Cells make many proteins that are eventually released outside the cell or inserted into the cell’s membrane. As these proteins are still being made, they are captured by a “signal recognition particle” (or SRP); this molecular machine then guides the newly forming protein to the cell’s membrane. SRPs are found in all living organisms on Earth and contain several different proteins and a short RNA molecule. However, a few species belonging to the archaeal domain of life did not seem to contain an identifiable gene for the RNA component of the SRP.

Now Plagens et al. have sought to solve the mystery of the “missing” component of this essential protein-targeting machine. This involved searching through the RNAs that are produced by an archaeon called Thermoproteus tenax, a single-celled microbe which grows in the absence of oxygen and at temperatures of up to 95°C.

Plagens et al. discovered that the “missing” SRP RNA gene had not yet been identified because rearrangements in this archaeon’s genome had swapped the left and right portions of the SRP RNA gene. Further experiments revealed that the correct sequence order is restored in mature SRP RNA molecules by the two ends of the molecule being linked to form a circle. These RNA circles are made by the cellular machinery that normally removes the unneeded sections from other RNA molecules (called transfer RNAs).

Circular RNA is much more stable at high temperatures and does not degrade easily, and Plagens et al. suggest that this particular arrangement is therefore especially advantageous for this species. Future work will now aim to work out which selective pressures favor the evolution of such fragmented RNAs.

All organisms contain signal recognition particles (SRPs) that bind to the ribosome and recognize signal peptides in secretory proteins (Luirink, 2004). SRP binding can arrest the elongation of nascent peptide chains and targets the ribosome-nascent chain complexes to the endoplasmic reticulum membrane (in Eukaryotes) or the plasma membrane (in Bacteria and Archaea). The SRPs consist of one central SRP RNA molecule and different SRP proteins. Eukaryotes harbor six SRP proteins, Bacteria have one SRP protein and archaeal SRPs are formed by an approximately 300 nt long SRP RNA and the two proteins SRP19 and SRP54 (Römisch et al., 1989; Bernstein et al., 1989; Bhuiyan, 2000; Cusack et al., 2000). The SRP RNA is pivotal for the structure and function of all SRPs. Archaeal and eukaryotic SRP RNAs are similar and usually contain eight helical elements that fold into a small Alu-domain and a large S-domain that are connected by an extended linker region (Batey, 2000; Gundelfinger et al., 1983) (Figure 1A). SRP19 binds to the tips of helices 6 and 8 in the S-domain, which form strictly conserved tertiary RNA interactions(Hainzl et al., 2002). The SRP RNA adopts a conformation that exposes a SRP54-binding site in helix 8 (h8) (Rose and Weeks, 2001) (Figure 1A). SRP54 is essential for (i) the recognition and binding of the signal sequence at the ribosome, and (ii) for the GTP-dependent interaction with the SRP receptor of the archaeal plasma membrane (Luirink et al., 1994). Intriguingly, few species of the archaeal genus Thermoproteus (e.g. Thermoproteus tenax and Thermoproteus uzoniensis) do not contain canonical SRP RNAs and SRP RNA genes are not annotated (Siebers et al., 2011). Genes encoding standard SRP19 (TTX_2083) and SRP54 (TTX_0615) proteins are present and signal sequences can be identified for secretory proteins (Supplementary file 1A). Thus, we aimed to search for the elusive SRP RNA genes in T. tenax to investigate potential alterations of one of the most conserved ribonucleoprotein complexes found in nature.

