellis and van der 
Yies, 1991). A preferred complex is a peptide To/Th antigen bound to the 
MRP-RNA transcript according to SEQ ID NO: 1 or 8. 
Recombinant vectors and recombinant cells transformed with the recombinant 
vectors are also contemplated as part of the present invention. Such 
vectors will include DNA segments which are transcribable to the disclosed 
mitochondrial import transcript. A preferred DNA encodes the mitochondrial 
import RNA transcript encoded within the mid-portion of mammalian MRP-RNA 
gene between coding region nt100 and nt200. Vectors may be prepared by any 
of numerous means well known to those of skill in the art. These vectors 
will include an appropriate signal sequence for mitochondrial import and 
transcriptional control elements appropriate to the type of cell one 
desires to transform or transfect. 
Suitable cell hosts include prokaryotic and eukaryotic cells. Preferred 
host cells are myoblast cells, such as C2C12 mouse myoblast cells. 
Prokaryotic cells are contemplated to be useful also; for example, one may 
employ E. coli or Salmonella cells transformed with an appropriate vector 
designed for expression in such prokaryotes. Convenient sources of 
commercially available vectors into which DNA may be cloned for 
transfection or transcription into appropriate cells include Strategene 
(La Jolla, Calif.), Mo Bi Tec (Wagenstieg, G ottingen, FRG). 
The inventors have identified particular RNA transcripts that direct import 
into the mitochondria. This discovery provides the basis for the disclosed 
method of importing biological molecules into the mitochondria. The method 
includes facilitating an association between an RNA import transcript and 
a biological molecule so that a complex is formed and introducing the 
complex into a targeted cell. The biological molecule may be a DNA, other 
RNA sequences or a polypeptide. In preferred embodiments, the transport 
RNA has the 58 bp sequence within SEQ ID NO: 1 or 8 which corresponds to 
nt118-nt175 in the MRP-RNA mammalian gene. Peptide RNA complexes are 
contemplated to be those which the peptide binds to the RNA transport 
molecule. 
RNA transport transcripts may be designed such that particular peptides 
specifically bind, thus not limiting binding to nucleolar peptides. Such a 
method is contemplated to be useful in the treatment of human diseases 
associated with deletions and point mutations in the mitochondrial genome. 
Diseases such as myoclonic epilepsy, ragged red fiber disease, 
mitochondrial myopathy and encephalomyopathy, lactic acidosis and 
stroke-like episodes are attributable to single-base substitutions in tRNA 
lysine and tRNA leucine. The tRNA lysine mutation causes a general 
reduction in mitochondrial protein synthesis. Mitochondrial dysfunction in 
cells of these patients may be correctable by linking a mitochondrial 
import signal to mitochondrial tRNA sequences expressed from nuclear 
transgenes, thereby eliminating a requirement for direct genetic 
transformation of mitochondria. 
The present invention also contemplates insertion of foreign nucleic acids 
into mitochondria. Thus, one combines a DNA segment which is complementary 
to a desired DNA with an RNA mitochondrial import transcript which may be 
selected for example from SEQ ID NO: 1 or 8 or from segments of DNA in 
accordance with SEQ ID NO: 1 or 8, preferably segments including the loop 
region between nt144-nt156. The complex is then introduced into a targeted 
cell by established methods; for example, by calcium phosphate 
co-precipation, liposomes or viral vectors. 
The present invention also embodies kits for use in detecting MRP-RNA 
transcripts. Kits for use in both Southern and Northern blotting are 
contemplated. Such kits will generally comprise a first container which 
includes one or more nucleic acid probes having a sequence in accordance 
with the sequences of the nucleic acid probes represented by SEQ ID NO: 4, 
SEQ ID NO: 5 and SEQ ID NO: 6 and a second container comprising one or 
more unrelated nucleic acids for use as a control. In preferred 
embodiments, such kits will include all such nucleic acid probes or 
segments. 
Kits including nucleic acid sequences in accordance with SEQ ID NO: 1 or 8 
or segments of Seq ID NO: 1 or 8 which include nt144-nt156 (SEQ ID NO: 3) 
are also contemplated. Such segments will be useful for targeting selected 
nucleic acids to the mitochondria. Modified sequences, designed to bind 
specific proteins may also be included, as may vectors into which selected 
sequences for coding a specific polypeptide may be inserted.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The present invention relates to the discovery of specific sequence 
elements that direct RNA to the mitochondria. By employing systematic 
mutations scanning through 90% of the coding region of the mouse MRP-RNA 
gene, a 58 nt region was identified. This RNA was found to be essential 
for import of the transcript into mitochondria. The same region, however, 
was not required for transcription, nuclear partitioning, or stability of 
the transcript in the cytoplasmic compartment. Certain other mutations 
within the MRP-RNA coding region did not affect import properties, 
indicating that specific sequence elements are required to direct RNA to a 
mitochondrial import pathway. 
The disclosed results indicate that transcriptional control elements 
sufficient to direct efficient transcription of the gene reside within the 
proximal 700 bp of 5' flanking DNA. This upstream region of the mouse 
MRP-RNA gene includes an array of regulatory motifs similar to those of 
the U6 and 7SK RNA genes (Chang and Clayton, 1989; Yuan et al., 1989; 
Topper and Clayton, 1990b). Deletions of either the putative distal or 
proximal sequence elements from the MRP-RNA promoter eliminate 
transcription. These results place the MRP-RNA gene with U6 and 7SK RNA as 
members of the set of RNA polymerase (Pol III)-transcribed genes that are 
controlled by upstream sequences without a requirement for internal 
elements governing transcription (Murphy et al., 1987; Carbon et al., 
1987; Das et al., 1988; Kunkel and Peterson, 1988; Waldschmidt et al., 
1991). 
