Prediction of signal recognition particle RNA genes

Marco Regalia
1
,
2
, Magnus Alm Rosenblad
1
and Tore Samuelsson
1
,
*
1
Department of Medical Biochemistry, Goteborg University, Box 440, SE-405 30 Goteborg, Sweden and
2
Department of General Physiology and Biochemistry, University of Milan, Via Celoria 20, Milan, I-20133, Italy
Received April 8, 2002; Revised and Accepted June 17, 2002
ABSTRACT
We describe a method for prediction of genes that
encode the RNA component of the signal recogni-
tion particle (SRP). A heuristic search for the
strongly conserved helix 8 motif of SRP RNA is
combined with covariance models that are based on
previously known SRP RNA sequences. By screen-
ing available genomic sequences we have identied
a large number of novel SRP RNA genes and we can
account for at least one gene in every genome that
has been completely sequenced. Novel bacterial
RNAs include that of Thermotoga maritima, which,
unlike all other non-gram-positive eubacteria, is pre-
dicted to have an Alu domain. We have also found
the RNAs of Lactococcus lactis and Staphylococcus
to have an unusual UGAC tetraloop in helix 8
instead of the normal GNRA sequence. An investi-
gation of yeast RNAs reveals conserved sequence
elements of the Alu domain that aid in the analysis
of these RNAs. Analysis of the human genome
reveals only two likely genes, both on chromosome
14. Our method for SRP RNA gene prediction is the
rst convenient tool for this task and should be
useful in genome annotation.
INTRODUCTION
Although there are many efcient tools for the identication of
protein genes in genome sequences, we lack tools for the
analysis of many non-coding RNAs. As a consequence most of
the nished genome sequences published today lack annota-
tion information about these RNAs. One difculty with the
identication of non-coding RNAs is that sequence tends to be
poorly conserved and, as a consequence, standard tools such as
BLAST (1) may be used only in the identication of orthologs
in fairly closely related organisms. On the other hand many
non-coding RNAs tend to have conserved secondary structure
elements that may be used for their identication. Methods
that have been used so far include the `covariance models'
developed by Eddy and Durbin (2) as well as `pattern
matching', which is based on regular expression matching
where base-pairing schemes are also taken into account. In the
latter category there is PatScan (http://www-unix.mcs.anl.
gov/compbio/PatScan/HTML/patscan.html) as well as the
`rnabob' tool of Sean Eddy (Washington University School
of Medicine, St Louis, MO; http://www.genetics.wustl.edu/
eddy/software/#rnabob).
In this work we have studied methods to identify the RNA
component of the signal recognition particle (SRP). The SRP
plays an important role in membrane targeting (3 and
references therein). Some of its structural and functional
features have been highly conserved during evolution and
SRP has been identied in all three kingdoms of life. In
eukaryotic cells, the SRP targets secretory proteins to the
endoplasmic reticulum (ER) membrane. Eukaryotic SRP
comprises one RNA molecule and six proteins, including
SRP54, a GTPase. As the signal sequence of the secretory
protein emerges from the ribosome it is recognized by the
SRP54 protein. In the resulting ribosomeSRP complex,
protein synthesis is arrested and the complex eventually docks
to the SRP receptor at the ER membrane. Here, SRP is
released, protein synthesis is resumed and translocation of the
nascent chain in the ER membrane is initiated. The SRP
receptor is composed of two subunits, SRa and SRb, which
are both GTPase proteins.
The SRP pathway in bacteria is similar to that in eukaryotes
although the major substrates for the bacterial SRP seem to be
integral membrane proteins (46). In Escherichia coli the SRP
has only two components: a 4.5S RNA and the Ffh protein,
homologous to SRP54. The bacterial FtsY protein is
homologous to SRa.
The overall design of SRP RNA is somewhat variable, as
shown in Figure 1. However, they all share a common motif
that will be referred to here as the helix 8 motif. The helix 8
region of the RNA binds to the M domain of the SRP54
protein, a domain that also harbors the signal sequence binding
activity. Many studies show that the SRP RNA has an
important biological role. For instance, the E.coli 4.5S RNA is
necessary for viability (7) and in an in vitro system the SRa
can stimulate the GTPase activity of SRP54 only when it is
bound to the RNA (8). It has been shown that the E.coli 4.5S
RNA plays an important role in the GTPase cycles of Ffh and
FtsY (9,10). Furthermore, the three-dimensional structure of
the helix 8/SRP54 domain of SRP has recently been elucidated
(11) and this structure suggests that the signal sequence
binding surface is composed not only of protein but also of
RNA elements.
