Microbiology Journal Club
www.mbio.ncsu.edu/MJC
9:10AM alternating Mondays, MB conference room
No specified topic
MB 810R, 1 credit hour
Microbiology Journal Club
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Microbiology Journal Club
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Basic MJC organization, grades, &c
Microbiology Journal Club
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Microbiology Journal Club
Jan 19 : MLK Day - no MJC
Feb 2 : Jim Brown
Feb 16 : Kristen Belanger
Mar 2 : Spring Break - no MJC
Mar 16 : Weaver Haney
Mar 30 : Jennifer Lonan
Apr 13 : Ahmet Bozdag
RNase P is the 5´pre-tRNA endoribonuclease
RNase P assay
- +
RNase P is present in all cells, and in mitochondria and plastids
RNase P in Bacteria - an RNA enzyme
1 large RNA
rnpB - 377nt (140kDa)
1 small protein
rnpA - 119aa (14kDa)
The RNA by itself is catalytically proficient in vitro
RNase P in the nucleus - also an RNA enzyme
1898
One large RNA
!
Rpr1 - 120kDa
!
This RNA is only generally
!
similar to the bacterial RNA
Nine assorted proteins
!
Pop1p - 100kD
!
Pop3p - 23kD
!
Pop4p - 33kD
!
Pop5p - 20kD
!
Pop6p - 18kD
!
Pop7p - 16kD
!
Pop8p - 15kD
!
Rpp1p - 32kD
!
Rpr2p - 16kD
!
None of these are at all
!
similar to the bacterial protein
The RNA is absolutely dependent on the proteins for activity
RNase P in Archaea - also an RNA enzyme
Ribonuclease P RNA
Methanobacterium thermoautotrophicum ΔH
Sequence : U42986, Pannucci 1999 PNAS 96:7803
Structure : Harris, et al., RNA (in press)
Image created 10/5/00 by JWBrown
G
U
A
G
UG
AU
A A
U
G
A
A
G CU
A bacterial-like RNA...
U C A CC
A
C
C
G AG
UG
G
A
U
A CA
CC
U
G
A
C
G
U
AC
C
A
GUC A
U
AC
A G U
GA
A
C
AG
A
G
P12
G
A
G CC A
A
G
C
U
G
G
A
A
G
A
GG
G
C A
C C U CU
A
G
P9 C
A
GG
P10/11
A
C U GA U U
GU
G
CG
C
AC
C A P7
CG
P15 A A A U G P16 G G U P17
P5
U
GG AUG C A A GG AC
CUGCC
GA U
A
G GU
A
GC
U
P8 C U
C
C
G
G
C
U
U
G
G
A
U
G
G
A
U
CU AC
A
G
G
C
G
A
G
A
AG
A
G
U
UGG
A
A CAU C
C
A
C
C
GUG
A
C
C
C
C
A
U
C
G
A
A
A
U
A
G
G
P6
G
A
P3
A
A
A
A GGGGCUG
U
U
C C C C U G G C G C G P4
U
A G C
A U
P2 C G
P1
G C
5´ A G C C G A A G G C G A A A C
A
CGGCU UC
G
3´ A
C A
A
A
UUGGGUGG
rnpB - 96kDa (293nt)
A catalytically-active “type A” RNA in most cases,
only distantly related to the eukaryotic RNA
but 4 eukaryotic-like proteins.
11
1618
688
687
MTH687p - 15kDa = Pop5p
MTH688p - 28kDa = Rpp1p
MTH11p
- 11kDa = Pop4p (C-terminal half)
MTH1618p - 17kDa = Rpr2p
Not at all like the single bacterial protein
Most of these have RNAs that are catalytically proficient in vitro
RNase P in primitive organelles
U
U
U
U
U
U
C
U
C
A
A
A
A
UA
AA
Ribonuclease P RNA
Reclinomonas americana 94 mitochondrion
A
G
A
G
U P12
U
U
UU
U
G
Sequence : AF007261, Lang, et al., 1997 Nature 387:493
Structure : Lang, et al., 1997 Nature 387:493
U
A
U
U
Image created 10/6/00 by JWBrown
A
U
A
A
A
A
A
A
A P13
G
AG A U A
A
A
U
U
CUA A
A
A
U
A
U
U
U
A
G
A P14
A UA
P11
UU
A
A U
U
UA
A A AG A
U
A G
A
A
C
AG
AA
P9
A
A C UC U A G
C P10
A
G
A
A
C
A
A G AG A C
C A P7
A
CU
P15
P5
G
U UG A G C A A A A C C A
G
U C UG
U
A
A
P8 C GA
U UG G A U
ACUC
A
C
G
U G
U
A
A
U
A
A UU
C
G
A
U
U
G
A A
A
U
P18 A U A CG
C
A
U
A AACAA
G
U
A
A
U UG U U
A
A
CU
A
A
G AG
A
G
G
G
A
P3
A
G
A UAUAAU
A
U
U UU
A P4
A AUAUUA
U
A
G
U AU
U
U A
U A
A
C G
A
A
U
A
P2 U A
U
A
G C
U
A
U A
U
A U A U U P19
5´A A A G U P1
A
UUAUU G G AC
G
G A CA
A
UUUCAAAUAA U
A
A A
3´
UU CG A C C UA
UA
U U