One of the mutants (pMRP-D) described herein produced transcripts that 
appeared to exhibit reduced stability. Notably, these transcripts lacked 
the region of MRP-RNA previously identified as containing the binding site 
for To/Th antigen, a nucleolar protein (Reimer et al., 1988) recognized by 
antisera from human patients with progressive systemic sclerosis and 
related autoimmune diseases (Yuan et al., 1991). Several lines of evidence 
indicated that binding of MRP-RNA to To/Th antigen may stabilize the RNA 
within the nucleus. The To/Th antigen has high affinity for MRP-RNA (Yuan 
et al., 1991), though the interaction is not completely selective among 
small nuclear RNAs. Immunoprecipitation with anti-To/Th antibody brings 
down both MRP-RNA and RNase P1-RNA (Gold et al., 1989; Yuan et al., 1991). 
Stabilization of small RNAs by protein binding has been documented 
previously in the interaction of 4S RNA with TFIIIA (Picard and Wegnez, 
1979). The deletion in MRP-D appears to limit stabilization of the 
transcript within the nucleus by impairing binding to To/Th antigen. 
Targeting signals that direct nuclear stabilization and mitochondrial 
partitioning of MRP-RNA appear to reside in distinct and separable domains 
of the transcript. At least 10 nuclear peptides are found to be associated 
with nuclear MRP-RNA in immunoprecipitation assays using anti-To sera 
(Karwan et al., 1991), suggesting that the repertoire of proteins binding 
to MRP-RNA may be large. 
The inventors have found that expression of the RNA subunit of RNase MRP 
(MRP-RNA) is subject to regulation by physiological stimuli that alter 
mitochondrial biogenesis. Both the rapidity (&lt;1 day) and magnitude 
(&gt;10-fold) of the induction of MRP-RNA within skeletal muscle subjected to 
motor nerve stimulation are notable in comparison to pre-translational 
responses of other genes that are regulated by this stimulus (Williams et 
al., 1986; Annex et al., 1991; Williams et al., 1987; Hood et al., 1989). 
Expression of transcripts of several nuclear genes encoding mitochondrial 
proteins, including citrate synthase and subunits of cytochrome oxidase 
and F.sub.1 F.sub.0 ATPase are induced by nerve stimulation, but the onset 
of these responses is not apparent until 3-10 days after the onset of 
nerve stimulation. Maximal induction of these nuclear genes requires 21 
days and does not exceed 5-fold. Among non-mitochondrial proteins, mRNA 
species encoding myoglobin and slow isoforms of contractile proteins are 
increased in abundance by 10-fold or greater, but these responses also 
require 3-10 days to emerge, and are not complete until 21 days. 
With respect to mitochondrial DNA (mtDNA) and expression of mitochondrial 
genes, the time course of the response of MRP-RNA in skeletal muscles 
subjected to continuous motor nerve stimulation is consistent with a role 
for MRP-RNA in modulating the rate of mtDNA replication. Previously, 
independent sets of experiments showed that copy number of mtDNA is 
increased by this stimulus. This response becomes measurable (&gt;2-fold 
after 3-10 days, increases markedly by 3-10-fold after 14-21 days, 
Williams et al., 1986; Williams, R. S., 1986; Annex et al., 1991), but 
declines thereafter to equilibrate at levels 1.5- to 2-fold above control 
values. Expression of rRNA and mRNA products of mitochondrial genes tracks 
almost identically with these adaptive responses of mtDNA, suggesting that 
variations in expression of mitochondrial genes in striated muscle are 
determined predominately by gene dosage in these cells, rather than by 
modulation of transcriptional efficiency (Williams et al., 1986; Williams, 
R. S., 1986). Stimulation-induced changes in abundance of MRP-RNA occur in 
parallel to, but temporally in advance of, increases in mtDNA, a time 
course predicted for a regulatory factor. 
A regulatory role for RNase MRP in controlling mtDNA replication is 
suggested by its putative function in cleaving nascent transcripts from 
the light strand promoter to generate short RNA primers for DNA synthesis 
originating from the heavy strand origin of replication (Chang and 
Clayton, 1987; Clayton, D. A., 1991). Thus, the activity of RNase MRP 
serves functionally to shift the mitochondrial genome from a 
transcriptional mode into a replicative mode. 
The presence of a relatively abundant cytoplasmic pool of MRP-RNA within 
mitochondria-rich skeletal and cardiac myocytes has been shown using in 
situ hybridization. Similarly, when myocytes rather than HeLa cells are 
examined, levels of MRP-RNA present in purified mitochondria exceed levels 
of U1 snRNA by at least an order of magnitude. 
Induction of MRP-RNA may contribute to the increase in rRNA and in 
translation of certain genes (Annex et al., 1991) that occur in the early 
phase (days 1-3) of the adaptive response of skeletal muscle to continuous 
nerve stimulation. The rise in U1 snRNA that accompanies the induction of 
MRP-RNA expression within this early period may indicate a general 
activation of genes encoding small RNAs involved in RNA processing during 
this early period. The disproportionate changes in MRP-RNA and U1 snRNA 
during the period of most rapid mitochondrial biogenesis (days 7-14) 
indicate, however, that these two genes are subject to selective 
regulation as well. 