The Alu domain is believed to be responsible for a
translational arrest activity of SRP and contains the Alu
domain of the RNA in complex with the heterodimer SRP9/
14. Also this complex has been studied in structural detail
(12). The SRP9/14 dimer is bound to a conserved core of the
RNA Alu domain, which forms a U-turn that connects two
*To whom correspondence should be addressed. Tel: +46 31 773 34 68; Fax: +46 31 41 61 08; Email: tore.samuelsson@medkem.gu.se
33683377 Nucleic Acids Research, 2002, Vol. 30 No. 15 2002 Oxford University Press
helical stacks. Based on the appearance of the SRP as a rod-
like structure, with the SRP54 binding site and Alu domain at
opposing ends, it has been speculated that the particle is able
to control translation by reaching from the ribosome
polypeptide exit site to a site of elongation factor or
aminoacyl-tRNA binding. Due to the limited size of the Alu
Figure 1. The three major categories of SRP RNA and conserved elements. (A) Eukaryotic and archaebacterial RNAs that contain helices 6 and 8 as well as
an Alu domain. The human SRP RNA is shown together with a highly conserved motif in helix 5 shared between most SRP RNAs that contain an Alu
domain, as shown in the present work. (B) Eubacterial RNAs that contain an Alu domain but that lack the helix 6 (Bacillus RNA shown). (C) Eubacterial
RNAs without an Alu domain (E.coli RNA shown). The consensus features of the helix 8 region of bacterial SRP RNA are indicated in the lower box.
Lactococcus and Staphylococcus have the UGAC tetraloop sequence. The shaded regions in the lower box correspond to bases in contact with the SRP54
protein as described by Batey et al. (11).
Nucleic Acids Research, 2002, Vol. 30 No. 15 3369
domain it could well enter into a tRNA or elongation factor
binding site.
There are three major classes of SRP RNA with respect to
its domain organization (Fig. 1). Archaebacterial RNAs are
eukaryote-like with an Alu domain as well as a helix 6
domain. A group of gram-positive bacteria, including
Bacillus, has an Alu domain but lacks the helix 6 region.
The remaining eubacteria-like proteobacteria have a simpler
RNA that lacks the Alu domain as well as the helix 6 domain.
The great diversity in terms of SRP RNA structure has
hampered the development of convenient methods for their
identication in genome sequences. In the context of the
Signal Recognition Particle Database (SRPDB) (13) we have
previously attempted to identify SRP RNA genes using a
combination of pattern searches and sequence similarity
searches. However, we have now improved our search
strategy considerably and developed an automated method
that is based on pattern matching and the COVE programs.
Using this method we have systematically searched for SRP
RNA genes in public sequence data and have discovered a
number of novel SRP RNAs as well as novel features of these
RNAs.
MATERIALS AND METHODS
Public sequence data were downloaded from the NCBI and
EBI ftp sites. Finished sequence data as well as HTG sections
were retrieved. Human expressed sequence tag (EST)
sequences were downloaded and used to obtain evidence of
expressed SRP RNA genes. Genomes of microorganisms were
also obtained from the TIGR CMR database at http://
www.tigr.org/tigr-scripts/CMR2/CMRGenomes.spl and sites
cited there. Sequences of Candida albicans were downloaded
from the Stanford Genome Technology Center website
at http://www-sequence.stanford.edu/group/candida (sequenc-
ing of C.albicans was accomplished with the support of the
NIDR and the Burroughs Wellcome Fund). Neurospora
sequences were from the Neurospora Sequencing Project,
Whitehead Institute/MIT Center for Genome Research (http://
www-genome.wi.mit.edu). The data set used in these studies
was neurospora_1.fasta.