A G
Ribonuclease P RNA
U
AA U
Porphyra purpurea chloroplast
C
A
A
G
A
G C
Sequence : U38804, Reith & Munholland 1995 Plant Mol. Biol. Rep. 13:333
A U
Structure : Harris, et al., RNA (in press)
C G
G
C
A
G U
Image created 10/8/00 by JWBrown
A
C G
GG U
C
A
G
A
C
A
A
U
A
A
A
A
GG
UA
A
AG U U
G
CC
A
AU
A
A
U
A
C
C
A
AC
A
U
G AA
A
A
U
C G
A
U
G
G
U
G
U
A
UA
U
C
A
C U A
U
U
G
A G C A
G
A
U
AG
C
A A
A C U A U G AG C
A
CU AA
CA
A G A UG U
AA AC
A
A
U
A
G
C
GG AG C A A AG G
UAAAUUUUUG
G
G
C U UU
U
A
U
G
CCUC
UCAU
A U U UG A UG A
A
C U
A
U
A
C G
AC
G
G
A
G
C
G
AU
C
U
U
A
U
C
A AC G
U
U
A
C
AA UC
A G
A
A
A
C
UG
A A
A A G U U A U UG G
C
C
U
G
UCA A UA A C AA
C
U
A
A
G
A
A
A
G
G
G UG
G
G
U
A
A
U
U AC
A
A U A A G U C UG
A
C
A
U
U
A
U
U UG U
G
U
U
U
A
G
A
C
G
U
A
UA
A
C G
A
A A U
G C
G
U
A U
C
C
U A
U GG
A
A U U
5´A A G U AA CG U A G
A
C C G
AA CA
3´
G
UGU
A UU UU U C A U
G A
A
A
UU C GGCCCA
These RNAs require protein for function, but the
native proteins have not been identified
RNase P in yeast mitochondria
Protein - Rpm2p
1202 a.a. = 132 kDa
Encoded in nucleus
Also a nuclear
transcription factor
RNA
Also required for P-body
turnover
mito encoded
Also involved in mito
import complex
null mutants petite
null mutants dead
What about human mitochondrial RNase P?
Why bother?
tRNA processing defects are a common cause of
maternally-inherited genetic neuromuscular disease:
Mitochondrial encephalomyopathy with lactic acidosis and
stroke-like episodes (MELAS) - various point mutations in
tRNALeu
myoclonic epilepsy with ragged-red fibers (MERRF) - various
point mutations in tRNALys
Animal mitochondrial genomics
Encodes 11 proteins:
integral membrane
proteins of ETS &
ATPase. All other
proteins imported
Encodes 2 rRNAs:
ssu-rRNA (12S) &
lsu-rRNA (16S)
Encodes a minimal
complete set of 22
tRNAs - no extras
Animal mitochondrial genomics
2 promoters for txn
of H and L strands
tRNA punctuation:
Each cistron is
separated by a tRNA
- processing creates
individual mRNAs
tRNA processing
defects affect gene
expression overall
Animal mitochondrial genomics
Each cell has many
mitochondria
Each mitochondrion
has a population of
chromosomes
Egg cells are also a
mixture (sperm mito
almost never are
retained)
Mitochondrial genetics
is a nightmare!
What about RNase P in human mitochondria?
THEJOURNAL
OF BIOLOGICAL CHEMISTRY
0 1985 by The American Society of Biological Chemists, Inc.
Vol. 260, No. 10, Issue of May 25, pp. 5942-5949,1985
Printed in U S A .
Characterization ofan RNase P Activity from HeLa Cell Mitochondria
COMPARISON WITH THE CYTOSOL RNase P ACTIVITY*
(Received for publication, October 26, 1984)
Claus-Jens Doersen$$, Cecilia Guerrier-Takadal, Sidney Altmanl,and Giuseppe AttardiS
From the tDivision of Biolom. California Institute of Technology, Pasadena, California 91125 ann‘ the TDepartment of Bwbgy,
Yale University, New Haven, Con&cticut 06520 ’
MATERIALS AND METHODS AND RESULTS’
Downloaded from www.jbc.org by on
A ribonuclease P-like activity waspartially purified configuration which shares the critical features of the strucfrom HeLa cell mitochondria by DEAE-cellulose and tures formed by the tRNA sequences, in the same way as E.
octyl-Sepharose chromatography. RNase P-like activ- coli RNase P might recognize the precursor to E. coli 4.5 S
ity can be quantitatively recovered from intact mito- RNA (9) and the bacteriophage 480-induced M3 RNA (10).
