[RNA Biology 4:3, 169-172, July–December 2007]; ©2007 Landes Bioscience
Brief Communication
Is Alba an RNase P Subunit?
J. Chris Ellis1
Jeffrey Barnes2
James W. Brown3,*
1Laboratory of Signal Transduction; NIEHS; Research Triangle Park, North
Carolina USA
2Applied Biosystems, Inc.; Foster City, California USA
3Department of Microbiology; North Carolina State University; Raleigh,
North Carolina USA
*Correspondence to: James W. Brown; Associate Professor, Department of
Microbiology; North Carolina State University; Campus Box 7615; Raleigh, North
Carolina 27695 USA; Tel.: 919.515.8803; Email: james_brown@ncsu.edu
Original manuscript submitted: 11/06/07
Manuscript accepted: 11/25/07
Previously published online as a RNA Biology E-publication:
http://www.landesbioscience.com/journals/rnabiology/article/5347
Key words
ribonuclease P, Archaea, ribozyme, Methanothermobacter thermoautotrophicus, Mth1483,
Rpp25
Acknowledgements
We thank Mark Foster, Ventkat Gopalan
and the Fermentation Facility at The Ohio
State University, for the gifts of recombinant
Mth1483 protein, unpublished data, and M.
thermoautotrophicus cell paste, respectively.
We also thank Danielle McLaurin and Alina
Lotstein for technical assistance. This work
was supported by a fellowship to J.C.E. from
the NCSU Biotechnology Training program,
funded by the NSF.
www.landesbioscience.com
Abstract
It has been suggested that Alba, a well‑established chromatin protein in Archaea,
is also a subunit of the archaeal RNase P holoenzyme, based on the observation that
the homolog of this protein in humans has been shown to be associated with RNase
P activity. Using the same biochemical methods we used previously to show that four
other proteins homologous to eukaryotic RNase P proteins are bona fide RNase P
subunits in Archaea, we could not detect any association of the Alba homolog in
Methanothermobacter thermoautotrophicus (Mth1483p) with the RNase P holoenzyme.
In addition, the presence of Mth1483p did not enhance the activity of RNase P holoen‑
zyme reconstituted from recombinant subunits. In conclusion, we find no evidence that
Alba is an RNase P subunit.
Abbreviations
RNase P, ribonuclease P; Mth1483, ORF 1483 of the Methanothermobacter thermoautotrophicus genome; Mth1483p, the protein encoded by Mth1483; Mth11/Mth11p,
the gene and encoded protein of ORF 11 of the M. thermoautotrophicus genome;
Mth1618/Mth1618p, the gene and encoded protein of ORF 1618 of the M. thermoautotrophicus genome; Mth687/Mth687p, the gene and encoded protein of ORF 687
of the M. thermoautotrophicus genome; Mth688/Mth688p, the gene and encoded
protein of ORF 688 of the M. thermoautotrophicus genome; pre-tRNA, precursor tRNA;
nt, nucleotide; aa, amino acid; Tris, tris-hydroxymethyl aminomethane; SDS-PAGE,
sodium dodecyl sulfate-denaturing polyacrylamide gel electrophoresis
Introduction
Ribonuclease P is a ribonuclease best known for the processing of pre-tRNA by
removing the 5' leader sequences (reviewed in refs 1–3). In all instances in which the
subunits have been identified, RNase P is composed of both RNA and protein subunits.
RNase P is best understood in bacteria, in which it is comprised of a large RNA
(ca. 400 nt/140 kDa) and a single small (ca. 120 aa/14 kDa) protein subunit. Bacterial
RNase P RNAs are capable in vitro (at elevated ionic strength) of cleaving pre-tRNAs in
the absence of protein.
In contrast, the eukaryotic nuclear RNase P contains a single RNA, which is a distant
homolog of the bacterial RNA, but many proteins.4,5 At least nine proteins in Homo
sapiens (hPOP1, Rpp29, hPOP5, Rpp20, Rpp30, Rpp21, Rpp40, Rpp25 and Rpp14) are
physically associated with RNase P, and 10 proteins in Saccharomyces cerevisiae (Pop1–8,
Rpp1 and Rpp2). The eukaryotic nuclear RNase P RNAs are dependent on protein
for activity in vitro (except at very low levels in some cases6) and, at least in
S. cerevisiae, in vivo as well.4 Nevertheless, as in bacteria, the RNA is the catalytic subunit
of the enzyme.6,7
RNase P in Archaea is composed of a single RNA and four conserved proteins.8,9
The RNA subunit is remarkably similar in secondary structure (less so in sequence) to
its bacterial homolog, and in most instances contain all of the structural and sequence
elements in the phylogenetically conserved “core” of the bacterial RNA;9 those that do
are likewise able in vitro to cleave pre-tRNA in the absence of protein at elevated (very
elevated) ionic strength.10 The four established RNase P proteins in Archaea are necessary
and sufficient for reconstitution of efficient enzymatic activity in vitro at moderate ionic
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Is Alba an RNase P Subunit?