The observation that MRP-RNA gene expression is induced more rapidly than 
other nuclear genes in response to nerve stimulation makes the MRP-RNA 
gene an attractive model for identification of transcriptional control 
pathways that link physiological activity of striated muscle to changes in 
gene expression. 
Findings from in vivo expression assays of plasmid constructions 
transfected into C2C12 myogenic cells permit two major conclusions. First, 
it was demonstrated that sequences sufficient to direct transcription of 
the mouse MRP-RNA gene in this cell background are included within the 
proximate 5' flanking region of the gene, and that intragenic 
transcriptional control elements appear not to be required. This finding 
places MRP-RNA within the class of small RNA genes transcribed by Pol III 
that includes U6, but distinguishes MRP-RNA from tRNA and 5S RNA genes 
that require intragenic sequence elements. Second, the deletional analysis 
indicates that an upstream region from -223 bp to -84 bp (relative to the 
transcription start site) contains sequences required for transcription. 
This upstream region of the MRP-RNA gene includes consensus binding sites 
for Sp1 and an octamer motif. Interestingly, the results indicate that an 
apparent binding site for nuclear respiratory factor 1 (NRF-1) located 
between -322 and -301 is not necessary for basal expression of MRP-RNA in 
these cultured cells. NRF-1 is implicated in transcriptional control of 
several nuclear genes encoding mitochondrial proteins (Evans and 
Scarpulla, 1989). This sequence motif and its cognate binding factor may 
be dispensable for basal expression of MRP-RNA in resting cells, but 
nevertheless may be involved in the induced expression of MRP-RNA that 
results from physiological stimuli such as nerve stimulation. 
Expression of MRP-RNA in specialized subtypes of rabbit striated muscles 
varies in proportion to respiratory activity. In addition, expression of 
MRP-RNA was induced in skeletal muscle by chronic stimulation of the motor 
nerve, a potent stimulus to mitochondrial biogenesis. It was also 
determined that the proximate 5' flanking region of the mouse MRP-RNA gene 
is sufficient to direct transcription in a muscle cell background. These 
results are consistent with a regulatory role for MRP-RNA and for RNase 
MRP in modulating mtDNA replication, and in maintaining the stoichiometry 
of subunits of mitochondrial enzymes that are derived from nuclear and 
mitochondrial genes. 
EXAMPLE 1 
A number of small RNA genes transcribed by RNA polymerase III (Pol III) 
require internal regulatory elements for efficient transcription 
(Bogenhagen et al., 1980; Sakonju et al., 1980; Galli et al., 1981; 
Hofstetter et al., 1981; Ullu and Weiner, 1985). In vitro transcription 
assays indicated that MRP-RNA also was transcribed by Pol III (Yuan and 
Reddy, 1991). With this information, internal deletions were made to 
determine if transcription was abolished. This was accomplished by 
constructing mutant plasmids designed to express mutant transcripts after 
transfection into mammalian cells. This resulted in the identification of 
a transcript that identified a specific mitochondrial transport RNA. 
Construction of Mutant MRP-RNA Genes 
The MRP-RNA gene was cloned by PCR amplification from mouse genomic DNA 
using primers based on the published sequence (Topper and Clayton, 1990b). 
The MRP-RNA gene clone, pMRP-A, consists of 273 bp of coding region, 700 
bp of 5' flanking DNA and the 3' transcriptional termination sequence. A 
unique Bgl II site was engineered near to the 3' terminus of the coding 
region using Bgl II linker PCR primers (FIG. 1, pMRP-A). Deletion mutants 
were generated by PCR primer-guided synthesis and Acc I and Esp I 
restriction to remove the selected segments of the MRP-RNA coding region 
(FIG. 1). All the constructs were cloned into pBluescript KS (Strateg ene, 
La Jolla, Calif.) and verified by restriction mapping and sequencing. 
Cell Culture and Transfection 
Mouse C2C12 myoblast cells were grown in Dulbecco's modified Eagle's medium 
with 10% fetal calf serum, 5% chick embryo extract and 20 units/ml 
penicillin-streptomycin. Calcium phosphate transfections were performed as 
described previously (Li et al., 1990). Thirty micrograms of plasmid DNA 
were added to each 100-mm dish. For transcriptional analysis, the 
duplicate plates of transfected cells were mixed. One half was used to 
extract transfected plasmid (Hirt, 1967) as a control for the efficiency 
of transfection, and the other half was employed for RNA isolation and 
Northern blot hybridization. 
In Vitro Transcription 
Nuclear and cytosolic extracts were prepared from Hela cells as described 
previously (Dignam et al., 1983). The in vitro transcription reactions 
were performed in 30 .mu.l of reaction volume containing 600 ng plasmid 
DNA and 7.5 .mu.l each of nuclear and cytosolic extract using a modified 
procedure (Ullu and Weiner, 1984). The reaction products were resolved by 
electrophoresis in 6% urea-potyacrylamide gels. 
Isolation of Mitochondria and Nuclei 
Cellular fractions enriched in mitochondria were isolated as shown in FIG. 