chondrial preparations treated with micrococcal nuWe tested mitochondrial fractions isolated from human
clease, strongly suggesting that the enzyme is localized cells for the presence of an RNase P-like activity using the
within the organelles. Mitochondrial RNase P (mt- precursor to E. coli suppressor tRNA* (11)as substrate. This
RNase P) cleaves the precursor to Escherichiacoli
approach has been previously used to detect RNase P-like
suppressor tRNATY’at the same site as E. coli RNase activities in subcellular fractions isolated from a variety of
P, producing the mature 5‘-end of tRNATy’. The sen- eukaryotic cells (12-17). This report describes the partial
sitivity of mtRNase P to pretreatment with nucleases
or Pronase indicates that the enzyme has essential RNA purification of an endoribonuclease from HeLa cell mitochonand protein components. Although the ionic require- dria, which is capable of processing the precursor to tRNA%
ments of mtRNase P are similar to those of the RNase with the same specificity as E. coli RNase P. The properties
P activity isolated from the post-mitochondrial cytosol of the partially purified mitochondrial RNase P-like enzyme,
fraction, the chromatographic properties of mtRNase designated mtRNase P, have been compared to those of the
P are distinct. Mitochondrial RNase P is probably a enzymatic activity isolated from the post-mitochondrial cypart of the mitochondrial RNA processing machinery tosol fraction of HeLa cells. The evidence indicates that
of mammalian mitochondria, being responsible for the mtRNase P probably represents the mitochondrial RNA procendonucleolytic cleavage of the RNA transcripts at the essing enzyme responsible for the endoribonucleolytic cleavage of mitochondrial RNA transcripts at the5’-side of tRNA
5’-side of the tRNA sequences.
sequences.
What about RNase P in human mitochondria?
What about RNase P in human mitochondria?
MOLECULAR AND CELLULAR BIOLOGY, Jan. 2001, p. 548–561
0270-7306/01/$04.00!0 DOI: 10.1128/MCB.21.2.548–561.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 21, No. 2
The RNase P Associated with HeLa Cell Mitochondria Contains
an Essential RNA Component Identical in Sequence
to That of the Nuclear RNase P
RAM S. PURANAM†
AND
GIUSEPPE ATTARDI*
Division of Biology, California Institute of Technology, Pasadena, California 91125
Received 17 July 2000/Returned for modification 16 August 2000/Accepted 19 October 2000
The unique mode of transcription of the mammalian mitochondrial DNA in the form of giant polycistronic molecules,
nuRNase P. Enzymatic activities involved in HeLa cell mitochondrial tRNA processing, in particular an RNase P-like ac-
Downloaded from mcb.asm.org by on
The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and
shown to reside in particles with a sedimentation constant ("17S) very similar to that of the nuclear enzyme
(nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNASer(UCN)
precursor [ptRNASer(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 "33 to "175 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.
What about RNase P in human mitochondria?
RNase P without RNA: Identification and
Functional Reconstitution of the Human
Mitochondrial tRNA Processing Enzyme
Johann Holzmann,1 Peter Frank,2 Esther Löffler,1 Keiryn L. Bennett,3 Christopher Gerner,2 and Walter Rossmanith1,*
1Center
for Anatomy & Cell Biology, Medical University of Vienna
of Cancer Research, Department of Medicine I, Medical University of Vienna
3Research Center for Molecular Medicine of the Austrian Academy of Sciences
1090 Vienna, Austria
*Correspondence: walter.rossmanith@meduniwien.ac.at
DOI 10.1016/j.cell.2008.09.013
2Institute
SUMMARY
tRNAs are synthesized as immature precursors, and
on their way to functional maturity, extra nucleotides
at their 50 ends are removed by an endonuclease
called RNase P. All RNase P enzymes characterized
so far are composed of an RNA plus one or more proteins, and tRNA 50 end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified
the components of human mitochondrial RNase P
and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that
does not require a trans-acting RNA component for
catalysis. Moreover, the mitochondrial enzyme turns
out to be an unexpected type of patchwork enzyme,
composed of a tRNA methyltransferase, a short-chain
dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary
origin, possibly representing the enzyme’s metallonuclease moiety. Apparently, animal mitochondria lost
the seemingly ubiquitous RNA world remnant after
reinventing RNase P from preexisting components.
INTRODUCTION
RNase P was shown to be the catalytic subunit of the enzyme, capable of catalyzing 50 leader removal from tRNA precursors even
in the absence of the protein subunit at elevated Mg2+ concentrations (Guerrier-Takada et al., 1983). More recently, RNase P
RNAs from archaea and eukarya were found capable of mediating cleavage in the absence of protein, too (Kikovska et al., 2007;
Pannucci et al., 1999). RNase P is therefore not only generally
considered to be a ribonucleoprotein, but tRNA 50 end maturation
in addition to ribosomal protein synthesis considered the only ribozyme-catalyzed cellular process universally persistent since
the hypothetical prebiotic RNA world (Gesteland et al., 2006).
Notwithstanding this general view, studies on RNase P activities from the organelles of some eukarya are at odds with a general ribonucleoprotein nature of the enzyme. Human mitochondrial and spinach chloroplast RNase P were both reported to
have a protein-like density, to be insensitive to micrococcal nuclease treatment, and, if sufficiently purified, to be devoid of putative RNase P RNAs (Rossmanith and Karwan, 1998a; Rossmanith et al., 1995; Thomas et al., 1995; Wang et al., 1988); similar
findings were reported for trypanosomal mitochondrial RNase P
(Salavati et al., 2001). However, because none of the protein
components of one of these enzymes had been identified, all evidence remained essentially indirect, and so the idea of an RNAfree, protein-only RNase P has always been met with considerable skepticism (reviewed in Walker and Engelke, 2006).
To finally resolve the enigma of one of these controversial RNAfree RNase P enzymes we identified the components of human
mitochondrial RNase P (mtRNase P), expressed the three identified proteins in E. coli, affinity-purified them to homogeneity, and
Figure 1. Partial
mtRNase P
Purification
of
Human
(A) Schematic overview of the five different purification
procedures employed.