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We isolated small RNA molecules from T. tenax cells and subjected them to Illumina Hiseq2000 RNA-Seq analysis. 13.45 million sequence reads were mapped to the reference genome to obtain insights into the RNome of T. tenax. Subsequently, we searched the sequence reads for minimal conserved SRP RNA features. SRP RNA molecules are highly structured and contain several conserved structural features but only few absolutely conserved sequence segments. These sequences include a conserved SRP54 binding motif in helix 8b and a GNAR tetraloop motif connecting helix 6. We identified these short motifs in an intergenic region between the genes TTX_0683 (pyruvate phosphate dikinase [ppdK]) and TTX_0684 (protein phosphatase [ppa]). The prediction of RNA folding for this intergenic sequence revealed a high potential for extended helical regions, which prompted us to analyze this region in more detail. We utilized the obtained RNA-Seq reads to identify the 5'- and 3'-termini of a 309 nt long highly structured RNA transcript. The alignment of this RNA molecule with other archaeal SRP RNA molecules that are annotated in the SRP database (Rosenblad, 2003) highlighted the presence of several conserved structural features (Figure 1B). However, the location of the 5'- and 3'-termini of this newly discovered SRP RNA was shifted from the standard location in helix 1 in the small Alu-domain to a region close to a functionally important loop in the large S-domain. This indicates that Thermoproteus species with ‘missing’ SRP RNA genes contain permuted SRP RNA sequences in which the transcripts’ termini have been shifted by over 100 nucleotides and the order of sequence motifs has been rearranged. We identified a similar permuted SRP RNA gene in T. uzoniensis and noticed that nucleic acid sequence variations do not occur in conserved regions of the SRP RNA and in most cases do not disrupt the structure of the SRP RNA (i.e. G-C base pairs are replaced by G-U or A-U basepairs) (Figure 1—figure supplement 1). Sequence reads that mapped to the termini regions of the T. tenax SRP RNA revealed unmapped parts consisting of sequences of the distant end, which indicates that these reads span ligated 5'-and 3'-termini (Figure 2A). The observed ligation sites are identical and restore the loop at the tip of helix 8 in the permuted SRP RNA. Thus, mature SRP RNAs exist as circular molecules. The RNA-Seq reads also revealed that SRP RNA precursor transcripts contain a 23 nt long leader sequence upstream of the ligated mature 5'-termini (Figure 2B). A TATA box promoter element (5′-TTAATA-3′) is present 26 nt upstream of the transcription start site in both T. tenax and T. uzoniensis. Additionally, we detected a C/D box sRNA gene adjacent of the permuted SRP RNA gene (Figure 2B). Northern Blot analyses verified the presence of the SRP in T. tenax total RNA preparations, and revealed three distinct bands that were reproduced with two different probes (Figure 2C). Full-length in vitro transcripts of the SRP RNA (linear and circularized with T4 RNA ligase) were run as size markers. The observed migration of this highly structured transcript was identical to the total RNA signal and suggests that the two prominent bands represent the circular form of the SRP RNA (Figure 2C).

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Next, we aimed to show that the permuted SRP RNA is able to bind SRP19 and SRP54 (Figure 3A). Recombinant SRP proteins were produced in Escherichia coli and variant S-domains of the SRP RNA were transcribed in vitro (Figure 3—figure supplement 1 and Figure 3—figure supplement 2). DEAE binding assays supported the presence of standard SRP protein recruitment. The interaction of SRP19 with the S-domain is required for the subsequent binding of SRP54 (Figure 3A). The open SRP RNA has a significantly reduced affinity for SRP19 and SRP54 (Figure 3B). The mutation of conserved SRP RNA motifs (Zwieb, 1992; Zwieb, 1994) abolished the binding of SRP19 (GNAR loop mutation) or SRP54 (h8b mutation) (Figure 3B). These assays verified that the conserved features of the identified SRP RNA molecule are required for SRP19 and SRP54 recognition and revealed that the lack of SRP RNA circularization impairs SRP protein binding (Figure 3B and Figure 3—figure supplement 2).

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Permutation of the SRP RNA sequence removes conserved termini and creates a novel ligation site. We investigated both sites in more detail. The canonical termini of archaeal SRP RNA molecules are located in helix 1 of the small Alu-domain. This helix is absent in the T. tenax SRP RNA. It has been suggested that the main function of this helix is to prevent the unfolding of the SRP RNA under the extreme growth conditions of hyperthermophiles (Zwieb, 2005). Thus, the replacement of this helix with a continuous sequence in a circular RNA molecule would provide improved RNA stability and a potential evolutionary advantage. The high stability of circular RNA molecules is also a likely explanation for the observed low RNA-Seq coverage as only few SRP RNA fragments were amenable to adapter ligation during RNA-Seq library preparation.

