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Structure and function of the archaeal exosome Elena Evguenieva-Hackenberg,1∗ Linlin Hou,1 Stefanie Glaeser2 and Gabriele Klug1 The RNA-degrading exosome in archaea is structurally very similar to the ninesubunit core of the essential eukaryotic exosome and to bacterial polynucleotide phosphorylase (PNPase). In contrast to the eukaryotic exosome, PNPase and the archaeal exosome exhibit metal ion-dependent, phosphorolytic activities and synthesize heteropolymeric RNA tails in addition to the exoribonucleolytic RNA degradation in 3′ → 5′ direction. The archaeal nine-subunit exosome consists of four orthologs of eukaryotic exosomal subunits: the RNase PH-domain-containing subunits Rrp41 and Rrp42 form a hexameric ring with three active sites, whereas the S1-domain-containing subunits Rrp4 and Csl4 form an RNA-binding trimeric cap on the top of the ring. In vivo, this cap contains Rrp4 and Csl4 in variable amounts. Rrp4 confers poly(A) specificity to the exosome, whereas Csl4 is involved in the interaction with the archaea-specific subunit of the complex, the homolog of the bacterial primase DnaG. The archaeal DnaG is a highly conserved protein and its gene is present in all sequenced archaeal genomes, although the exosome was lost in halophilic archaea and some methanogens. In exosome-containing archaea, DnaG is tightly associated with the exosome. It functions as an additional RNA-binding subunit with poly(A) specificity in the reconstituted exosome of Sulfolobus solfataricus and enhances the degradation of adenine-rich transcripts in vitro. Not only the RNA-binding cap but also the hexameric Rrp41–Rrp42 ring alone shows substrate selectivity and prefers purines over pyrimidines. This implies a coevolution of the exosome and its RNA substrates resulting in 3′ -ends with different affinities to the exosome. © 2014 John Wiley & Sons, Ltd. How to cite this article:

WIREs RNA 2014, 5:623–635. doi: 10.1002/wrna.1234

INTRODUCTION

T

he eukaryotic RNA-degrading exosome was discovered in 1997 as an essential and highly conserved protein complex responsible for processing and degradation of RNA in 3′ → 5′ direction.1,2 Soon after the sequencing of several archaeal genomes, it was ∗ Correspondence to: [email protected]

1 Institute

of Microbiology and Molecular Biology, University of Giessen, Giessen, Germany

2 Institute

of Applied Microbiology, University of Giessen, Giessen,

Germany Conflict of interest: The authors have declared no conflicts of interest for this article. Additional Supporting Information may be found in the online version of this article.

Volume 5, September/October 2014

recognized that they harbor genes for orthologs of subunits of the eukaryotic exosome.3,4 Three of the genes (encoding orthologs of the eukaryotic Rrp4, Rrp41, and Rrp42 proteins) were found in a conserved superoperon strongly suggesting the existence of an archaeal exosome.3 In addition to these three proteins, the purified archaeal exosome contains a fourth predicted subunit, which is an ortholog of the eukaryotic Csl4, and an archaea-specific subunit, the homolog of the bacterial primase DnaG5–7 (Figure 1), which are encoded in separate operons. Archaea are prokaryotic microorganisms with molecular features defining them as the third domain of life.8–10 They are ubiquitous, but most extremophiles and all methanogens belong to the domain of archaea. Based on 16S rRNA comparisons, archaea show a closer phylogenetic relationship

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(a)

(b) kDa 47.5

32.5

M CoIP DnaG

Rrp41

Rrp42

Rrp42

Rrp41 Rrp4

Rrp4

25 Cs14 16

Cs14 DnaG

RPD RPD SI

N SI

N

KH Zn

N

TOPRIM

C

FIGURE 1 | Core components of the archaeal exosome. (a) Exemplary silver-stained sodium dodecyl sulfate-polyacrylamide gel with the exosome purified from Sulfolobus solfataricus cell-free extract. Rrp41-directed serum was used for the coimmunoprecipitation (CoIP). M, marker proteins, the sizes in kDa are indicated. CoIP, elution fraction, the exosomal subunits are marked. (b) Schematic representation of the protein domains in the subunits of the archaeal exosome. RPD, RNase PH domain; N, N-terminal domain; C, C-terminal domain; S1, S1 domain; KH, KH domain; Zn, Zn-ribbon domain. The asterisk marks the active RPD of Rrp41.