(B) Specific activity of the partially purified mtRNase P
preparations relative to the starting material in each
case.
(C) mtRNase P preparations separated by SDS-PAGE
and stained by Coomassie Brilliant Blue (gels shown
were subsequently processed for ion-trap mass spectrometry). Preparations run in different gels, from left to
right: molecular weight standards and mitochondrial
extract (detergent lysate), 10% gel; Mono Q/glycerol
gradient-purified mtRNase P, 8% gel; Mono Q/heparin
sepharose-purified mtRNase P, 10% gel; phenyl
sepharose/heparin sepharose-purified mtRNase P,
10% gel; Mono P/glycerol gradient-purified mtRNase
P, 10% gel; Mono P/heparin sepharose-purified
mtRNase P, 8% gel. Molecular weight standards
comigrated in each gel and indicated by short bars
are the same as in the leftmost gel; the migration front
is indicated by the long bar.
(D) Number of proteins identified in above indicated
preparations (see also Table S7).
recovery. In order to avoid this apparent obstacle for a conventional purification-to-homogeneity approach we designed
a strategy based on partial purification only, using a minimal
number of purification steps and requiring only moderate
amounts of starting material. Outlined in brief, this strategy identifies the overlap proteome of different partially purified enzyme
heparin affinity matrix or by rate-zonal sedimentation in glycerol
gradients. The five different two-step purification procedures
(Figure 1A) increased the specific activity of the peak fraction
of the second step between 5- and 30-fold (Figure 1B). In line
with the rationale of our PPOP strategy the SDS polyacrylamide
gel electrophoresis (SDS-PAGE) patterns of the five preparations
Table 1. Partial Purification Overlap Proteome
Rank (Group #) in mtRNase P Preparationsb
KIAA0391
Mono Q
Phenyl
Sepharose
Mono P
Mono P
UniProt
IPI
Glycerol
Gradient
Heparin
Sepharose
Heparin
Sepharose
Glycerol
Gradient
Heparin
Sepharose
Trifunctional enzyme subunit a
(hydroxyacyl-CoA dehydrogenase/
3-ketoacyl-CoA thiolase/enoyl-CoA
hydratase subunit a)
P40939
IPI00031522
106; 98
1; 2
2; 6
1; 1
4; 7
Peroxisomal bifunctional enzyme
(enoyl-CoA hydratase/3-hydroxyacyl
CoA dehydrogenase)
Q08426
IPI00216164
46; 168
2; 3
3; 5
2; 6
1; 2
RNA (guanine-9-)methyltransferase
domain containing 1
Q7L0Y3
IPI00099996
14; 19
13; 14
17; 15
5; 2
2; 3
Carbamoyl-phosphate synthetase 1
P31327
IPI00011062
3; 2
35; 58
16; 42
11; 34
7; —
Acetyl-CoA acetyltransferase 1
(acetoacetyl CoA thiolase)
P24752
IPI00030363
183; 87
17; 18
8; 10
12; 9
12; 10
Heat-shock 70 kDa protein 9
(stress-70 protein, mortalin)
P38646
IPI00007765
4; 6
14; 23
9; 12
—; 111
25; —
Hydroxysteroid (17-b) dehydrogenase
10 (3-hydroxyacyl-CoA dehydrogenase
type 2)
Q99714
IPI00017726
21; 36
12; 29
18; 35
7; 15
11; 15
ATP synthase subunit a
P25705
IPI00440493
29; 66
8; 24
29; 61
8; 8
66; —
Mitochondrial transcription factor B2
(mitochondrial dimethyladenosine
transferase 2)
Q9H5Q4
IPI00034069
102; —
6; 4
42; 52
39; 39
9; 8
Eukaryotic translation elongation
factor 1 a-like 3
Q5VTE0
IPI00472724
137; 147
30; 49
70; 39
29; 31
34; 11
Poly(A) RNA polymerase
(PAP-associated domain containing 1)
Q9NVV4
IPI00470416
205; 160
37; 19
71; 37
17; 11
85; —
40S ribosomal protein S3
P23396
IPI00011253
84; —
18; 55
36; 55
152; 136
10; 16
Translation elongation factor Tu
P49411
IPI00027107
13; 16
—; 162
41; 49
151; —
96; —
Peroxisomal trans-2-enoyl-CoA
reductase
Q9BY49
IPI00744627
123; 152
23; 41
118; 89
46; 75
36; 67
Mitochondrial histidyl-tRNA
synthetase 2
P49590
IPI00027445
114; —
27; 39
—; 267
63; 64
29; 48
Uncharacterized protein C17orf42
Q96QE5
IPI00170503
172; —
56; 47
203; 198
43; 54
39; 44
Leucine-rich repeat containing 59
Q96AG4
IPI00396321
133; 221
33; 37
59; 34
163; 125
106; —
ATP-dependent RNA helicase DEAD
box 5
P17844
IPI00017617
223; —
—; 103
89; 136
73; 45
32; 22
peptidyl-prolyl cis-trans isomerase B
(cyclophilin B)
P23284
IPI00646304
226; —
68; 122
212; —
140; 84
13; 14
Mitochondrial single-stranded
DNA-binding protein
Q04837
IPI00029744
111; 137
57; 60
178; 124
95; 70
103; 54
Protein (Common Synonyms)a
+ ORF
Mono Q
a
Gene
Proteins identified in all five mtRNase P preparations. Four obvious contaminants/artifacts (Keratins, Titin) excluded (see Table S8 for a complete
listing and Tables S1–S9 for further details not listed here).
b
Ranking based on the number of shared peptides per protein for each preparation and mass spectrometry analysis. Group number from ion-trap
mass spectrometer and Spectrum Mill analysis listed first; group number from quadrupole time-of-flight (QTOF) mass spectrometer and Mascot/
EpiCenter analysis listed after the semicolon; —, not identified by one of the mass spectrometry platforms (depending on position).