Rnabob. Rnabob source code was obtained from http://www.
genetics.wustl.edu/eddy/software/#rnabob. Rnabob descrip-
tors were constructed for the different categories: (i) archae-
bacteria and eubacteria with GRRA loop; (ii) eubacteria with
TRRC loop; (iii) plants; (iv) yeasts; and (v) metazoans. They
are listed and described in more detail at the website http://
bio.lundberg.gu.se/srpscan/. The rnabob source code was
slightly modied to produce output with sequences anking
the descriptor pattern and to be able to produce sequence
output in fasta format. It was used to scan the sequence
databases with the appropriate descriptors for the different
classes of SRP RNA genes and to produce outputs in fasta
format using the -f option. The -s (skip) option was used to
avoid non-signicant hits in regions of `N' repeats.
COVE. The COVE programs (version 2.4.2) were obtained
from http://www.genetics.wustl.edu/eddy/software/#cove.
COVE statistical models were built from the SRP RNA
genes alignments available on the SRPDB website at http://
bio.lundberg.gu.se/dbs/SRPDB/srprna.html using the -a
option of the covet (train) program. The same sequences,
unaligned, were used as initial training data for the models.
The -m (maximum likelihood) option was used to give more
reliable results. Statistical models were built for the following
categories: (i) prokaryotes without Alu domain; (ii) pro-
karyotes with Alu domain; (iii) yeasts; and (iv) non-yeast
eukaryotes. Each model was trained on the subset of
sequences in the SRPDB belonging to organisms of that
particular category. Once novel sequences were found, models
were rebuilt on the new set of sequences to obtain a higher
accuracy. The covels (local score) program was used to search
rnabob fasta outputs for SRP RNA gene candidates. A list was
produced with the sequences with the best match to the
statistical model used. The -w (window) option was set to a
value slightly larger than the expected maximum size for an
SRP RNA. Alignments of the sequences were constructed
using the covea (align) program and the appropriate statistical
model for each category. The -o (outle) and -s (scorele)
options were used to produce le outputs with alignments and
scores of similarity to the model. SRP RNA genes alignments
and COVE covariance statistical models are available at the
website http://bio.lundberg.gu.se/srpscan/.
Mfold. Mfold version 3.1 was generously provided by
Dr M. Zuker (Rensselaer Polytechnic Institute, Troy, NY)
(14). It was used to predict the secondary structure of
predicted SRP RNAs. To obtain a folding consistent with
the secondary structure constraints implicit in the rnabob
descriptors or in the COVE models, we used these constraints
as input to mfold in many instances.
Automated procedure. Perl scripts were used to produce an
automatic procedure where genomic sequences are searched
using rnabob with descriptors of the helix8 motif in combin-
ation with the COVE programs. As an alternative, rnabob may
be used alone, where one makes use of descriptors for helix8
as well as for the Alu domain to obtain candidates for the full
RNA sequence.
RESULTS AND DISCUSSION
A protocol for identication of SRP RNA genes
As many other non-coding RNAs, SRP RNA is poorly
conserved with respect to sequence and effective methods for
RNA identication have to incorporate secondary structure
information. The region most highly conserved in SRP RNA is
the helix 8 region, which is characterized by a specic base-
pairing scheme in combination with primary sequence
elements, as indicated in Figure 1. It is interesting to note
that there is a strong correlation between the consensus
features and the sites of protein interaction as revealed by the
structure of an SRP54RNA complex (Fig. 1C) (11).
We have developed a procedure to identify potential SRP
RNA genes in genome sequences where the rst step relies on
the identication of the helix 8 motif. We search for this motif
using a slightly modied version of the RNA motif nder
rnabob (http://www.genetics.wustl.edu/eddy/software). The
helix 8 motif is retrieved together with appropriate anking
sequences. As a second step, the sequence(s) retrieved in the
3370 Nucleic Acids Research, 2002, Vol. 30 No. 15
rst step may be further analyzed using rnabob with a more
detailed descriptor that also takes into account the Alu domain
motif. An example of such a descriptor for plant RNAs is
shown in Figure 2. However, a more accurate, although
slower, method is to use the probabilistic covariance models
implemented in the COVE programs (2) to analyze the
sequences retrieved in the rst pattern matching step where a
model is produced on the basis of previously known SRP RNA
sequences. The COVE programs were also highly useful in
producing multiple alignments of SRP RNA sequences, to
identify conserved motifs in these RNAs and to predict the 5
and 3 ends of the RNA, as will be described in more detail
below. It should be noted that COVE is computationally
demanding and therefore it is not feasible to analyze complete
genome sequences without a rst heuristic step. It is possible
that the pattern-matching approach as a rst step will exclude
sequences that would obtain a high score from COVE, but we
have tried to minimize this problem by using relatively
degenerate descriptors in the rnabob search.