2. For each experiment, 20 plates of C2C12 cells were transfected with a 
mutant plasmid. One thirtieth of harvested cells were lysed directly to 
isolate total cellular RNA. The remaining cells were presolubilized with 
digitonin to facilitate isolation of mitochondria (Moreadith and Fiskum, 
1984; Howell et al., 1986). Briefly, the cells were suspended in 4 volumes 
of mitochondria homogenization buffer (MtHB: 210 mM mannitol, 70 mM 
sucrose, 5 mM HEPES pH 7.3, 0.5% BSA) after three washes. Digitonin (5%) 
was added to a final concentration of about 0.5 mg/ml. The cells were 
washed once, resuspended in 4 volumes of MTHB and homogenized with a 
Dounce homogenizer (6-15 strokes). The lysate was diluted with 0.5.times. 
MTHB and centrifuged at 8,000 g for 10 minutes. After resuspending the 
pelleted mitochondria, nuclei and cell debris in 15 volumes of MTHB, the 
lysate was subjected to three consecutive centrifugations at 1000-1080 x g 
for 5 min each (2100-2200 rpm, Sorvall RT6000B centrifuge, H1000B rotor). 
An aliquot was removed from the supernatant of each low speed 
centrifugation step and centrifuged at 8000 x g for 10 min to produce 
increasingly purified mitochondrial fractions for RNA extraction (FIG. 2). 
Nuclei were isolated in a parallel procedure. The nuclear pellets from the 
initial low speed centrifugation step were washed once with MTHB and once 
with 1 ml ionic buffer (10 mM HEPES pH 7.9, 150 mM KCl, 10 mM MgCl.sub.2). 
RNA Isolation and Northern Blot Hybridization 
RNA was isolated by a modification of the guanidinium thiocyanate procedure 
(Sambrook et al., 1989). RNA samples were electrophoresed in 1.5% (for U1 
and MRP-RNAs) and 1.1% (for ribosomal RNAs) denaturing agarose gels. The 
gel buffer contained 20 mM HEPES pH 7.5, 5 mM NaCl, 1 mM EDTA and 2.2M 
formaldehyde. After transfer to nylon membranes, Northern blot 
hybridizations were performed by standard techniques (Overhauser et al., 
1987). Control MRP-RNA was synthesized in vitro from Hind III-linearized 
pMRP-A using T7 RNA polymerase, yielding a 1 kb RNA transcript. 
Synthetic oligonucleotides were used as probes specific for detection of 
MRP-RNA, U1 RNA, 28S cytoplasmic ribosomal RNA, and 16S mitochondria 
ribosomal RNA. Three different oligonucleotide probes (see FIG. 1) were 
used to detect mutant and endogenous MRP-RNA transcripts: 
##STR1## 
The 5 bases underlined in probe 3 were replaced in mutant constructs by 
the 3 bases underlined and indicated in lower case in probe 1. 
RNA Quantitation and Data Analysis 
Hybridization of .sup.32 P-labelled probes to specific bands in Northern 
blots was measured quantitatively using ImagerQuant or PhosphoImager 
(Molecular Dynamics, Sunnyvale, Calif.). Mitochondrial import of mutant 
MRP-RNA transcripts was calculated as follows: 
##EQU1## 
Nuclear partition ratios were calculated in a similar manner. Mutant 
MRP-RNA sequences were analyzed with the computer program SQUIGGLES (Zuker 
and Stiegler, 1981) to assess thermodynamic predictions of internal 
pairing and folding. 
Plasmid Constructs for Expression of Mutant Transcripts 
To identify sequences within the MRP-RNA required for mitochondrial 
targeting, plasmid constructions designed to express mutant transcripts 
after transfection into mammalian cells were prepared. A linker mutation 
plasmid, pMRP-A, was engineered with a 3 bp insertion and a 5 bp deletion 
at nt 251-255, yielding a 273 nt RNA transcript, two nucleotides shorter 
than wild type MRP-RNA. Three other plasmid constructions carried 
deletions designed to scan 90% of the coding region for putative import 
signals. The deletion in pMRP-B removed most of the 3' end of the gene 
from nt 181 to nt 255, while most of the 5' region (nt 6 to nt 115) was 
removed in pMRP-D, including the To/Th antigen binding domain (Yuan et 
al., 1991). The deletion in pMRP-F extended from nt 118 to nt 175 (SEQ ID 
NO: 9) and disrupted a sequence resembling an intragenic transcriptional 
control region (Box A) found within some small RNA genes transcribed by 
RNA polymerase III (Pol III) (Sakonju et al., 1980; Galli et al., 1981; 
Ullu and Weiner, 1985). All the mutant plasmids contained identical 5' 
promotor sequences and 3' flanking signals for transcriptional termination 
(FIG. 1). 
Transcription of MRP-RNA Mutants 
Mutant MRP-RNA plasmids were transfected into C2C12 myogenic cells and 
transient expression was detected by Northern blot hybridization with 
specific oligonucleotide probes. Probe 1 hybridized selectively to mutant 
MRP-RNAs transcribed from pMRP-A, pMRP-D and pMRP-F, but not to endogenous 
MRP-RNA (FIG. 3a). Probe 2 hybridized to both mutant and endogenous 
MRP-RNAs, but distinguished deleted forms by differences in size (FIG. 
3b). 
The results indicated that all of the mutant MRP-RNA constructs were 
transcribed. Plasmid pMRP-A, pMRP-F and pMRP-B produced transcript levels 
similar to each other (FIG. 3), and equivalent to the endogenous MRP-RNA 
band (FIG. 3b; FIG. 5a, lane 1; and FIG. 9c, lane 1). Disruption of the 
Box A-like element by the deletion in pMRP-F had no effect on the relative 
abundance of the resulting transcript relative to other constructs in 
which the box A-like element was undisturbed. 