9
C1 MT
7o D1
rf
42
Figure 2. Affinity Purification of Presumptive
mtRNase P Proteins Overexpressed in 293
Cells
HS
=
0
B1
7
B1
HS
B1
7B
1
0
1
TD
9M
RG
9M
KIAA0391 was stopped when the association of RG9MTD1 with mtRNase P was
identified.
A protein of approximately 26 kDa copurified with RG9MTD1 (Figure 2B, lanes 11 and
13). The apparent association was confirmed by overexpression of RG9MTD1
with a FLAG- instead of a 63 His-tag and
subsequent anti-FLAG immunoprecipitation
instead of immobilized metal affinity chromatography (IMAC) (Figures 2C and 2D).
Mass spectrometry revealed the 26 kDa protein to be hydroxysteroid (17-b) dehydrogenase 10 (HSD17B10), in the literature
more frequently referred to as 3-hydroxyacyl-CoA dehydrogenase type 2 (HADH2). In fact HSD17B10 was a constituent of
the PPOP (Table 1), but although its rank distribution in the five
subproteomes was compatible with the relative specific activity
of mtRNase P, we did not initially consider it as a mtRNase P proRG
TD
1
RG
(A) mtRNase P activity of mitochondrial extracts from
293 cells overexpressing His-tagged RG9MTD1 (I) or
C17orf42 (II) and immobilized metal affinity batch
chromatography of those; substrate, (mt)pre-tRNATyr.
(B) 8% SDS-PAGE and silver staining of samples assayed in (A).
(C) mtRNase P activity of mitochondrial extracts from
293 control cells (c) or cells overexpressing FLAGtagged RG9MTD1 (I) and fractions of an anti-FLAG immunoprecipitation; substrate, (mt)pre-tRNATyr.
(D) 10% SDS-PAGE and silver staining of samples assayed in (C).
metabolism (database annotation and/or specific domains/
motifs), (2) they were predicted to be mitochondrial, and
(3) they showed a rank distribution in the five subproteomes
roughly consistent with the relative specific activity of mtRNase
P in the respective preparations. The three proteins selected for
further testing were RNA (guanine-9-)methyltransferase domain
containing 1 (RG9MTD1; UniProt Q7L0Y3), the uncharacterized
Table 1. Partial Purification Overlap Proteome
Rank (Group #) in mtRNase P Preparationsb
“MRPP2”
X
X
+ ORF
KIAA0391
Mono Q
Phenyl
Sepharose
Mono P
Mono P
UniProt
IPI
Glycerol
Gradient
Heparin
Sepharose
Heparin
Sepharose
Glycerol
Gradient
Heparin
Sepharose
Trifunctional enzyme subunit a
(hydroxyacyl-CoA dehydrogenase/
3-ketoacyl-CoA thiolase/enoyl-CoA
hydratase subunit a)
P40939
IPI00031522
106; 98
1; 2
2; 6
1; 1
4; 7
Peroxisomal bifunctional enzyme
(enoyl-CoA hydratase/3-hydroxyacyl
CoA dehydrogenase)
Q08426
IPI00216164
46; 168
2; 3
3; 5
2; 6
1; 2
RNA (guanine-9-)methyltransferase
domain containing 1
Q7L0Y3
IPI00099996
14; 19
13; 14
17; 15
5; 2
2; 3
Carbamoyl-phosphate synthetase 1
P31327
IPI00011062
3; 2
35; 58
16; 42
11; 34
7; —
Acetyl-CoA acetyltransferase 1
(acetoacetyl CoA thiolase)
P24752
IPI00030363
183; 87
17; 18
8; 10
12; 9
12; 10
Heat-shock 70 kDa protein 9
(stress-70 protein, mortalin)
P38646
IPI00007765
4; 6
14; 23
9; 12
—; 111
25; —
Hydroxysteroid (17-b) dehydrogenase
10 (3-hydroxyacyl-CoA dehydrogenase
type 2)
Q99714
IPI00017726
21; 36
12; 29
18; 35
7; 15
11; 15
ATP synthase subunit a
P25705
IPI00440493
29; 66
8; 24
29; 61
8; 8
66; —
Mitochondrial transcription factor B2
(mitochondrial dimethyladenosine
transferase 2)
Q9H5Q4
IPI00034069
102; —
6; 4
42; 52
39; 39
9; 8
Eukaryotic translation elongation
factor 1 a-like 3
Q5VTE0
IPI00472724
137; 147
30; 49
70; 39
29; 31
34; 11
Poly(A) RNA polymerase
(PAP-associated domain containing 1)
Q9NVV4
IPI00470416
205; 160
37; 19
71; 37
17; 11
85; —
40S ribosomal protein S3
P23396
IPI00011253
84; —
18; 55
36; 55
152; 136
10; 16
Translation elongation factor Tu
P49411
IPI00027107
13; 16
—; 162
41; 49
151; —
96; —
Peroxisomal trans-2-enoyl-CoA
reductase
Q9BY49
IPI00744627
123; 152
23; 41
118; 89
46; 75
36; 67
Mitochondrial histidyl-tRNA
synthetase 2
P49590
IPI00027445
114; —
27; 39
—; 267
63; 64
29; 48
Uncharacterized protein C17orf42
Q96QE5
IPI00170503
172; —
56; 47
203; 198
43; 54