As SRP RNAs are very different in different taxonomic
groups, it was not possible to use a single helix 8 descriptor for
rnabob or a single COVE model for an efcient identication
of all SRP RNAs. For instance, the helix 8 motif is different in
plants and certain yeasts that have a 6 nt (GYUUCA) loop
instead of the normal tetraloop sequence, and a few bacteria
have a UGAC tetraloop (see below). Therefore, for the
problem of gene identication we had to consider different
classes of SRP RNA such as archaebacteria, eubacteria with
and without Alu domain, plants, yeasts and metazoans.
A web server for the prediction of SRP RNA genes is
available at http://bio.lundberg.gu.se/srpscan/. These pages
also present more detailed information on the work described
here. The web service also presents the potential secondary
structure of the predicted RNA sequence according to mfold
(14). In this procedure the folding is constrained by the base
pairing as specied by the rnabob descriptor or by the COVE
model used.
Identifying the helix 8 motif in bacterial genome
sequences
As mentioned above, the rst step of our SRP RNA gene
nding procedure is one where we search for the helix 8 motif.
As a starting point we designed a pattern based on all
previously known SRP RNA sequences as collected from the
SRPDB (13). A helix 8 motif NNAGGxxxGNRAyyy
AGCAG (where xxx pairs with yyy) was derived from
available SRP RNA genes. We then used rnabob to identify
this pattern in genome sequence data. From a majority of
bacterial genomes, this motif identied one gene that could be
successfully folded into a hairpin structure characteristic of
SRP RNAs.
However, in a few genomes, such as in Vibrio cholerae and
Lactococcus lactis, no SRP RNA could be identied using this
motif. To resolve this problem we made BLAST (1) or
FASTA (15) searches using SRP RNA sequence from a
closely related organism. Hence, the E.coli sequence was used
as a query in a BLAST or FASTA search of the V.cholerae
genome. This result revealed one homologous sequence in
Vibrio that could be folded into a hairpin structure and that had
the consensus features of the helix 8, except that in one of the
bulges it had NNAGA instead of the normal NNAGG. We
then made a rnabob search in bacterial genomic sequences
with a revised pattern, NNAGRxxxGNRAyyyAGCAG.
As expected, this search revealed the SRP RNA gene
candidate in Vibrio but also in Deinococcus radiodurans and
Buchnera spp., two other strains for which we previously
failed to identify an SRP RNA.
The pattern as described still failed to identify an RNA in
the strains Staphylococcus aureus and L.lactis. A FASTA
search of the Lactococcus genome using the Bacillus subtilis
SRP RNA revealed a homolog that had the characteristics of
SRP RNA but, instead of the normal GGAA tetraloop, it had
the unusual tetraloop sequence UGAC (Fig. 3). This unex-
pected nding led us to search for a helix 8 pattern where we
had substituted the GNRA tetraloop for UGAC. Using rnabob
with such a pattern also revealed SRP RNA candidates in
S.aureus (Fig. 3) and Staphylococcus equi. Thus, both
Lactococcus and Staphylococcus have the helix 8 motif but
with the UGAC tetraloop. Even though this is a very unusual
tetraloop sequence we believe that we have identied the true
SRP RNA gene homologs. This is because the sequence
similarity between these RNAs and SRP RNAs of other
Figure 2. Characteristics of plant SRP RNAs. Consensus features of helix 8
and Alu domains are shown schematically, as well as descriptor for rnabob
based on these features. N represents any nucleotide and an asterisk indi-
cates an optional nucleotide. The numbers refer to allowed number of bases
of Alu domain loops and spacers between the Alu and helix 8 domains.
Nucleic Acids Research, 2002, Vol. 30 No. 15 3371
closely related bacteria is highly signicant. Furthermore,
there are no other obvious SRP RNA gene candidates in
Lactococcus and Staphylococcus. Finally, the SRP RNA gene
candidates with the UGAC loop are not part of any known
protein-coding regions.