However, the deletion in pMRP-D resulted in reduced levels of transcript in 
the total cellular RNA pool (FIG. 3a). This difference was not 
attributable to reduced efficiency of transfection (FIG. 3c). In vitro 
transcription assays revealed that all of the mutant constructs were 
transcribed at an equivalent rate, suggesting that the reduced abundance 
of MRP-D transcripts resulted from more rapid degradation rather than from 
disruption of an internal control element important for transcription. 
Assessment of Mitochondrial Partitioning 
MRP-RNA was present mainly in the nucleus, and only a fraction of the total 
cellular pool was found in the mitochondrial compartment. For assessment 
of mitochondrial partitioning of MRP-RNA, mouse C2C12 myoblast cells were 
chosen because of their high mitochondrial content relative to other cell 
lines. The mitochondrial partitioning of heterologous MRP-RNA was examined 
through sequential mitochondrial preparations segregated from nuclei and 
cytosol. The final mitochondrial fraction was devoid of nuclear 
contamination as assessed by staining with Trypan Blue. A series of 
controls confirmed the authenticity of the mitochondrial fractions (FIG. 
2) isolated from these cells. As shown in FIG. 4, U1 RNA (Carmo-Fonseca et 
al., 1991), chosen as a nuclear marker, thereby serving as a negative 
control for mitochondrial import, was abundant in the whole cell 
homogenate but was depleted during purification of mitochondria. Likewise, 
cytosolic (28S) ribosomal RNA was removed by the fractionation procedure. 
In contrast, 16S mitochondrial ribosomal RNA was markedly enriched in the 
mitochondrial fractions. 
Mitochondrial Import of Foreign and Endogenous MRP-RNA 
To examine mitochondrial import of foreign RNA sequences, the partitioning 
of MRP-A transcripts was compared with that of endogenous MRP-RNA. The 
small linker mutation within MRP-A permitted it to be distinguished 
readily from the endogenous gene product while introducing only minor 
alterations in the sequence of the transcript. Such minimal deviation from 
the endogenous sequence was considered unlikely to interfere with 
mitochondrial import of the pMRP-A transcript, thus permitting this 
construct to serve as a positive control for mitochondrial partitioning of 
products of other mutant MRP-RNA genes. 
FIG. 4a demonstrates that the foreign MRP-A transcript partitions into 
mitochondria in parallel with endogenous MRP-RNA (FIG. 4b). Quantitative 
analysis (FIG. 4f) indicated that mitochondrial partitioning of the pMRP-A 
transcript was marginally lower than that of endogenous MRP-RNA, but this 
may not necessarily reflect less efficient mitochondrial import of the 
foreign gene product. It has been reported that transfection of cells in 
culture by calcium phosphate coprecipitation results in expression of 
foreign genes in less than 20% of the cell population (Sambrook et al., 
1989). 
Since foreign MRP-RNA transcripts were present in approximately equal 
abundance to the wild type transcripts in RNA extracted from the entire 
cell population, these results indicated that trans-gene expression was at 
least 5-fold greater than that of the endogenous gene within the fraction 
of cells that took up the plasmid DNA. Such high level expression in a 
small fraction of cells may saturate carriers in the import pathway and 
create the appearance of less efficient import. 
Mitochondrial Partitioning of Deleted Forms of MRP-RNA 
Using the same assay system, mitochondrial partitioning of deleted forms of 
MRP-RNA was examined. Transcripts derived from pMRP-B partitioned into 
mitochondria in a manner indistinguishable from pMRP-A and the endogenous 
gene product (FIG. 5), indicating that sequences from nt 181 to nt 255 are 
not involved in mitochondrial targeting. Although pMRP-D transfection 
resulted in a lower abundance of transcripts in the total cellular RNA 
pool, increasing gel loads and extending exposure time revealed that 
pMRP-D transcripts also partitioned similarly to pMRP-A, pMRP-B and 
endogenous MRP-RNA (FIG. 6). This indicated that the region from nt 6 to 
nt 115 also is not essential for identifying RNA for mitochondrial import. 
In distinct contrast, the mutation present in transcripts derived from 
pMRP-F severely impaired its partitioning into mitochondria (FIG. 7). 
Although the pMRP-F transcripts accumulated to high levels within the 
cell, its fractionation pattern was similar to that of U1 RNA, indicating 
that removal of a 58 nucleotide region of RMR-RNA between nt 118 and 175 
(SEQ ID NO: 9) rendered the resulting transcript defective for 
mitochondrial import. 
FIG. 8 summarizes the results of this mutational analysis in terms of a 
standardized mitochondrial partition ratio. Transcripts from pMRP-A, 
pMRP-B and pMRP-D remained competent for mitochondrial import, while the 
mitochondrial partition ratio of MRP-F decreased by an order of magnitude. 