39; 44
Leucine-rich repeat containing 59
Q96AG4
IPI00396321
133; 221
33; 37
59; 34
163; 125
106; —
ATP-dependent RNA helicase DEAD
box 5
P17844
IPI00017617
223; —
—; 103
89; 136
73; 45
32; 22
peptidyl-prolyl cis-trans isomerase B
(cyclophilin B)
P23284
IPI00646304
226; —
68; 122
212; —
140; 84
13; 14
Mitochondrial single-stranded
DNA-binding protein
Q04837
IPI00029744
111; 137
57; 60
178; 124
95; 70
103; 54
Protein (Common Synonyms)a
“MRPP1”
Mono Q
a
Gene
Proteins identified in all five mtRNase P preparations. Four obvious contaminants/artifacts (Keratins, Titin) excluded (see Table S8 for a complete
listing and Tables S1–S9 for further details not listed here).
b
Ranking based on the number of shared peptides per protein for each preparation and mass spectrometry analysis. Group number from ion-trap
mass spectrometer and Spectrum Mill analysis listed first; group number from quadrupole time-of-flight (QTOF) mass spectrometer and Mascot/
EpiCenter analysis listed after the semicolon; —, not identified by one of the mass spectrometry platforms (depending on position).
his-tagged MRPP1 extracts on IMAC column
60kDa
f12 contains something released
from iMAC-MRPP1(:MRPP2) that can
reconstitute activity!
17 proteins, including KIAA0391
Figure 3. Identification of a Further Subunit by Functional Reconstitution of mtRNase P from Recombinant MRPP1 and MRPP2
and Mitochondrial Fractions
(A) Reconstitution of mtRNase P activity from recombinant MRPP1 and MRPP2 and from mitochondrial extract (mtE) fractionated by 10% SDS-PAGE. Schematic
indication of the fractionation shown on top; a similar, but unstained and unfixed, gel, fractionated according to a prestained molecular weight standard, was used
for protein elution; substrate, (mt)pre-tRNAIle.
(B) Chromatogram of an IMAC of extract from 293 cells overexpressing His-tagged MRPP1. Mitochondrial extract was loaded with 100 mM NaCl and column
developed with 50 mM NaCl steps (indicated in red). Peak fraction from reconstitution in (C) indicated by gray shading.
(C) mtRNase P activity of mitochondrial extract (mtE) and of indicated IMAC fractions from (B) assayed in the absence or presence of recombinant MRPP1 and
MRPP2; substrate, (mt)pre-tRNATyr.
SDS-PAGE-fractionated mitochondrial extract in the 60 kDa
were identified (Table S10), two of which were also present in
Table 1. Partial Purification Overlap Proteome
Rank (Group #) in mtRNase P Preparationsb
“MRPP2”
X
+ ORF
KIAA0391
“MRPP3”
Mono Q
Phenyl
Sepharose
Mono P
Mono P
UniProt
IPI
Glycerol
Gradient
Heparin
Sepharose
Heparin
Sepharose
Glycerol
Gradient
Heparin
Sepharose
Trifunctional enzyme subunit a
(hydroxyacyl-CoA dehydrogenase/
3-ketoacyl-CoA thiolase/enoyl-CoA
hydratase subunit a)
P40939
IPI00031522
106; 98
1; 2
2; 6
1; 1
4; 7
Peroxisomal bifunctional enzyme
(enoyl-CoA hydratase/3-hydroxyacyl
CoA dehydrogenase)
Q08426
IPI00216164
46; 168
2; 3
3; 5
2; 6
1; 2
RNA (guanine-9-)methyltransferase
domain containing 1
Q7L0Y3
IPI00099996
14; 19
13; 14
17; 15
5; 2
2; 3
Carbamoyl-phosphate synthetase 1
P31327
IPI00011062
3; 2
35; 58
16; 42
11; 34
7; —
Acetyl-CoA acetyltransferase 1
(acetoacetyl CoA thiolase)
P24752
IPI00030363
183; 87
17; 18
8; 10
12; 9
12; 10
Heat-shock 70 kDa protein 9
(stress-70 protein, mortalin)
P38646
IPI00007765
4; 6
14; 23
9; 12
—; 111
25; —
Hydroxysteroid (17-b) dehydrogenase
10 (3-hydroxyacyl-CoA dehydrogenase
type 2)
Q99714
IPI00017726
21; 36
12; 29
18; 35
7; 15
11; 15
ATP synthase subunit a
P25705
IPI00440493
29; 66
8; 24
29; 61
8; 8
66; —
Mitochondrial transcription factor B2
(mitochondrial dimethyladenosine
transferase 2)
Q9H5Q4
IPI00034069
102; —
6; 4
42; 52
39; 39
9; 8
Eukaryotic translation elongation
factor 1 a-like 3
Q5VTE0
IPI00472724
137; 147
30; 49
70; 39
29; 31
34; 11
Poly(A) RNA polymerase
(PAP-associated domain containing 1)
Q9NVV4
IPI00470416
205; 160
37; 19
71; 37
17; 11
85; —
40S ribosomal protein S3
P23396
IPI00011253
84; —
18; 55