It is interesting to note that the UGAC tetraloop is found in
two species that both have close relatives with the normal loop
sequence. Thus, Staphylococcus is most closely related to
Bacillus, and Lactococcus is most closely related to
Streptococcus. A phylogenetic tree with these sequences is
shown in Figure 4. It would therefore seem as if the change
from GGAA to UGAC loop sequence occurred at least twice
during evolution. This suggests that the UGAC loop confers
some biological advantage in these gram-positive bacteria.
Recently, evidence has been presented that the tetraloop
sequence of E.coli SRP RNA (4.5S RNA) is important for the
interaction of FtsY, the receptor for bacterial SRP (16). In the
context of this evidence it will be interesting to test the
function of the UGAC tetraloop experimentally as well as to
study its structural role in the helix 8 domain.
For every prokaryotic genome we examined we identied
only one possible SRP RNA gene candidate. When we used
rnabob alone for prediction there was sometimes more than
one gene candidate to one genome, but most often false
positives could be identied because they did not fold into the
hairpin structure characteristic of SRP RNAs or because they
Figure 3. Comparison of SRP RNAs with GGAA and UGAC tetraloops. Streptococcus pyogenes and Lactococcus are closely related in sequence and
secondary structure but differ with respect to their tetraloop sequence. Similarly, the Bacillus and Staphylococcus RNAs, both with an Alu domain, are closely
related but differ in their tetraloop sequence. Also shown is the predicted RNA of T.maritima, which was unexpectedly found to have an Alu domain like the
Bacillus group of RNAs.
3372 Nucleic Acids Research, 2002, Vol. 30 No. 15
were part of protein-coding regions. At the same time, gene
predictions for bacterial genomes with the COVE models were
always very specic, i.e. for each genome tested we observed
only one gene candidate. When we used a COVE model with
the helix 8 domain only to score potential SRP RNA gene
candidates obtained from an rnabob search we sometimes
observed 16S rRNA genes with a relatively high score.
However, these hits are not likely to be signicant as the
regions in 16S RNA identied by COVE are not the same in
the different RNAs.
Bacterial Alu domains
Archaebacteria and certain eubacteria have SRP RNAs with
domains that seem to be analogous to the Alu domains of
eukaryotic SRP RNAs. Such eubacterial RNAs with Alu
domains have been identied in Bacillus, Listeria and
Clostridium. We now wanted to know which RNAs that we
identied using rnabob and the helix 8 motif descriptor also
contain an Alu domain. We used rnabob with a descriptor
accounting for consensus features of the Alu domain together
with a variable spacer between the helix 8 and the Alu domain.
The features of the Alu domain descriptor were based on a
careful inspection of available Bacillus, Listeria and
Clostridium sequences. The resulting descriptor was used to
analyze all the sequences that we identied using the helix 8
motif as described above. In addition to the Bacillus, Listeria
and Clostridium sequences that we expected to nd, we also
identied an Alu domain in the RNA of Thermotoga maritima
(Fig. 3). A similar result was obtained when we used the
COVE programs with gram-positive Alu domain RNAs in the
model. This was a somewhat unexpected nding as
Thermotoga is clearly distinct from the Firmicutes group.
The Thermotoga RNA does not contain a helix 6 region, and in
this respect it belongs to the Bacillus group of RNAs. This
nding raises interesting questions as to the relationship
between Thermotoga and the Bacillus branch. One possible
explanation for the presence of an Alu domain RNA in
Thermotoga is a horizontal gene transfer event. However, we
nd no evidence of such an event, as phylogenetic trees based
on SRP RNA sequences are very similar to trees based on 16S
rRNA sequences (data not shown).
In a search among the archaebacterial genomes we found as
expected an RNA with an Alu domain in all of the species
examined, also in the recently sequenced Thermoplasma
volcani and Thermoplasma acidophilum. All the sequences we
identied also had a helix 6 region according to the structure
predicted by mfold. The Aeropyrum pernix RNA differs from
the other archaebacterial RNAs in that it seems to have a helix
inserted in the Alu domain between the helices 3 and 4 (http://
bio.lundberg.gu.se/srpscan).
We noted that the sequence from Sulfolobus solfataricus
reported by Kaine (17) is very different from the sequence
predicted from the genome sequence of the same bacterium
(18). In fact, it is as distant to the genomic sequence as to the
gene predicted from the genome of Sulfolobus tokodaii (19).