The occurrence of RNA termini near the tip of helix 8 is more problematic for the functionality of the SRP RNA. Ligation of these RNA ends guarantees the presence of a canonical loop in this region. However, this ligation reaction requires the presence of clearly defined termini as all sequenced circularization junctions were found to be identical. Thus, we investigated if the observed precursor transcripts might be subjected to endonucleolytic processing to generate mature RNA ends. We observed that the 23 nt long leader sequence of the primary transcript can fold with extended trailer sequences into a so-called bulge helix bulge (BHB) motif (Figure 4A). A similar motif is also present at the termini of the permuted T. uzoniensis SRP RNA (Figure 4—figure supplement 1). BHB elements are structural motifs found in intron-containing transfer RNAs (tRNAs) (Thompson and Daniels, 1988). A BHB consists of a central four base pair helix that is flanked by at least one three nt unpaired bulge. These structures are recognized by the tRNA splicing endonuclease that cleaves the bulge sequences and recruits the tRNA ligase RtcB to join the tRNA sequences and circularize the intron sequence (Popow et al., 2011; Englert et al., 2011). The presence of a BHB motif suggests that RtcB could be responsible for SRP RNA circularization, if the T. tenax splicing endonuclease specifically recognized the identified BHB motif. It should be noted, that T. tenax contains a record number of 48 introns in 46 tRNAs (Chan and Lowe, 2009). These introns are processed by a tRNA splicing endonuclease that belongs to a family of heterotetrameric enzymes (Randau et al., 2005). This family shows the most relaxed BHB substrate recognition mechanism and does not rely on the recognition of tRNA sequences for cleavage (Yoshinari et al., 2009). We produced recombinant T. tenax splicing endonuclease (TTX_1594, catalytic subunit and TTX_1893 structural subunit, Figure 3—figure supplement 1) and could verify specific cleavage of the proposed BHB motif (Figure 4B). This suggests that the tRNA splicing machinery (i.e. tRNA splicing endonuclease & tRNA ligase RtcB) has a moonlighting activity and processes and circularizes permuted SRP RNA molecules.

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Finally, we asked why the SRP RNA gene was permuted in some Thermoproteus species and believe that this observation correlates with the emergence of increased tRNA gene fragmentation (Fujishima et al., 2010; Sugahara et al., 2008; Randau et al., 2005). It has been argued that the fragmentation of tRNAs genes can provide a selective advantage during evolution as tRNA genes frequently serve as viral integration sites (Randau and Söll, 2008). The presence of efficient tRNA splicing machinery that can deal with large-scale tRNA intron transposition is required to maintain functionality of a permuted SRP RNA. Thus, it is plausible that permuted SRP RNA genes are derived from an ancestral variant with simple BHB introns. The mechanism of archaeal genome rearrangement events is not known. However, it should be noted that C/D box sRNA genes have previously been observed in the vicinity of split genes and are also located near the permuted SRP RNA gene (Randau, 2012). The potential mobility of C/D box sRNA gene sequences will be subject of future investigations. In conclusion, genome rearrangement events near non-coding RNA genes can result in novel maturation pathways of universal RNA molecules. One example is the observed occurrence of permuted and circularized SRP RNA molecules whose increased stability should provide a selective advantage.

1. 1. Daume M
   2. Plagens A
   3. Randau L

   (2015) Identification of the Thermoproteus tenax small Rnome

   Publicly Available at the NCBI Gene Expression Omnibus (Accession no: GSE72127).

   http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE72127

1. 1. Batey RT

   (2000) Crystal structure of the ribonucleoprotein core of the signal recognition particle

   Science 287:1232–1239.

   https://doi.org/10.1126/science.287.5456.1232

   • Google Scholar
2. 1. Bernstein HD
   2. Poritz MA
   3. Strub K
   4. Hoben PJ
   5. Brenner S
   6. Walter P

   (1989) Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

   Nature 340:482–486.

   https://doi.org/10.1038/340482a0

   • Google Scholar
3. 1. Bhuiyan SH

   (2000) Assembly of archaeal signal recognition particle from recombinant components

   Nucleic Acids Research 28:1365–1373.