36; 55
152; 136
10; 16
Translation elongation factor Tu
P49411
IPI00027107
13; 16
—; 162
41; 49
151; —
96; —
Peroxisomal trans-2-enoyl-CoA
reductase
Q9BY49
IPI00744627
123; 152
23; 41
118; 89
46; 75
36; 67
Mitochondrial histidyl-tRNA
synthetase 2
P49590
IPI00027445
114; —
27; 39
—; 267
63; 64
29; 48
Uncharacterized protein C17orf42
Q96QE5
IPI00170503
172; —
56; 47
203; 198
43; 54
39; 44
Leucine-rich repeat containing 59
Q96AG4
IPI00396321
133; 221
33; 37
59; 34
163; 125
106; —
ATP-dependent RNA helicase DEAD
box 5
P17844
IPI00017617
223; —
—; 103
89; 136
73; 45
32; 22
peptidyl-prolyl cis-trans isomerase B
(cyclophilin B)
P23284
IPI00646304
226; —
68; 122
212; —
140; 84
13; 14
Mitochondrial single-stranded
DNA-binding protein
Q04837
IPI00029744
111; 137
57; 60
178; 124
95; 70
103; 54
Protein (Common Synonyms)a
“MRPP1”
Mono Q
a
Gene
Proteins identified in all five mtRNase P preparations. Four obvious contaminants/artifacts (Keratins, Titin) excluded (see Table S8 for a complete
listing and Tables S1–S9 for further details not listed here).
b
Ranking based on the number of shared peptides per protein for each preparation and mass spectrometry analysis. Group number from ion-trap
mass spectrometer and Spectrum Mill analysis listed first; group number from quadrupole time-of-flight (QTOF) mass spectrometer and Mascot/
EpiCenter analysis listed after the semicolon; —, not identified by one of the mass spectrometry platforms (depending on position).
All 3 proteins
required
All 3 proteins
required
Correct
cleavage
site
Correct
cleavage site
5´-phosphate
5´-phosphate
No other E.coli RNA involved (note that tRNA will inhibit enzyme)
nuclease relieves inhibition by E.coli P RNA
E.coli P RNA actually inhibits activity (binds substrate, but can’t cleave at the ionic strength?)
nuclease resistant (no RNA)
nuclease destroys E.coli P RNA, but boosts activity
nuclease destroys contaminating RNA, but no loss of activity
Figure 5. Accumulation of Mitochondrial tRNA Precursors after RNAi-Mediated Silencing of MRPP1, MRPP2, or MRPP3 Gene Expression
MRPP1, MRPP2, or MRPP3 gene expression in HeLa cells was transiently knocked down by transfection of specific siRNAs. mRNA and (mt)pre-tRNA levels were
determined by quantitative real-time RT-PCR (forward primers for (mt)pre-tRNA analysis were located in the rRNA or mRNA sequence preceding the respective
tRNAs’ 50 end in the mitochondrial primary transcript, while reverse primers were located in the tRNA sequence). RNA quantities were normalized for 18S rRNA
and GAPDH mRNA levels and expressed relative to those of untreated cells from the same experiment and time after transfection. Mean and SEM of triplicate
analyses are shown.
(A) Relative quantity of MRPP1 mRNA after RNAi-mediated silencing of MRPP1 gene expression (semi-logarithmic plot).
(B) Relative quantity of MRPP2 mRNA after RNAi-mediated silencing of MRPP2 gene expression (semi-logarithmic plot).
(C) Relative quantity of MRPP3 mRNA after RNAi-mediated silencing of MRPP3 gene expression (semi-logarithmic plot).
(D) Relative quantity of (mt)pre-tRNAVal after RNAi-mediated silencing of MRPP1 gene expression.
(E) Relative quantity of (mt)pre-tRNAVal after RNAi-mediated silencing of MRPP2 gene expression.
(F) Relative quantity of (mt)pre-tRNAVal after RNAi-mediated silencing of MRPP3 gene expression.
(G) Relative quantity of (mt)pre-tRNAIle after RNAi-mediated silencing of MRPP1 gene expression.
(H) Relative quantity of (mt)pre-tRNAIle after RNAi-mediated silencing of MRPP2 gene expression.
(I) Relative quantity of (mt)pre-tRNAIle after RNAi-mediated silencing of MRPP3 gene expression.
moreover been linked to a form of X-linked mental retardation
MRPP1/MRPP2 complex via its NAD+-binding domain; this do-
Figure 6. Loss of mtRNase P Activity after RNAi-Mediated Silencing
of MRPP1, MRPP2, or MRPP3 Gene Expression
(A) mtRNase P activity in mitochondrial extracts of siRNA-treated HeLa cells.
