Kinship in the SRP RNA family

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Jul 7, 2009 - The pre-SRP is transported to the cytosol and binds SRP54 in order to complete the assembly of a functional particle.27. SRP54 (named Ffh or ...
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RNA Biology 6:5, 508-516; November/December 2009; © 2009 Landes Bioscience

Kinship in the SRP RNA family Magnus Alm Rosenblad,1 Niels Larsen,2 Tore Samuelsson3 and Christian Zwieb4,* Department of Cell and Molecular Biology; University of Gothenburg; Göteborg, Sweden; 2Danish Genome Institute; Aarhus C, Denmark; Department of Medical Biochemistry and Cell Biology; Institute of Biomedicine; Sahlgrenska Academy of University of Gothenburg; Göteborg, Sweden; 4 Department of Molecular Biology; University of Texas Health Science Center at Tyler; Tyler, TX USA 1 3

T

Key words: signal recognition particle, SRP, SRP RNA, comparative sequence analysis Submitted: 07/07/09 Revised: 08/04/09 Accepted: 08/07/09 Previously published online: www.landesbioscience.com/journals/ rnabiology/article/9753 *Correspondence to: Christian Zwieb; Email: [email protected]

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he signal recognition particle (SRP) is a ribonucleoprotein complex which participates in the targeting of protein to cellular membranes. The RNA component of the SRP has been found in all domains of life, but the size of the molecule and the number of RNA secondary structure elements vary considerably between the different phylogenetic groups. We continued our efforts to identify new SRP RNAs, compare their sequences, discover new secondary structure elements, conserved motifs, and other properties. We found additional proof for the variability in the apical loop of helix 8, and we identified several bacteria which lack all of their SRP components. Based on the distribution of SRP RNA features within the taxonomy, we suggest seven alignment groups: Bacteria with a small (4.5S) SRP RNA, Bacteria with a large (6S) SRP RNA, Archaea, Fungi (Ascomycota), Metazoa group, Protozoa group, and Plants. The proposed divisions improve the prediction of more distantly related SRP RNAs and provide a more inclusive representation of the SRP RNA family. Updates of the Rfam SRP RNA sequence collection are expected to benefit from the suggested groupings. This review of the phylogenetically shared and divergent features of the SRP RNA molecule guides in the extraction of new SRP RNA sequences from the genomic data and promotes a more inclusive representation of the SRP RNAs. This increase in knowledge will help to better understand the evolution of the SRP RNA molecule and its function in protein targeting.

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SRP in Protein Targeting The signal recognition particle (SRP) is a ribonucleoprotein complex which binds to the signal peptide of to-be-targeted proteins upon their appearance at the ribosomal exit site. In eukaryotic systems where this process has been studied extensively, the SRP delays translation of the protein until the ribosome-bound SRP has an opportunity to associate with the membrane resident SRP receptor (SR). The SRP is released from the ribosome upon GTP hydrolysis, mediated by the mutually activating SRP and SR GTPases, allowing translation and translocation to proceed. The protein transverses the membrane co-translationally and thereby enters another cellular compartment or the extracellular space.1-5 In eukaryotes, the proteins are targeted to the membrane of the endoplasmic reticulum (ER). In archaea, SRP delivers proteins to the plasma membrane.6 Multiple protein targeting pathways exist in bacteria where SRP is primarily involved in the incorporation of inner membrane proteins.7 The plastid SRPs of photosynthetic organisms direct proteins to the thylakoid membrane.8 So far, no SRP or SR components have been identified in mitochondria. Besides the above-described classical pathway of SRP-mediated co-translational transport of proteins, SRP has been shown to participate also in post-translational protein sorting. In eukaryotes, SRP uses this mode to deliver tail-anchored proteins with a hydrophobic C-terminal insertion sequence to the ER.9 Similarly, the SRP assists post-translationally in the import of nuclear encoded proteins to the thylakoid membrane of chloroplasts.8