strength,11‑13 although low levels of activity can be reconstituted
from subsets of the proteins.13 These four proteins were originally
identified as potential RNase P subunits based on their distant
but discernible similarity to four of the eukaryotic nuclear RNase
P proteins: Pop5/hPop5 (=Mth687p), Pop4/Rpp29 (=Mth11p),
Rpp1/Rpp30 (=Mth688p) and Rpr2/Rpp21 (=Mth1618p).8,11,14
One of these proteins, Mth687p (the homolog of eukaryotic Pop5p)
has a three‑dimensional fold that resembles the conserved structure of the single bacterial RNase P protein, although there is no
recognizable sequence similarity between these bacterial proteins
and the archaeal/eukaryotic proteins.15,16 In addition to these four
proteins, ribosomal protein L7 improves the thermostability of the
reconstituted holoenzyme in Pyrococcus horikoshii;17 this is probably
an idiosyncrasy of the enzyme in the Pyrococcus/Thermococcus
lineage. In addition to its role in the ribosome, L7 in Archaea is a
component of box C/D snoRNPs.18
H. sapiens RNase P protein Rpp25 has only tenuous similarity to
the S. cerevisiae RNase P protein Pop8p,19 but there is clear similarity
to an archaeal protein known as Sso10b or “Alba.”20 This protein has
been extensively characterized in Sulfolobus acidocaldarius, in which
it is a dimeric, highly basic protein that binds cooperatively and at
high density to DNA, inducing negative supercoiling and compacting
the DNA.21‑23 DNA binding affinity of Alba is regulated by Sir2,
which deacetylates a key lysine, modulating gene expression.24‑26
The structure of the N‑terminus of Alba is similar to the DNA
binding domain of DNase I, followed by a region of similarity to
initiation factor domain IF3 at the C‑terminus.21,27 Although the
role of Alba as a DNA binding protein is clear, it has been suggested
that it has a dual role in Archaea: one in DNA binding, and the other
in RNase P.20 The role of Alba in RNase P function was proposed
the basis of its similarity in sequence to the H. sapiens RNase P
protein Rpp25, and the presence of both DNA‑ and RNA‑binding
structural motifs, rather than experimental evidence. It was further
suggested that Alba may originally have been an RNA binding
protein that has retained this function, but subsequently evolved
DNA binding properties in Archaea.
Is Alba a component of the RNase P holoenzyme in Archaea? We
examined this question using the same approaches that were used to
confirm the presence of the four previously identified proteins in the
archaeal RNase P, but could find no evidence for either the physical
association of this protein with the native RNase P holoenzyme nor
enhancement of catalytic activity of enzyme reconstituted in the
presence of this protein.
Materials and Methods
Methods were substantially the same as previously described for
the confirmation of Mth11p, Mth687p, Mth688p and Mth1618p
as RNase P protein subunits.8
RNase P cleavage assays. Enzyme samples were assayed for RNase
P activity in 10 mL reactions containing 50 mM Tris‑Cl pH 8.0,
20 mM MgCl2, 1 M NH4Cl and ca. 2 nCi 32P‑labeled pre-tRNA.
Reactions were incubated at 50°C for 30 minutes, and products were
separated by electrophoresis in 8% urea‑polyacrylamide gels and
visualized by autoradiography.
Production of antisera to Mth1483p. Antiserum against
Mth1483p was produced by Cocalico Biologics from recombinant protein provided by Mark Foster, Ohio State University.
Antisera against the other RNase P proteins has been previously
described.8
170
Figure 1. Mth1483p does not copurify with RNase P activity. The level of
RNase P activity in each fraction is shown above the corresponding level
of Mth1483p detected in the western blot below. r1483p = 50 ng purified
recombinant his‑tagged Mth1618p, 25–39 = 5 mL of glycerol gradient
fractions (numbered from the top of the gradient), probed with a 1:1000 of
anti‑Mth1483p antiserum.
Purification of M. thermoautotrophicus RNase P. M. thermoautotrophicus cell extracts were fractionated using DEAE‑Trisacryl Plus
M (Sigma‑Aldrich). Active fractions were pooled and samples were
fractionated in 10–40% glycerol gradients. Fractions collected from
the bottom of the tubes were assayed for RNase P activity.