Mitochondrial extracts were prepared from cells transfected with control
siRNA, MRPP1 siRNA #1, MRPP2 siRNA #2, or MRPP3 siRNA #2 after the indicated time in parallel with untransfected cells (no siRNA). Extracts were adjusted for protein content and assayed for mtRNase P activity; substrate, 2 nM
(mt)pre-tRNATyr.
(B) Same as (A) but substrate (mt)pre-tRNAIle.
similar C-terminal spacing (Figure S1;
ther comments). In the largest block o
all putative homologs (Figure S1B; 476
the identity of the only four amino ac
most striking: (three) aspartate(s) and
of which are most commonly involved i
ordination of protein metallonuclease
thus seductive to speculate that MRPP
lonuclease domain and enables RNA h
basically identical to those proposed
(Steitz and Steitz, 1993).
Whereas all three mtRNase P prote
RNA-binding potential, their exact st
tions, their individual contribution to s
specificity, and their precise role in hy
to be determined. Nonetheless, some o
their identification warrant extra ment
MRPP2 constitute a stable subcomp
a limiting building block of the holoenzy
pression alone was sufficient to incre
and (3) interaction of MRPP3 with the M
plex is so weak that it is apparently brok
as low as 150 mM. The last observ
whether mtRNase P is at all a stable p
the concerted action of two more dyn
units on a common substrate. In any ca
of the holoenzyme finally turned out to
countered during enzyme purification,
and increase in specific activity. In fac
a conventional purification-to-homoge
all have been successful, and we be
proach is a more generally useful stra
of the components of fragile enzymatic
mtRNase P’s Relation to Other RNa
As exotic as the composition of human
A Protein-Only RNase P in Human
Mitochondria
Scott C. Walker1 and David R. Engelke1,*
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606, USA
*Correspondence: engelke@umich.edu
DOI 10.1016/j.cell.2008.10.010
1
In bacteria, archaea, and the eukaryote nucleus, the endonuclease ribonuclease P (RNase P)
is composed of a catalytic RNA that is assisted by protein subunits. Holzmann et al. (2008)
now provide evidence that the human mitochondrial RNase P is an entirely protein-based
enzyme.
Ribonuclease P (RNase P) is responsible for the 5! maturation of precursor
transfer RNAs (pre-tRNAs). The catalytic RNA subunit of bacterial RNase P
was one of the first examples of RNAbased catalysis. All forms of RNase P
characterized to date have a fundamen-
tally similar RNA subunit that retains
catalytic activity. This catalytic RNA is
widely presumed to be a remnant of
the hypothesized “RNA world” in which
RNA is thought to have been the original
functional macromolecule preceding the
evolution of protein. Although the nature
of the RNase P catalytic RNA seems
evolutionarily conserved, the number
of proteins associated with the RNA
actually increases with the complexity
of the organism—ranging from one in
bacteria to at least four in archaea and
to at least nine in eukaryotes (Figure 1)
Figure 1. The Evolution of RNase P
(Left) The compositions of characterized RNA-based RNase P enzymes from bacteria, archaea, and eukarya show an increase in protein content with increased
complexity of the organism. The sites of interaction between RNase P subunits are not known in most cases and are represented schematically. The structure
of the proposed ancestral RNA-only RNase P is not known and is assumed to have the critical structural elements conserved in all forms of RNase P RNA.
(Right) The composition of the fully characterized mitochondrial RNase P is shown for yeast (S. cerevisiae) and human (H. sapiens). Human mtRNase P is
composed only of proteins (mitochondrial RNase P proteins 1, 2, 3) (Holzmann et al., 2008). The third subunit of the human mtRNase P (MRPP3) binds to the
two-protein subcomplex weakly and may associate dynamically (arrow). Although key structural elements of the RNA subunit are preserved in various yeast
mtRNase P enzymes (solid line), the entire RNA structure is not well defined (dashed line).
412
Cell 135, October 31, 2008 ©2008 Elsevier Inc.
Are there any other potential non-RNA RNase P’s?
Trypanosomes?
Green plant
chloroplasts?
Aquifex
aeolicus?
Pyrobaculum?
NO!
Putative RNase P RNA
Pyrobaculum aerophilum
AAA
U
Rough draft secondary structure
A
G
A
7/13/06 Jim Brown / Todd Lowe
G C
A A GG
P10
CG G C
A
G
A
G C CG
GC
P7
CC
C
GG
P8
G
G G C GG
GG
P5
P15 A A G C A P16 U G
CU
GC
U
G G GC C G
CU
GC GG C C C C C
A GC
CG
G
C
G
C C C GG
C GC C
GG G
A
G
G
G
A
A
C A
G
C
U
G
C
G
G
P17 G G
C
C
G
GCG
G
U
C
G
P6
A
C
G
UC
A
C
U
C
A
A
A
G
P3
G
A
A CGGGC C C C UU C U G
G
P4
U
A GC C C G G G GG G G C G A
G
A
A G G C P2
G C
P1
G
A
5´ G
A
G GG C G C C G
A
C
A
C
C
G
C
G
G
C
C
G
C
A
3´
G
A
C
AU GGGGGC G
Nanoarchaeum?
No P!
Next MJC