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RNA Families

article addendum

Discovery of SRP RNA SRP RNA was first detected in avian and murine oncornavirus particles, but was then found to be also a stable component of uninfected HeLa cells where it associated with membrane and polysome fractions.10-12 Initially, SRP RNA was named 7SL RNA due to its sedimentation coefficient and in order to distinguish it from the functionally unrelated 7SK RNA. The search for an SRP RNA function came to an end when cell biologists discovered an 11S signal recognition “protein” complex (fortuitously also abbreviated “SRP”) in canine pancreatic tissue which promoted the translocation of secretory proteins across microsomal membranes.13-16 Eventually, an RNA molecule was found to be part of the 11S complex.17 The terminal regions of mammalian SRP RNA were recognized as being related to the dominant Alu family of repetitive sequences in the human genome. It is understood that Alu-DNA originated from SRP RNA by excision of the SRP RNA-specific (S) fragment, followed by reverse transcription and integration into multiple chromosomal sites.18,19 The Alu and S domains of the SRP can be separated by mild digestion with micrococcal nuclease and are synonymously called the small and large domain, respectively (Fig. 1A).20,21 The Role of the SRP RNA in Assembly of the SRP The SRP RNA component provides the framework for the proper arrangement of the SRP proteins.22-26 The assembly of the small bacterial SRPs, composed of an RNA (4.5S RNA) and one protein molecule (the SRP54 homologue Ffh), appears to be controlled by the expression levels and binding affinity between the two components. In eukaryotes, the SRP RNA was found to be located in a specialized region of the nucleolus where it combines with the imported SRP proteins SRP9/14, SRP19 and SRP68/72. The pre-SRP is transported to the cytosol and binds SRP54 in order to complete the assembly of a functional particle.27 SRP54 (named Ffh or p48 in the bacteria) is a component of every SRP and has an important role in signal peptide

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binding and release. The complex composed of SRP54 and SRP RNA helix 8 has co-evolved into a highly conserved molecular entity (Fig. 1A) due to its essential function in protein sorting and cellular integrity.28 Typically, SRP54 is encoded by a single gene. However, there are exceptions, such as in Arabidopsis thaliana where three divergent SRP54 genes have been identified.29 This heterogeneity may be related to the observed diversity of the plant SRP RNAs.30,31 SRP19 of the archaeal and eukaryotic SRPs initiates assembly of the particle by mediating the formation of a non-canonical hydrogen bonded A-A pair between the adenosine of the helix 6 GNAR tetraloop and the 3'-adenosine of the apical helix 8 loop (Fig. 1C). In the archaea, this tertiary interaction may take place without assistance of SRP19 and may be followed by the SRP19-dependent stabilization of the parallel arrangement of helices 6 and 8.25,32-34 The SRP68/72 heterodimer, together with SRP19, SRP54, and approximately 150 SRP RNA residues, constitute the large (S) domain of the SRP. About one third of the human SRP68 protein was shown to be required for binding to the SRP RNA.35 A relatively small region of protein SRP72 binds to an SRP RNA section which contains the conserved 11-nucleotide 5e motif (Fig. 1A).36 In archaea, which lack protein SRP72, the 5e motif may contribute to the formation of an interdomain hinge or kink as observed by cryo-electron microscopy of the elongation-arrested ribosome bound canine SRP.37,38 In contrast to the detailed structural information of complexes containing SRP9/14, SRP19 and SRP54, high-resolution data about SRP68/72 are unavailable.39-43 Assembly of SRP9/14 with the small (or Alu) SRP RNA domain is dependent on the presence of the UGU(NR) motif and might engage the loops of helices 3 and 4 in an RNA tertiary interaction (Fig. 1B). SRP9/14 is absent in the bacteria, but HBsu, a protein originally identified as being histone-like, has been proposed to be part of the Bacillus SRP.44 A tRNA-like molecule has been suggested to serve functions of the missing SRP9/14 in Trypanosomatids.45 Yeast and other