   https://doi.org/10.1093/nar/28.6.1365

   • Google Scholar
4. 1. Brock TD
   2. Brock KM
   3. Belly RT
   4. Weiss RL

   (1972) Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature

   Archiv Mikrobiologie 84:54–68.

   https://doi.org/10.1007/BF00408082

   • Google Scholar
5. 1. Chan PP
   2. Lowe TM

   (2009) GtRNAdb: a database of transfer RNA genes detected in genomic sequence

   Nucleic Acids Research 37:D93–D97.

   https://doi.org/10.1093/nar/gkn787

   • Google Scholar
6. 1. Cusack S
   2. Weichenrieder O
   3. Wild K
   4. Strub K

   (2000) Structure and assembly of the alu domain of the mammalian signal recognition particle

   Nature 408:167–173.

   https://doi.org/10.1038/35041507

   • Google Scholar
7. 1. Englert M
   2. Sheppard K
   3. Aslanian A
   4. Yates JR
   5. Soll D
   6. 3rd

   (2011) Archaeal 3'-phosphate RNA splicing ligase characterization identifies the missing component in tRNA maturation

   Proceedings of the National Academy of Sciences 108:1290–1295.

   https://doi.org/10.1073/pnas.1018307108

   • Google Scholar
8. 1. Fujishima K
   2. Sugahara J
   3. Tomita M
   4. Kanai A

   (2010) Large-scale tRNA intron transposition in the archaeal order thermoproteales represents a novel mechanism of intron gain

   Molecular Biology and Evolution 27:2233–2243.

   https://doi.org/10.1093/molbev/msq111

   • Google Scholar
9. 1. Gowda K

   (1997) Binding site of the m-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA

   Nucleic Acids Research 25:388–394.

   https://doi.org/10.1093/nar/25.2.388

   • Google Scholar
10. 1. Gundelfinger ED
    2. Krause E
    3. Melli M
    4. Dobberstein B

    (1983) The organization of the 7SL RNA in the signal recognition particle

    Nucleic Acids Research 11:7363–7374.

    https://doi.org/10.1093/nar/11.21.7363

    • Google Scholar
11. 1. Hainzl T
    2. Huang S
    3. Sauer-Eriksson AE

    (2002) Structure of the SRP19–RNA complex and implications for signal recognition particle assembly

    Nature 417:767–771.

    https://doi.org/10.1038/nature00768

    • Google Scholar
12. 1. Luirink J
    2. Ten Hagen-Jongman CM
    3. van der Weijden CC
    4. Oudega B
    5. High S
    6. Dobberstein B
    7. Kusters R

    (1994)

    An alternative protein targeting pathway in escherichia coli: studies on the role of FtsY

    The EMBO Journal 13:2289–2296.

    • Google Scholar
13. 1. Luirink J

    (2004) SRP-mediated protein targeting: structure and function revisited

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1694:17–35.

    https://doi.org/10.1016/j.bbamcr.2004.03.013

    • Google Scholar
14. 1. Popow J
    2. Englert M
    3. Weitzer S
    4. Schleiffer A
    5. Mierzwa B
    6. Mechtler K
    7. Trowitzsch S
    8. Will CL
    9. Luhrmann R
    10. Soll D
    11. Martinez J

    (2011) HSPC117 is the essential subunit of a human tRNA splicing ligase complex

    Science 331:760–764.

    https://doi.org/10.1126/science.1197847

    • Google Scholar
15. 1. Randau L
    2. Calvin K
    3. Hall M
    4. Yuan J
    5. Podar M
    6. Li H
    7. Soll D

    (2005) The heteromeric nanoarchaeum equitans splicing endonuclease cleaves noncanonical bulge-helix-bulge motifs of joined tRNA halves

    Proceedings of the National Academy of Sciences 102:17934–17939.

    https://doi.org/10.1073/pnas.0509197102

    • Google Scholar
16. 1. Randau L
    2. Münch R
    3. Hohn MJ
    4. Jahn D
    5. Söll D

    (2005) Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5′- and 3′-halves

    Nature 433:537–541.