Nuclear Partitioning of Mutant and Endogenous MRP-RNA 
Following nuclear transcription, many mature RNAs reach the cytoplasm 
through controlled pathways through the nuclear envelope (Hamm and Mattaj, 
1990; and reviewed by Nigg et al., 1991). The nuclear partitioning of 
mutant and endogenous MRP-RNAs was measured in order to determine if there 
was a blockade in the pathway out of the nucleus. Equal amounts of nuclear 
and total cellular RNA were probed with appropriate oligonucleotide 
probes. As shown in FIG. 9, all mutant MRP-RNAs partitioned to nuclei 
somewhat less efficiently than endogenous MRP-RNA. Nuclear partition 
ratios ranged from 0.28-0.42 (pMRP-A=0.28, pMRP-B=0.42, pMRP-D=0.41 and 
pMRP-F=0.32). These results suggested that carriers that stabilize MRP-RNA 
within the nuclear compartment, like those involved in mitochondrial 
import, may be saturable, so that transcripts accumulate preferentially in 
the cytoplasm when expression of heterologous MRP-RNA's is increased 
several fold above ambient levels. Additionally, the results indicated 
that defective mitochondrial import of MRP-F is not a result of failure of 
this transcript to exit from the nucleus. 
EXAMPLE 2 
The intracellular location of MRP-RNA was further supported by the results 
of in situ hybridization and electron microscopy studies on adult mouse 
myocardial cells. Cardiac myocytes manifest a high fractional volume of 
mitochondria and a high ratio of mtDNA to nuclear DNA, suggesting that if 
present, intramitochondrial MPR-RNA should be more readily detectably than 
in cells of lower respiratory activity. As shown in this example, MRP-RNA 
is preferentially localized both to nucleoli and to mitochondria in 
cardiomyocytes. These experiments therefore document mitochondrial 
partitioning of MRP-RNA by a method that avoids cell fractionation. This 
is important because of the virtually unavoidable contamination of 
purified mitochondrial fractions with small nuclear RNAs. 
Localization of MRP-RNA to Nucleoli and Mitochrondia in Cardiomyocytes 
In situ hybridization of an antisense .sup.35 S-labelled RNA probe to 
sections of adult mouse heart was analyzed by light microscopy and 
revealed prominent nuclear clustering of MRP-RNA within both 
cardiomyocytes and non-myocyte cells of the cardiac wall (not shown). This 
result was consistent with prior cell fractionation studies that localized 
the bulk of the intracellular pool of MRP-RNA to the nuclear pool of 
MRP-RNA to the nuclear compartment (Chang and Clayton, 1987). It was also 
found that hybridization of the antisense MRP-RNA probe within the 
cytoplasm exceeded that of an irrelevant RNA probe, suggesting that 
cardiomyocytes also contain a distinct cytoplasmic pool of MRP-RNA. 
Cytoplasmic binding of the antisense MRP-RNA probe was less apparent in 
non-muscle cells. The resolution obtainable by light microscopy was, 
however, insufficient to distinguish between intra-mitochondrial and 
extra-mitochondrial locations of cytoplasmic MRP-RNA. 
Higher resolution was obtained by in situ hybridization using a 
biotinylated complementary RNA probe in ultrathin cryosections of adult 
mouse heart, followed by immunogold labeling and electron microcopy. 
Results are illustrated in FIG. 16, and show preferential binding of the 
antisense MRP-RNA probe to nucleoli and mitochondria. 
The intracellular distribution of the antisense MRP-RNA probe and an 
irrelevant RNA control probe were determined quantitatively using a 
systematic random sampling technique (Gunderson and Jensen, 1987) by an 
experienced observer blinded to the identity of the probes. Results of a 
representative experiment employing this quantitative analysis are shown 
in FIG. 17. When results from three independent experiments were combined, 
binding of the antisense MRP-RNA signal to nucleoli and mitochondria was 
significantly greater than binding to myofibrils (Student's t test, 
p&lt;.01). Hybrids formed between the antisense MRP-RNA probe and its 
cellular targets in nucleoli and mitochondria were resistant to RNAase H 
(not shown), indicating that the results were not confounded by 
hybridization of the antisense MRP-RNA probe to complementary sites in 
nuclear or mitochondrial DNA. 
The presence of an authentic intramitochondrial pool of MRP-RNA indicated 
the existence of a pathway for mitochondrial import of RNA in mammalian 
cells and supports the involvement of RNAase MRP in intramitochondrial RNA 
processing and priming of mtDNA replication. The low abundance of MRP-RNA 
in mitochondrial factions from HeLa cells (Kiss and Filipowicz, 1992) may 
be attributable to the low respiratory activity of these cells in 
comparison to highly aerobic cells such as cardiomyocytes. 
EXAMPLE 3 
Although the exact role and function of MRP-RNA is not defined, the 
following experiments indicate one of many possible regulatory functions 
with respect to mtDNA replication and mitochondrial biogenesis. Expression 
of MRP-RNA in specialized subtypes of mammalian striated muscles was 
examined. The results showed that changes in abundance of MPR-RNA 
correlated with specific activity of citrate synthase, a marker of 
mitochondrial proliferation. 
Induction of MRP-RNA by Contractile Activity in Striated Muscle 
Animal Surgery and Tissue Preparation 
Adult New Zealand White rabbits weighing 2.3-3.5 kg were anesthetized by 
isoflurane inhalation. Under sterile conditions, pulse generations were 
implanted and their electrodes placed adjacent to the common peroneal 
nerve of one hind limb according to the procedure described originally by 
Salmons and Vrbova (1969). Nerves were stimulated continuously at 6-10 Hz 
for 1, 3, 7, 14, or 21 days. The rabbits were anesthetized with 
pentobarbital sodium (50 mg/kg, iv) for removal of muscles under sterile 
conditions. Muscle tissue was rinsed in cold sterile saline, frozen in 
liquid nitrogen, and stored at -80.degree. C. All protocols were reviewed 
and approved by the Institutional Review Board for Animal Research and 
were conducted in accordance with the NIH Guide for the Care and Use of 
Laboratory Animals. 