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Ascomycota possess a reduced small domain which lacks SRP RNA helices 3 and 4 (Suppl. Table 1). These organisms also lack protein SRP9 and contain instead the structurally related protein SRP21. Despite this relationship, SRP21 does not seem to participate in the formation of a complex with SRP14. Instead, the yeast SRP RNA forms a complex with a SRP14 homodimer.46 Identification of SRP RNA Sequences Automated BLAST searches were used for identifying SRP RNAs with similarity to known sequences in the SRP database (SRPDB, see Materials and Methods). While this approach was useful for finding closely related sequences, it failed to identify distantly related SRP RNAs with unexpected or new properties. Since SRP RNA contained only a small number of strictly conserved residues embedded in the context of difficult to predict secondary structures, we combined a heuristic pattern-based search for conserved features of the helix 8 region with a covariance model (CM) SRP RNA model.47,48 When applicable, the computational predictions took into account the locations of promoter and termination signals, presence of helices according to phylogeny, consideration of helix insertions, and the presence of other SRP related genes. For example, identification of protein SRP19 would encourage the search for an SRP RNA featuring helix 6. In contrast to automated CM searches which were dependent on cutoff scores, manual inspection was used to identify certain idiosyncratic SRP RNAs. Comparative Analysis of SRP RNA Sequences Due to the growing availability of numerous genome sequences, comparative sequence analysis continued to be the prominent approach for determining SRP RNA secondary structure and tertiary interactions. Related sequences were represented as alignments to distinguish, among other features, the variable portions from the more conserved regions. RNA helices were identified by observing covariances and compensating

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Figure 1. SRP RNA nomenclature and tertiary interactions. (A) The human SRP RNA secondary structure is outlined in gray. Helices are numbered from 1 to 12 and helical sections are labeled with letters a to f. The 5'- and 3'-ends are indicated. Dotted numbers (e.g., 6.1, 9.1 and 12.1) are for insertions in helices 6, 9 and 12, respectively. The approximate boundaries of the large (S) and the small (Alu) domains are shown. Dark gray are the UGU(NR) motif (labeled UGU) in the small domain, the 5e motif within helical section 5e, the GNAR apical tetraloop of helix 6 (N is for any nucleotide, R is for a purine) and the SRP54 binding motif of helix 8 in the large domain. Dashed lines suggest the tertiary interaction in the small and large domain. (B) Examples of the tertiary interaction (dashed lines) between the loops of helices 3 and 4. MG: Metazoa group, AR: Archaea and LB: Bacteria with a large (6S) SRP RNA. (C) Details of the tertiary interaction between the loops of helices 6 and 8 across the phylogenetic spectrum. The dashed line connects the conserved adenosine residues. MG, Metazoa group; FA, Fungi (Ascomycota); PL, Plants; PG, Protozoa group; SB, Bacteria with a small (4.5S) SRP RNA.

bases changes (CBCs) which supported the existence of a base pair because, during evolution, random mutations would not have been corrected unless required for function. Using this approach, eight helices had been identified in the initial alignment of 39 SRP RNA sequences from the three phylogenetic domains.49 The updated SRPDB collection contained

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262 representative SRP sequences (a total of 964 sequences) which were distributed throughout the three domains of life (Suppl. Table 1). These sequences are available at the internet address http:// rnp.uthct.edu. Using the goffice viewer, sequence alignments can be zoomed and colored to highlight conservation, covariations and other properties.28

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SRP RNA Nomenclature With advances in the identification of new SRP RNAs, particularly in the genomes of protozoa and fungi, a nomenclature for all SRP RNAs was proposed in 2005.21,50 The originally established numbering system for helices 1 to 8 remained unchanged, and this is a testimony to the predictive