Unless there are very large differences between different
isolates of S.solfataricus, it seems likely that there is a mistake
as to the identity of the strain.
Prediction of 5 and 3 ends
We want our method to predict as accurately as possible the 5
and 3 ends of the RNA gene product. For the RNAs with an
Alu domain we expect that the ends should be close to the Alu
domain. For the eubacterial RNAs without an Alu domain the
prediction of ends cannot be guided by such an element, and
another difculty is that there is a variation in size between
different species. A few such RNAs have been sequenced,
such as those of E.coli (20), a number of mycoplasmas
(2123) and Pseudomonas aeruginosa (24), with a variation in
size from 77 to 114 nt. However, analysis by COVE can also
make use of the fact that a majority of genes have a T-rich
sequence immediately downstream of the 3 end of the mature
sequence. Figure 5 shows an alignment of a number of
sequences of RNAs without Alu domain that was produced by
COVE and where the starting point was an alignment where
the T-rich regions had been manually aligned. However, a
similar result was obtained when unaligned sequences were
used as input to the program. In spite of the variation in size
between these bacterial RNAs, it seems that the COVE model
can predict the 5 and 3 ends reasonably well. For the RNAs
that have been sequenced and for which we have information
about the correct ends, these have the predicted site <23 bp
from the correct one. Of course we cannot exclude the
possibility that the ends of a few RNAs are incorrectly
predicted because these RNAs have a structure which is
different from the expected hairpin structure.
The T-rich sequence downstream of the gene is highlighted
in Figure 5. The hairpin structure of the mature RNA sequence
in combination with the T-rich sequence is highly reminiscent
of the rho-independent transcription termination signal. It
seems likely that this signal is the actual transcription
termination signal and that very little, if any, 3 end processing
is needed to obtain the mature 3 end.
Analysis of eukaryotes
Whereas prokaryotic genomes have only one SRP RNA gene,
many eukaryotes contain multiple genes as listed in Table 1.
As for the analysis of prokaryotes, it was necessary to dene
different descriptors and cove models for different taxonomic
Figure 4. Relationship of gram-positive SRP RNAs with unusual UGAC
tetraloop. Shown in the gure is a part of a phylogenetic tree based on an
alignment with COVE of all available SRP RNA sequences from this study.
The tree was based on UPGMA and was drawn using Phylodraw (33). As
indicated in the gure, the Lactococcus RNA is more closely related to the
Streptococcus RNA than to the Staphylococcus RNA, and the Staphylo-
coccus RNA is more closely related to the Bacillus RNA than to the
Lactococcus RNA. Furthermore, the Staphylococcus/Bacillus RNAs have an
Alu domain.
Nucleic Acids Research, 2002, Vol. 30 No. 15 3373
groups. For COVE analysis, we divided eukaryotes into yeasts
and non-yeasts.
Yeasts. The yeast SRP RNA genes are difcult to identify and
analyze as they show a low degree of conservation between
different yeast species, and because they are distinct from
other eukaryotic RNAs. Previously, SRP RNAs have been
identied in Yarrowia (25), Schizosaccharomyces pombe (26)
and Saccharomyces cerevisiae (27). The S.cerevisiae se-
quence is unusual in that the total length of the RNA is 519 nt,
whereas most eukaryotic RNAs are ~300 nt. All the yeast
sequences have a helix 8 motif like the metazoan counterparts.
However, the Alu domain is very different from the bacterial
and metazoan counterparts. It has been suggested that the
S.pombe, S.cerevisiae and Yarrowia lipolytica RNAs have a
simplied Alu domain that has the consensus features
indicated in Figure 6 (28).