Enzyme Assays 
Citrate synthase activity was measured in whole muscle homogenates as 
described previously (Williams et al., 1986). Activities were expressed 
relative to muscle wet weight. 
RNA Analysis 
Total RNA was extracted from tissue samples by homogenization with 
guanidine isothiocyanate and centrifugation through 5.7M cesium chloride, 
followed by phenol-chloroform extraction and ethanol precipitation. RNA 
concentrations were measured spectrophotometrically from absorbance 
measurements made at 260 nm. Northern blots of total RNA were prepared on 
MagnaGraph Nylon Transfer Membranes by capillary blotting following 
electrophoresis in formaldehyde-agarose gels. RNA was immobilized by 
photocrosslinking (Stratolinker). Blots were prehybridized at 60.degree. 
C. for 2-3 hours in 0.25M sodium phosphate (pH 7.2), 1 mM EDTA, 0.25M 
sodium chloride, 10% polyethylene glycol, 7% SDS, 1% bovine serum albumin, 
and 120 .mu.g/ml of denatured salmon sperm DNA. 
Oligonucleotides 
Oligonucleotides were synthesized based on the published sequences of the 
coding regions for mouse MRP-RNA (Chang and Clayton, 1989) and U1 snRNA 
(Carmo-Fonseca et al., 1991) and included the sequences 
GAGAATGAGCCCCGTGTGGTTG (SEQ ID NO: 6) and GGTCTAAACCCAGCTCACGTTC (SEQ ID 
NO: 7), respectively. The resulting oligonucleotides were end-labeled with 
[.sup.32 P] ATP by T4 kinase. Hybridizations were performed at 60.degree. 
C. for 16-24 hours in a shaking water bath. Filters were washed in 
0.5.times. SSC, 0.1% SDS for 15 minutes twice at room temperature (MRP) or 
for 15 minutes twice at room temperature and 15 minutes twice at 
40.degree. C. (U1). The filters were placed on film (Amersham 
Hyperfilm-MP) and exposed for 24-96 hours. Autoradiograms were scanned 
using a Molecular Dynamics 300A Computing Densitometer. 
Promoter Analysis 
The MRP-RNA gene was cloned by PCR amplification from mouse genomic DNA 
using primers based on the published sequence (Topper and Clayton, 1990). 
The MRP-RNA gene construct, pMRP-A, consisted of 273 bp of coding region, 
700 bp of 5' flanking DNA and the 3' transcriptional termination sequence. 
A unique Bgl II site was engineered near the 3' terminus of the coding 
region using Bgl II linker PCR primers. Deletion mutants were generated by 
PCR primer-guided synthesis and by restriction at unique sites to remove 
selected segments of the MRP-RNA promoter and coding regions. All the 
constructs were cloned into pBluescript KS (Stratagene, La Jolla, Calif.) 
and verified by restriction mapping and sequencing. 
Mouse C2C12 myoblast cells were grown in Dulbecco's modified Eagle's medium 
with 10% fetal calf serum, 5% chick embryo extract and 20 units/ml 
penicillin-streptomycin. Calcium phosphate transfections were performed as 
described previously (Li et al., 1990). Thirty micrograms of plasmid DNA 
were added to each 100-mm dish. After two days, the duplicate plates of 
transfected cells were mixed. One half was used to extract transfected 
plasmid (Hirt, B., 1967) as a control for the efficiency of transfection, 
and the other half was employed for RNA isolation and Northern blot 
hybridization. The probe for these experiments was a synthetic 
oligonucleotide with sequence GAATGAGATCTGTGGTTGGTGCG (SEQ ID NO: 4), end 
labelled with [.sup.32 P] ATP. This probe is complementary to transcripts 
derived from plasmid constructions that contain the BglII linker within 
the MRP-RNA coding region (pMRP-A and derivatives), but fails to hybridize 
to endogenous MRP-RNA under the stringency conditions that were chosen for 
these experiments. 
Effects of Nerve Stimulation on Citrate Synthase Activity 
Continuous electrical stimulation of the motor nerve at 6-10 Hz led to 
increases in specific activity of citrate synthase in tibialis anterior 
muscles that were detectable by 7 days (1.5-fold over contralateral 
control muscles) and progressive up to 21 days (3.5-fold increase) of 
motor nerve stimulation (FIG. 12). These results were similar to those of 
extensive prior studies using this model (Henriksson et al., 1986; Seedorf 
et al., 1986; Williams et al., 1986; Williams R. S., 1986; Annex et al., 
1991). 
Effects of Nerve Stimulation on U1 and MRP-RNA 
As shown in FIG. 13, expression of both U1 and MRP-RNA was induced within 
the first day of motor nerve stimulation. Longer periods of stimulation 
further increased the abundance of MRP-RNA, which peaked at levels 
approximately 14-fold greater than those seen in contralateral 
unstimulated muscles after 14 days of stimulation. By 21 days, MRP-RNA 
remained elevated (3.5-fold) above levels present in contralateral 
muscles, but declined from the apogee noted at earlier time points. Part 
of this late decline in stimulation-induced abundance of MRP-RNA between 
days 14 and 21 was attributable to small but consistent increases in 
MRP-RNA levels in contralateral unstimulated muscles in comparison to 
muscles from naive animals. 