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power of comparative sequence analysis.49 Helices 9 to 12 were added to accommodate features of the large SRP RNAs of the Saccharomycetes and its relatives.21 The helical sections within helices 9 to 12 were referred to with dotted numbers (e.g., 9.1 and 12.1, Figs. 1A and 2C). The “extra” helix of the Onygenales SRP RNAs, now more accurately positioned as an insertion into the 5'-portion of helix 6, was referred to as helix 6.1.48 Since computations depended on a single character for each column in the alignment, helix names beyond single digits were assigned capital letters A to I in the alignment pairing mask. This is indicated by the small table shown in Figure 2C. SRP RNA Motifs Conserved features, or motifs, play an crucial role in the identification of SRP RNA sequences and the investigation of SRP structure and function. The asymmetric loop between helical sections 8a and 8b and the adjacent base paired 8b section have been known for some time to be a prominent property of every SRP RNA. 8b contains an invariant A-C as well as adjacent highly conserved G-G and G-A non-canonical pairings. These nucleotides contribute to the formation of a flat minor RNA groove which is required for the binding of protein SRP54 (this protein is called Ffh in the bacteria). The SRP54/ Ffh-RNA complex is a target for intense investigations in attempts to explain its role in signal peptide binding and release.51-56 It has been suggested that helix 8 may interact directly with the N-terminal portion of the signal peptide.39 Recently, evidence has been provided that this region of the SRP RNA releases the autoinhibition of the mutually controlled GTPase activities between the signal-bound SRP and the SRP receptor (SR).57,58 We found that the apical loop of helix 8 was less conserved than previously thought. Depending on taxonomic membership it was composed of four, five or six residues. The loop typically contained a highly conserved guanosine as its first, and an adenosine as its last residue. Both residues are probably required for a near coplanar orientation with the conserved adenosine residue of the helix 6 GNAR

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tetraloop (Fig. 1C). While the apical helix 8 tetraloop was shown to be usually GNRA in bacteria, the increasing number of identified bacterial SRP RNAs revealed a lower than anticipated level of conservation. The most common variant appeared to be URRC (R is for a purine residue) which was represented in many bacterial groups.48 Examples of similar tetraloop sequences (e.g. URRU) were observed in several Bacteroides species and Onion yellows phytoplasma. The URRC tetraloop was found only once in the eukaryotes (Volvox carterii). The pentaloop in helix 8 of the SRP (4.5S) RNA of the gram-negative bacterium Gemmatimonas aurantiaca represented the first example of a deviation from a tetraloop in the bacterial SRP RNAs. However, a sequencing mistake could not be excluded (Suppl. 1; Fig. 1C). The 11-nucleotide 5e motif was composed of four symmetrically arranged base pairs which are interrupted by a threenucleotide loop.48 The first nucleotide of the loop was shown to be a conserved adenosine residue in eukaryotes. This adenosine (A240 in human SRP RNA) was found to be essential for the binding of protein SRP72.36 The UGU(NR) motif (abbreviated as UGU in Fig. 1A) of the small domain, either connected helices 3 and 4 or was part of the helix 2 hairpin in the fungal SRP RNAs (Fig. 2C). The motif was shown previously to form a U-turn which was required for the binding of the SRP9/14 heterodimer or a SRP14 homodimer.40,46 Because UGU(NR) was present in the SRP RNAs of organisms which lack genes for SRP9 or SRP14, it might also serve an unknown SRP14-independent function. SRP RNA Size Distribution, Secondary Structures and Tertiary Interactions The identified SRP RNAs spanned a wide spectrum with respect to size and the number of structural features (Fig. 2 and Suppl. Table 1). Although the precise termini of the SRP RNA molecules were sometimes difficult to determine, the smallest functional SRP RNAs likely exist in Mycoplasma and related species.59 The prototypical Escherichia coli SRP