We now also analyzed unnished genome sequence data of
Neurospora crassa (http://biology.unm.edu/biology/ngp/
home.html) and C.albicans (http://www-sequence.stanford.
edu/group/candida/) and were able to use our method to detect
SRP RNA candidates in these genomes. This was possible by
Table 1. Eukaryotic SRP RNA genes as predicted from genomic sequences
Chromosomal
location
Number of genes GenBank accession
numbers
Neurospora crassa Unknown 1
Candida albicans Unknown 1
Arabidopsis thaliana Chromosome 1 2 AC011807
AC013453
Chromosome 2 2 AC005662
AC005311
Chromosome 4 3 AC005275
AF069442
Chromosome 5 1 (+ 2 pseudogenes?) ATF17I14
AB026661
Drosophila Chromosome 3R 2 AC095014
Caenorhabditis elegans Chromosome 3 4 CEB0285
U00064
U23515
CEB0284
Chromosome 5 1 (pseudogene?) U41993
Homo sapiens Chromosome 14 2 CNS01DX7
AC020711
The Drosophila genes are identical, in an opposite orientation, and separated by ~2500 nt. The genes on
C.elegans chromosome 3 have 5 start positions 3172520, 3162782, 3822816 and 4011623, respectively.
Figure 5. Alignment of eubacterial RNAs without Alu domain. All eubacterial SRP RNAs without an Alu domain identied in this study were aligned using
a COVE model. The gure shows selected sequences from this alignment. The starting point for the alignment was one where T-rich regions at the 3 end
were manually aligned to each other, but a similar result was obtained when unaligned sequences were used as input. The T-rich region is highlighted as well
as conserved elements of the helix 8 region (see also Fig. 1).
3374 Nucleic Acids Research, 2002, Vol. 30 No. 15
using in the rst screen relatively degenerate helix 8 descrip-
tors for rnabob. In the case of Neurospora it has a 6 nt loop in
the helix 8 part like S.cerevisiae.
Interestingly, both C.albicans and N.crassa RNAs have a
sequence that is consistent with the simplied Alu motif,
suggesting that this motif is highly conserved in yeasts.
Figure 6 shows a tentative alignment with the yeast sequences
obtained by COVE. It highlights the homology in the Alu
domain as well as a predicted structural homology of part of
the molecule with the helices 6 and 8. On the basis of this
alignment it seems likely that yeast RNAs basically have the
same folding as other other SRP RNAs although there are
large insertions in the S.cerevisiae RNA with unknown
folding. It must be noted that it is difcult to predict the 5
and 3 ends of the yeast RNAs and experimental studies will
have to be carried out in order to determine these as well as to
fully understand the folding of the different yeast RNAs.
Plants. The plants tend to have fairly conserved SRP RNAs.
The predictions of plant RNAs might therefore be considered
more reliable than for the other categories of eukaryotes. In
Arabidopsis thaliana we have identied eight different gene
candidates, as shown in Figure 7. Of these, only two were
previously reported (13,29). It will require experimental work
to see if all these genes are actually expressed or if some of
them are pseudogenes. It has previously been shown that two
Arabidopsis SRP RNA genes contain promoter elements, USE
and TATA, upstream of the coding region that are identical to
the promoters of pol III-specic plant U-snRNA RNA genes
(30) and that are important for transcriptional activity. It has
been proposed that the plant SRP RNA transcription mainly
relies on these elements and that internal promoter elements
play a minor role. These upstream elements are indicated in
Figure 7 and it is interesting to note that they are found in
virtually all the novel genes. We therefore speculate that the
majority of these genes are not pseudogenes. However, two of
the genes on chromosome 5 do not have these elements (Fig. 7)
and may therefore be pseudogenes.
Metazoans. In Drosophila and Caenorhabditis elegans we
found two and ve genes, respectively (Table 1). The coding
sequences of the Drosophila genes are identical and the genes
are located in an opposite orientation, separated by ~2500 nt.
In C.elegans the genes on chromosome 3 were identied
before whereas the one on chromosome 5 is a novel nding.
However, it might be a pseudogene as the promoter sequence
is very different from the genes on chromosome 3. As with the
Drosophila genes, the genes on chromosome 3 are in pairs
with the genes in opposite orientation (Table 1).
The prediction of SRP RNA genes in the human genome is
complicated by the presence of a large number of Alu repeats
evolutionarily related to SRP RNA. As a result there is a large
background from hits to Alu repeats in the list of high-scoring
hits from COVE. The strongest candidate genes are three
genes on chromosome 14 (one gene in CNS01DX7 and two
genes in AC020711; see Table 1) and one on chromosome 10
(in GenBank accession no. AC091810, not shown in Table 1).