By contrast, the early induction of U1 snRNA was not sustained, and 
expression of U1 snRNA was maintained at levels approximately 1.5 times 
those seen in contralateral unstimulated muscles throughout the 21-day 
period of nerve stimulation. Like MRP-RNA, small but consistent increases 
in U1 snRNA were observed in contralateral unstimulated muscles of animals 
undergoing nerve stimulation for extended periods in comparison to naive 
animals. 
The apparent increases in U1 and MRP-RNA in contralateral, unstimulated 
muscles between days 14 and 21 could be attributable to any of several 
factors, including: variations among those non-inbred animals in basal 
expression of small RNAs; recovery from stress-induced depression of basal 
expression of these small RNAs associated with the surgical procedure; 
systemic effects of chronic nerve stimulation on unstimulated muscles. The 
basis for these variations in expression of U1 and MRP-RNA among control 
muscles is immaterial in the described experiments in which each animal 
serves as its own control. 
Relative Abundance of MRP-RNA in Specialized Muscle Subtypes 
MRP-RNA was expressed to higher levels in mitochondria-rich cardiac and 
slow skeletal muscles than in glycolytic fast skeletal muscles of adult 
rabbits (FIG. 14). These results complement the analysis of effects of 
nerve stimulation and suggest a consistent relationship between 
respiratory activity and expression of MRP-RNA among striated muscles of 
this species. 
Promoter Analysis 
The pMRP-A construct that includes the proximal 700 bp of 5' flanking 
region and coding region of the mouse MRP-RNA gene was expressed to high 
levels following transfection into C2C12 myogenic cells (FIG. 15A). 
Efficient transcription in this cell background was independent of 
intragenic sequences, including a motif similar to the Box A elements of 
tRNA genes, as determined by the continued high expression of constructs 
with internal deletions collectively spanning more than 90% of the coding 
region. Likewise, sequences upstream of position -223, relative to the 
transcriptional start site, appeared to be dispensable for transcription 
in this transient expression system. By contrast, when removed either by 
5' or internal deletions, sequences between -223 and -84 were essential 
for promoter activity (FIG. 15C). Variations in expression of the reporter 
gene (MRP-RNA carrying the BglII linker) were not attributable to 
differences in efficiency of transfection, as assessed by recovery of 
plasmid constructs from transfected cells (FIG. 15B). 
EXAMPLE 4 
The region between nt 118 and nt 175 (SEQ ID NO: 9) of mouse MRP-RNA was 
analyzed by an algorithm to determine features of secondary structure. A 
stable stem-loop was found to be preserved in all forms of MRP-RNA 
efficiently imported into mitochondria. 
Secondary Structure of Mouse MRP-RNA 
Analysis of the nt 118 to nt 175 (SEQ ID NO: 9) region of mouse MRP-RNA was 
made using an algorithm to predict secondary structure. 
REFERENCE AND BRIEF DETAILS OF HOW USED 
The nt 118-175 region (SEQ ID NO: 9) contained an evolutionarily conserved 
sequence (nt 144 to nt 156) within a flexible base-pairing region, as well 
as a stable stem-loop (FIG. 10). This was preserved in all of the forms of 
MRP-RNA that were efficiently imported into mitochondria. 
EXAMPLE 5 
A simplified model, illustrated in FIG. 11, is consistent with the data 
concerning the intracellular trafficking of MRP-RNA. The impaired 
mitochondrial partitioning of pMRP-F may be attributable to failure of its 
transcript to be recognized by a protein carrier(s) of a mitochondrial 
import apparatus. Transcripts of pMRP-D are likely to be labile due to 
defective binding to a protein(s) that stabilize the transcript within the 
nuclear compartment (possibly To/Th antigen). 
While the compositions and methods of this invention have been described in 
terms of preferred embodiments, it will be apparent to those of skill in 
the art that variations may be applied to the methods, compositions, and 
in the steps or sequences of steps of the methods described herein without 
departing from the concept, scope and spirit of the invention. More 
specifically, it will be apparent that certain agents which are 
biologically or physiologically related may be substituted for the for the 
agents described herein with the same or similar results achieved. All 
such similar substitutes and modifications apparent to those skilled in 
the art are deemed to be within the scope, spirit and concept of the 
invention as defined by the appended claims. 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 100 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCGTGCACACGCGCGTAGACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACC60 
CCTCCGGGGCGAGCTGAGCGGCGTGGCGCGGGGGCGTCAT100 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: RNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GACUUCCCCCGCAAGUC17 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: RNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CCGCCAAGAAGCG13 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GAATGAGATCTGTGGTTGGTGCG23 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CATGTCCCTCGTATGTAGCCTAG23 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GAGAATGAGCCCCGTGTGGTTG22 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GGTCTAAACCCAGCTCACGTTC22 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 101 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ACGTGCATACGCACGTAGACATTCCCCGCTTCCCACTCCAAAGTCCGCCAAGAAGCGTAT60 
CCCGCTGAGCGGCGTGGCGCGGGGGCGTCATCCGTCAGCTC101 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ACTTCCCCCGCAAGTCACTCGTTAGCCCGCCAAGAAGCGACCCCTCCGGGGCGAGCTG58 
__________________________________________________________________________