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RNA (4.5S RNA) is known to be composed of 114 nucleotides. Its RNA forms a hairpin which corresponded to helix 8 and the more conserved helix 5 sections (Fig. 2A). The gram-positive bacteria encoded not only the smallest, but also the majority of the larger bacterial SRP RNAs. For example, the 6S Bacillus subtilis SRP RNA (scRNA) displayed a fully extended helix 5, paired terminal regions (helix 1), helices 2 to 4 (see Suppl. Fig. 2A), and a pronounced tertiary interaction in the small domain (Fig. 1). Thus, the large bacterial SRP RNAs resembled in many aspects their archaeal equivalents but lacked helix 6. Thermotogae was the only family of gram-negative bacteria containing a large SRP RNA. In contrast to the variable size of the bacterial and eukaryotic SRP RNAs, relatively minor differences were observed among the archaeal SRP RNA sequences and secondary structures. This may be due to slow evolutionary rates observed previously in a comparison of archaeal protein sequences.60 All archaeal SRP RNAs contained helices 1 to 8, but lacked helix 7 (Suppl. Tab. 1). The tertiary interaction between the apical loops of helix 3 and helix 4 was supported by additional CBCs suggesting a compactly folded small domain (Fig. 1B). This structure appeared to be similar to the experimentally determined structure of the homologous eukaryotic (Alu) domain in complex with the protein SRP9/14 heterodimer.40 The crystal structure of SRP19-bound to the Methanococcus jannaschii SRP RNA from the large domain demonstrated binding between the apical tetraloops of helix 6 and helix 8.33 The conservation of the hydrogen bonded adenosines suggested stabilization of the parallel arrangement of the helices. This feature appeared to be shared among the archaeal and eukaryotic SRP RNAs (Fig. 1C). The eukaryotic SRP RNAs were highly variable with respect to sequence and secondary structure. Saccharomycetes represented the largest SRP RNAs known to date having acquired helices 9 to 12 as insertions into helix 5, as well as an extended helix 7 (Fig. 2C). Because many eukaryotic sub-groups were represented by a small number of sequences (Suppl. Table 1), certain secondary structure

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Figure 2. SRP RNA secondary structures of increasing complexity. Shown are the secondary structures of the (A) small (4.5S) SRP RNA of Eschericia coli, the (B) prototypical 301-nucleotide human SRP RNA, and the (C) 502-nucleotide Saccharomyces cerevisiae SRP RNA. Helices and their sections are labeled according to Zwieb et al. (2005) (see also Fig. 1A). Residues are labeled in increments of ten and the 5'- and 3'-ends as such. The legend in panel c shows the naming conventions (A to H) for double digit helices. Additional secondary structure drawings are provided in Supplements 2 and 3. 512

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Table 1. Proposed alignment groups Abbreviation

Alignment group

Properties

Species examples

AR

Archaea

h1-h6, h8, no h7

Sulfolobus solfataricus, Archaeoglobus fulgidus, Candidatus Korarchaeum cryptofilum

SB

Bacteria with a small (4.5S) SRP RNA

one hairpin composed of h8 plus a portion of h5

Escherichia coli, Mycobacterium tuberculosis, Mycoplasma mycoides, Cyanidioschyzon merolae chloroplast

LB

Bacteria with a large (6S) SRP RNA

h1-h5, h8, no h6

Bacillus subtilis, Clostridium perfringens, Thermoanaerobacter tengcongensis, Thermotoga maritima

FA

Fungi (Ascomycota)

h2, h5-h8 may possess h9-12, 6.1

Aspergillus oryzae, Coccidioides immitis, Saccharomyces cerevisiae, Neurospora crassa

MG

Metazoa group

h2, h5-h8, small (Alu) domain may be reduced, may possess h10

Phakopsora pachyrhizi, Homo sapiens, Monosiga brevicollis, Rhizopus oryzae

PL

Plants

h2-h8, diverse SRP RNA sequences

Galdieria sulphuraria, Chlamydomonas reinhardtii, Arabidopsis thaliana

PG

Protozoa group

h2-h8, small (Alu) domain may be reduced or absent

Tetrahymena thermophila, Dictyostelium discoideum, Trypanosoma brucei, Giardia lamblia

Groups with their prevalent properties are listed: alphabetically. AR, Archaea; FA, Fungi (Ascomycota); LB, Bacteria with a large (6S) SRP; RNA; MG, Metazoa group; PL, Plants; PG, Protozoa group; SB, Bacteria with a small (4.5S); SRP RNA. Species examples are given in the column on the right.