Western blots for detection of Mth1483p. Samples of purified
RNase P and recombinant Mth1483p were separated by SDS‑
PAGE and transferred to nitrocellulose membranes. Blots were probed
with anti‑Mth1483 serum and subsequently with HRP‑conjugated
goat anti‑rabbit IgG (Supersignal West Pico Rabbit IgG Detection
Kit, Pierce #34083), followed by visualization according to the
manufacturers instructions.
Immunoprecipitation of RNase P using antisera to Mth1483p.
Protein‑A agarose was incubated with immune or preimmune serum,
and cross‑linked with sodium borate and dimethyl pimelimidate.
Purified RNase P was added and mixed overnight at 4°C. The beads
were pelleted, and the supernatant (“flow‑through”) collected. Beads
were washed and eluted with three washes of elution buffer at 72°C.
Samples of the final bead slurry, flow‑through, and elutions were
tested for RNase P activity.
Reconstitution of active RNase P-holoenzyme. His‑tagged
recombinant RNase P proteins dissolved in 8 M urea were diluted,
bound to Ni‑NTA agarose (Qiagen),washed, and eluted by treatment
with Factor Xa (Pierce). Aliquots of these proteins were added to
RNase P assay buffer (above) containing M. thermoautotrophicus
RNase P RNA, heated to 65°C for 5 min, cooled to 50°C and then
tested for RNase P activity.
Results and Discussion
The Mth1483p does not co-purify with RNase P-activity.
Mth1483p was detected in western blots of glycerol gradient fractions of partially‑purified RNase P from M. thermoautotrophicus,
but the peaks of RNase P and Mth1483 protein are well‑separated,
with Mth1483p sedimenting more rapidly even than the RNase P
holoenzyme (Fig. 1). We have shown previously that all four bona
fide RNase P proteins copurify exactly with RNase P activity in these
gradients.9 Mth1483p could not be detected in the purified RNase P
post‑glycerol‑gradients, whereas Mth11p and Mth1618p were readily
detected in this purified enzyme preparation (data not shown).
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2007; Vol. 4 Issue 3
Is Alba an RNase P Subunit?
Figure 2. Anti‑Mth1483p sera does not immunoprecipitate RNase P activity.
Immune and preimmune sera to Mth1483p was bound to protein‑A agarose
beads and mixed with glycerol gradient purified RNase P. Reactions were
washed and eluted with three times with elution buffer heated to 72°C. Beads
and elutions were assayed for RNase P activity.
An additional line of evidence against a role for Alba homologs
in Archaea as RNase P subunits is that deletion of the gene for Alba
(albA) in Methanococcus voltae does not affect growth, but does affect
the overall protein pattern in a way similar to deletion mutants
of the other chromatin protein genes hmvA and hstB, encoding
a histone‑like protein and a histone, respectively.28 In contrast,
deletion of any of the ten nuclear RNase P proteins in S. cerevisiae
is lethal,4 as is elimination of the single RNase P protein in Bacillus
subtilis.29
In conclusion, although there is evidence that the H. sapiens
homolog of the archaeal chromatin protein Alba is physically
associated with RNase P, the biochemical evidence does not support
a second role for Alba in the archaeal RNase P holoenzyme. Given
that other eukaryotic nuclear RNase P enzymes (e.g., yeast) do not
contain clear homologs of this protein, if the Rpp25 protein really
is a subunit of the human enzyme, it seems more likely that this
represents a recruitment of this protein to the RNase P holoenzyme
in a recent (in evolution time scales) ancestor of humans, rather than
an ancient link between chromatin structure and RNA processing.
References
Figure 3. Mth1483p does not inhance reconstitution of RNase P activity
from recombinant components. Recombinant RNase P RNA and protein
components indicated (each at ca. 100 nM) were reconstituted and
assayed for RNase P activity. Native = partially‑purified M. thermoautotrophicus RNase P.
Anti‑Mth1483 does not immunoprecipitate RNase P activity.
RNase P activity was not retained by protein-A agarose treated
with anti‑Mth1483p immune serum (Fig. 2). In contrast, parallel
reactions using anti‑Mth11p serum efficiently retained RNase P
activity compared to the preimmune sera (data not shown). We have
previously shown that antisera against all four bona fide RNase P
proteins immunoprecipitates RNase P activity from partially purified
or purified RNase P preparations from M. thermoautotrophicus.9
Mth1483p does not enhance reconstitution of RNase P activity.
The RNase P RNA and the four known RNase P proteins are all
necessary and sufficient to reconstitute efficient RNase P activity
in the M. thermoautotrophicus, Pyrococcus horikoshii and Pyrococcus
furiosus systems; Mth1483p or the Alba homologs in the Pyrococcus
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poor solubility of two of the proteins12), the inclusion of Mth1483p
in reconstitution assays does not improve activity (Fig. 3).
www.landesbioscience.com
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