    https://doi.org/10.1038/nature03233

    • Google Scholar
17. 1. Randau L
    2. Söll D

    (2008) Transfer RNA genes in pieces

    EMBO Reports 9:623–628.

    https://doi.org/10.1038/embor.2008.101

    • Google Scholar
18. 1. Randau L

    (2012) RNA processing in the minimal organism nanoarchaeum equitans

    Genome Biology 13:R63.

    https://doi.org/10.1093/bioinformatics/btp250

    • Google Scholar
19. 1. Rose MA
    2. Weeks KM

    (2001) Visualizing induced fit in early assembly of the human signal recognition particle

    Nature Structural Biology 8:515–520.

    https://doi.org/10.1038/88577

    • Google Scholar
20. 1. Rosenblad MA

    (2003) SRPDB: signal recognition particle database

    Nucleic Acids Research 31:363–364.

    https://doi.org/10.1093/nar/gkg107

    • Google Scholar
21. 1. Römisch K
    2. Webb J
    3. Herz J
    4. Prehn S
    5. Frank R
    6. Vingron M
    7. Dobberstein B

    (1989) Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

    Nature 340:478–482.

    https://doi.org/10.1038/340478a0

    • Google Scholar
22. 1. Siebers B
    2. Zaparty M
    3. Raddatz G
    4. Tjaden B
    5. Albers S-V
    6. Bell SD
    7. Blombach F
    8. Kletzin A
    9. Kyrpides N
    10. Lanz C
    11. Plagens André
    12. Rampp M
    13. Rosinus A
    14. von Jan M
    15. Makarova KS
    16. Klenk H-P
    17. Schuster SC
    18. Hensel R
    19. Fairhead C

    (2011) The complete genome sequence of thermoproteus tenax: a physiologically versatile member of the crenarchaeota

    PLoS ONE 6:e24222.

    https://doi.org/10.1371/journal.pone.0024222.s010

    • Google Scholar
23. 1. Sugahara J
    2. Kikuta K
    3. Fujishima K
    4. Yachie N
    5. Tomita M
    6. Kanai A

    (2008) Comprehensive analysis of archaeal tRNA genes reveals rapid increase of tRNA introns in the order thermoproteales

    Molecular Biology and Evolution 25:2709–2716.

    https://doi.org/10.1093/molbev/msn216

    • Google Scholar
24. 1. Thompson LD
    2. Daniels CJ

    (1988)

    A tRNA(Trp) intron endonuclease from halobacterium volcanii. unique substrate recognition properties

    The Journal of Biological Chemistry 263:17951–17959.

    • Google Scholar
25. 1. Yoshinari S
    2. Shiba T
    3. Inaoka D-K
    4. Itoh T
    5. Kurisu G
    6. Harada S
    7. Kita K
    8. Watanabe Y.-I

    (2009) Functional importance of crenarchaea-specific extra-loop revealed by an x-ray structure of a heterotetrameric crenarchaeal splicing endonuclease

    Nucleic Acids Research 37:4787–4798.

    https://doi.org/10.1093/nar/gkp506

    • Google Scholar
26. 1. Zwieb C

    (1992)

    Recognition of a tetranucleotide loop of signal recognition particle RNA by protein SRP19

    The Journal of Biological Chemistry 267:15650–15656.

    • Google Scholar
27. 1. Zwieb C

    (1994) Site-directed mutagenesis of signal-recognition particle RNA identification of the nucleotides in helix 8 required for interaction with protein SRP19

    European Journal of Biochemistry 222:885–890.

    https://doi.org/10.1111/j.1432-1033.1994.tb18936.x

    • Google Scholar
28. 1. Zwieb C

    (2005) A nomenclature for all signal recognition particle RNAs

    RNA 11:7–13.

    https://doi.org/10.1261/rna.7203605

    • Google Scholar

Author details

1. André Plagens

   Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   AP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Michael Daume

   Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   MD, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Julia Wiegel

   Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

   Contribution

   JW, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

4. Lennart Randau

   1. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
   2. LOEWE Center for Synthetic Microbiology, Synmikro, Marburg, Germany

   Contribution

   LR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   lennart.randau@mpi-marburg.mpg.de

   Competing interests

   The authors declare that no competing interests exist.

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