features, particularly in helices 9 to 12, were relatively weakly supported. However, experimental evidence has been provided for their existence.61 Accelerated sequencing and faster methods for identifying SRP RNA genes might allow in the future to more firmly establish or disprove the proposed base pairings. The Ascomycota SRP RNAs were characterized by a reduction or loss of helices 3 and 4. An insertion into helix 6 (previously named “extra helix”,28 now referred to helix 6.1) had occurred in the Onygenales (Table 1). SRP RNAs in the “animal” group displayed a low level of diversity and resembled in their overall design the archaea SRP RNAs. The interaction between the loops of helix 3 and helix 4 involved a smaller number of base pairs than the equivalent tertiary interaction of the archaeal SRP RNAs (Fig. 1B). Direct evidence for their existence has been provided by crystallography.40 Helices 3 and 4 were extended in Plasmodium, but helix 4 was diminished

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or lost in several other protozoans. A small set of phylogenetically diverse protists such as Giardia lamblia lacked a small domain (Avesson L et al., unpublished). The small (Alu) domain may be dispensable in organisms with slow protein synthesis rates. This idea is supported by the recent elucidation of how proteins are targeted to the ER of mammalian cells.62 Seed plants were shown to express numerous highly divergent SRP RNAs. Because these RNAs had been extracted from membrane-bound SRPs, they are apparently functional in an individual plant.30,31 We speculate that this variability might reflect differentially optimized SRPs. Evidence accumulated that the size of the apical loop of SRP RNA helix 8 varies between four and six residues. While the first and last loop residues were preserved as G and A, respectively, pyrimidines were inserted into the plant and yeast SRP RNAs. Examples of pentaloops were found in the protozoan group (N. gruberi, E. histolytica and T. vaginalis)

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and, possibly, in the bacterium G. aurantiaca (Fig. 1C). These variations should be taken into account when attempting to identify SRP RNAs sequences. Absent SRP Components Chloroplasts of seed plants lacked SRP RNA component but contained a homologue of SRP54 (cpSRP54). The cpSRP54 diverged enough from other SRP54 homologues to loose its ability to bind to the bacterial (4.5S) SRP RNA. Besides being part of the co-translational SRP, cpSRP54 was found to bind to protein cpSRP43 to form the post-translationally active chloroplast SRP (cpSRP).63,64 Arabidopsis cpSRP54 and cpFtsY (the chloroplast homologue of eukaryotic SR) were shown to function without an RNA component.65 It is unclear when, in evolution, chloroplasts lost the SRP RNA gene.66 SRP RNA was shown to be essential for survival of Eschericia coli.51,52 Other bacteria, such as Streptococcus mutans, remained viable without an SRP RNA gene and suffered only impaired growth and increased sensitive to environmental stress.67 A survey of completed eubacterial genomes revealed nine organisms which lacked all SRP components, including the bacterial SR homologue FtsY (Rosenblad MA, unpublished). An absence of SRP had previously only been noted in the obligate symbiont Nanoarchaeum equitans, an archaea with a significantly reduced genome, as well as in the extremely small 160-kilobase genome of the bacterial symbiont Candidatus Carsonella ruddii.68 SRP RNA Alignment Groups The observed divergent properties and widespread phylogenetic distribution of the SRP RNA necessitated separate groupings for the identification of SRP RNA sequences through automated procedures as employed, for example, by the Rfam project. Table 1 lists the seven proposed alignment groups with their properties and example species. The separation between bacteria with a small (4.5S) SRP RNA and bacteria with a large (6S) SRP RNA was decided upon in order to fully represent both groups and help in the identification of additional large bacterial