The predicted RNAs of these genes can all be folded into the
structure characteristic of eukaryotic RNAs. The genes on
chromosome 14 have similar sequences immediately upstream
of the coding sequence whereas the gene on chromosome 10 is
different in this region. We also carried out BLAST searches
Figure 6. Tentative alignment of yeast RNAs. RNAs shown are from S.cerevisiae, S.pombe and Y.lipolytica as well as RNAs predicted in this study from
genome sequence data of N.crassa and C.albicans. The alignment was produced by COVE. COVE had difculties in correctly aligning helix 8 of the
S.cerevisiae RNA to the corresponding part of the other RNAs, using the unaligned sequences as a starting point. Therefore, the starting point for the COVE
alignment shown in the gure was one where the helix 8 domain of the S.cerevisiae RNA had been manually aligned with the helix 8 domain of the other
RNAs. A conserved motif at the 5 end is shown with the consensus sequence RCUGURAUGGY, with base pairing as indicated by the arrows. The `~'
symbols indicate insertions in the S.cerevisiae RNA relative to the other RNAs.
Nucleic Acids Research, 2002, Vol. 30 No. 15 3375
of human EST database sequences to obtain evidence of
expression of these genes. The results show that the major
species in EST databases are CNS01DX7 (~50%) and one of
the genes in AC020711 (~50%). These two variants also
correspond to the sequences reported by Ullu et al. (31). The
fact that only one gene on the AC020711 segment is expressed
also has support from previous experimental studies (32). In
conclusion, the likely predominating human SRP RNA genes
are the one contained in CNS01DX7 and one of the genes in
AC020711 on chromosome 14.
When COVE was used to produce an alignment of all our
predicted eukaryotic SRP RNAs, including those of yeasts, the
UGU consensus sequence of the predicted Alu domain of the
yeast RNAs, as indicated in Figure 6, aligned well with the
corresponding sequence in the Alu domains of the metazoans.
This gives further support to the structural relationship
between the metazoan and yeast Alu domains. The result of
this alignment also shows that it is possible to use only one
COVE model for the prediction and analysis of all eukaryotic
SRP RNAs, with the possible exception of the S.cerevisiae
RNA. This model will now be very useful in the identication
of SRP RNA genes in genomes that become available in the
future.
Alignment of all the eukaryotic SRP RNAs also revealed
conserved elements. In addition to the helix 8 and Alu domain
motifs already referred to, the most prominent one is the motif
shown in Figure 1, which is located in helix 5 near the helices
6 and 8. This region of the RNA is involved in the interaction
with the SRP68/72 protein dimer. Possibly, the conserved
motif is an important recognition element for these proteins,
just as the conserved parts of helix 8 and the Alu domain are
recognized by the SRP9/14 dimer and the SRP54 protein,
respectively.
Conclusion
Non-coding RNA genes vary in their degree of primary and
secondary structure conservation. It is obvious that the lower
the degree of conservation, the more difcult is the prediction
of such genes from genomic sequence information. Certain
RNAs, like rRNAs, are so conserved that they may be
effectively detected by primary sequence homology only.
Other RNAs, like tRNA and SRP RNA, have to incorporate a
search for secondary structure features. With both tRNA and
SRP RNA, the degree of conservation in terms of combined
primary and secondary structure features is sufcient to
develop an accurate prediction method. However, there are
more difcult cases, like the yeast SRP RNAs, that quite
strongly diverge from a consensus structure. The predictions
of yeast RNAs will therefore have to be veried by experi-
mental studies. Experimental work is also required to deter-
mine the exact location of the 5 and 3 ends of mature RNAs
and to distinguish pseudogenes from genes that give rise to
functional RNAs.
Nevertheless, the methods described are in general very
efcient in identifying SRP RNA gene candidates. In this
method we combine a heuristic pattern screen for the
conserved helix 8 and Alu domain motifs with the COVE
programs. We account for SRP RNA genes in all organisms
that have been completely sequenced. We have detected a
number of novel RNAs as well as novel features of SRP RNAs
such as the unusual tetraloop sequence of the Lactococcus and
Staphylococcus RNAs. In particular, our method is very
sensitive and specic in the prediction of bacterial RNA genes.
For the rst time, we now have an automated procedure to
detect SRP RNA genes that may be used as a routine tool
during genome annotation.
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