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SRP RNAs. The archaeal SRP RNAs, although similar to the “animal” and the large bacterial SRP RNAs, deserved separate attention due to their prominent phylogenetic distance from the bacterial and eukaryotic domains.69 The Fungi (Ascomycota) SRP RNAs varied considerably in size, and refining their SRP RNA secondary structures required special focus in order to account for the unique SRP RNA features in this group. The Metazoa and Protozoa shared many SRP RNA properties, but their large phylogenetic distance motivated separation into two groups. Plant SRP RNAs were highly diverse, apparently even in a single organism, and thus had to be maintained separately. If desired, the alignments could be easily merged and regrouped because they shared one pairing mask. Discussion and Future Directions The discovery of closely related SRP RNAs has become routine, but tools which can identify more distantly related SRP RNAs with unexpected properties are not only computationally intensive but also require manual inspection. The proposed alignment groupings are expected to improve the automated discovery process using CM searches employed, for example, by Rfam. As a result, we expect that the time required for manual inspections will be significantly reduced and can be used more productively to investigate suspected SRP RNA candidates. Separating the large bacterial SRP RNAs from their smaller cousins will be a significant advance as to the representation of all bacterial SRP RNAs. An important addition are the exceptionally diverse sequences in the Fungi (Ascomycota) group. Their number is expected to grow soon as several fungal genomes are nearing completion. Our broad survey demonstrates that SRP RNA features which were thought to be well established may require modifications when attempting to extract sequences from distantly related genomes. We anticipate that earlier assumptions about the properties of certain SRP RNA secondary structure motifs will have to be adjusted as we fathom the biological diversity and discover new SRP features. Particularly within the Saccharomycetes,

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related sequences will be welcomed to identify more CBCs in helices 9 to 12. Established methods and tools can then be applied to build three-dimensional models of these diverse SRPs and achieve a better understanding of protein targeting across the phylogenetic spectrum.70 We found that some bacteria lack all their SRP component genes. This raises the question of how these organisms manage to target their proteins by alternative mechanisms. Access The data are freely accessible for research purposes at the internet address http:// rnp.uthct.edu/rnp/SRPDB/SRPDB.html or the mirror sites at http://bio.lundberg. gu.se/dbs/SRPDB/SRPDB.html and http://genome.ku.dk/resources/srpdb/. Alignments can be browsed at http://rnp. uthct.edu:8000/. Materials and Methods Identification and maintenance of SRP RNA sequences. SRP RNA genes were predicted as described previously by applying a combination of pattern searches using rnabob (www.genetics.wustl.edu/ eddy/software/#rnabob) and COVE or Infernal version 0.81.47,48 RNA secondary structure predictions were carried out by COVE/Infernal and MFOLD.71 Identifications were evaluated by taking into account transcription signals, SRP protein genes, and taxonomic relationships. All data, including the gapped SRP RNA sequences, were stored as tables with the same RNA secondary structure pairing mask, but separated into alignment groups as described within the text of this communication. Representative sequences were submitted to the blastcl3 client at NCBI. Perl scripts (available from the last author upon request) were used to format the search results. The data were filtered by their accession numbers to remove any already known sequences as well as previously noted false positive hits. For each alignment group, sequences were arranged according to NCBI taxonomy. FASTA formatted alignments were extracted and examined with the semiautomated editor SARSE.72 Gaps were introduced into the new sequences, adjustments to the overall

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alignment were made as required, and the realignments were stored in tabular format. Data shown at the SRPDB (rnp.uthct. edu) were extracted to include alphabetically and phylogenetically ordered organism lists, corresponding FASTA formatted gapped sequence files, as well as RNA secondary structures in the Connect format, usable by various of RNA secondary structure analysis programs. Browsers. The alignments can be navigated with a web based browser developed by the Danish Genome Institute, partly sponsored by the Nucleic Acid Function and Technology group at Aarhus University, Denmark, and downloaded via links posted at http://www.rnai.dk. Acknowledgements

We thank Paul Gardner at Rfam for carrying out preliminary tests with the covariation models of the proposed SRP RNA alignment groups, Fatemeh Kaveh for investigating certain features of the bacterial SRP RNAs, and Jan Gorodkin for providing the SRPDB mirror at http:// genome.ku.dk/resources/srpdb. This work was supported by NIH grant GM-49034 to C.Z. Note

Supplementary materials can be found at: www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup.pdf www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup01.txt www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup02.txt www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup03.txt www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup04.txt www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup05.txt www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup06.txt www.landesbioscience.com/supplement/ RosenbladRNA6-5-Sup07.txt

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