to eukarya than to bacteria.8,9 Accordingly, many key proteins and protein complexes in archaea like those responsible for replication, transcription, and translation are simplified versions of their eukaryotic counterparts.10–12 It turned out, however, that the nine-subunit core of the archaeal exosome, which is composed entirely of eukaryotic orthologs, shows higher similarity in structure and function to bacterial polynucleotide phosphorylase (PNPase) than to the non-catalytic core of the eukaryotic exosome.13 This correlates with the structure of the mRNA substrates of the archaeal exosome: in contrast to eukaryotic mRNAs, archaeal and bacterial mRNAs are intron-less, do not possess a methylguanosine cap and do not carry long, stabilizing poly(A) tails.14 The exosome is considered as a major 3′ → 5′ exoribonuclease6 and the only RNA-tailing enzyme15 in archaea with similarly broad spectrum of cellular substrates like its eukaryotic counterpart.16 Despite this, not all archaea possess an exosome. Some methanogenic and all halophilic Euryarchaeota lack genes for the exosomal core proteins Rrp41, Rrp42, Rrp4, and Csl43,15,17 (Figure 2). It was noted previously that the loss of the exosome was a case of concerted gene loss, as not only the three genes rrp41, rrp42, and rrp4, which are co-localized as a conserved triad, but also the csl4 gene, which is located elsewhere on the chromosome, are missing in exosome-less archaea.3 As these archaea belong to the two distantly related clades Methanococci and Halobacteria/Methanomicrobia in the phylogenetic subtree of Euryarchaeota (Figure 2), most probably two independent gene loss events occurred during evolution. This suggests the existence of a selection pressure for exosome elimination. Why was the exosome lost in certain archaea? The most prominent exosome-less archaea are extremophiles—extreme halophiles or hyperthermophilic methanogens. However, it should be noted 624

that there are mesophilic, exosome-less methanogens, whereas many hyperthermophiles and even hyperthermophilic methanogens have an exosome. It is tempting to speculate that the exosome was lost in extremophilic ancestors of the today’s exosome-less phylogenetic lines, and that this loss was an adaptation to the harsh environmental conditions, to which the cells were exposed. Such conditions probably hamper the regulation of a phosphorolytic protein complex with a dual function in tailing and in degradation of RNA. Possibly to avoid the regulation of this dual function, in eukaryotes, the phosphorolytic activity of the exosome was lost and the function of RNA tailing in the process of RNA degradation was taken over by other proteins.2 Interestingly, archaea without exosome still harbor the homolog of the bacterial primase DnaG. The high conservation of DnaG in archaea may be due to its recently proposed function in the cell as a primase.18 Alternatively or in addition, the archaeal DnaG protein may play an important role in RNA metabolism even in the absence of exosome. In agreement with the exclusive role of the exosome in RNA tailing, the exosome-less archaea do not have any posttranscriptional modifications at the 3′ -end of RNA.15,17 RNA degradation in methanogenic exosome-less archaea obviously relies exclusively on cleavage and polyadenylation specificity factor (CPSF) homologs with 5′ → 3′ exoribonucleolytic and/or endoribonucleolytic activity,19,20 whereas the halophiles harbor a 3′ → 5′ exoribonuclease with homology to bacterial RNase R in addition to several CPSF homologs17,20 (Figure 3). Exosome-containing archaea like Sulfolobus solfataricus also have CPSF homologs with exoribonucleolytic and probably also endoribonucleolytic activities. Like in most bacteria and in eukarya, their RNA is degraded exoribonucleolytically in both directions—in 5′ → 3′ direction by aCPSF2 (formerly

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Structure and function of the archaeal exosome

Bacteria

Nitrosopumilus maritimus (CP000866) Candidatum Korarchaeum cryptofilum (CP0009689) Nanoarchaeum equitans (AE017199) Methanosarcina barkeri (AJ012094) Methanosarcina mazei (AE008384) Methanosarcina acetivorans (M59137) Methanococcoides burtonii (CP000300) Methanohalophilus mahii (M59133) Methanosalsum zhilinae (FJ224366) Methanohalobium evestigatum (FR733675) Methanosaeta concilii (CP002565) Methanosaeta thermophila (AB071701) Methanosaeta harundinacea (CP003117) Methanocella paludicola (AB196288) Methanocella conradii (JN048683) Methanocella arvoryzae (AM114193) Methanoregula boonei (DQ282124) Methanospirillum hungatei (AY196683) Methanosphaerula palustris (EU156000) Methanocorpusculum labreanum (AY260436) Methanoculleus marisnigri (M59134) Methanoplanus petrolearius (U76631)

*

18 3

**

* *

*

Halobacteria

Thaumarchaeota Korarchaeota Nanoarchaeota

Methanomicrobia

9960

*

Euryarchaeota

Archaeoglobi

9

Methanobacteria

10 Thermococci 5

Thermoplasmata

Aciduliprofundum boonei (CP001941) 15

*

Methanococci

Methanopyrus kandleri (AE009439) 16 11 9

Sulfolobaceae Crenarchaeota

Thermoproteales Desulfurococcales - acidilobales

0.01

FIGURE 2 | The exosome was lost in some phylogenetic lineages of archaea. All genome-sequenced archaea harbor the dnaG gene. In the presented phylogenetic distribution, lineages or strains which do not possess genes for the core subunits of the exosome rrp41 and rrp42 are highlighted in bold and marked with an asterisk. The maximum-likelihood tree is based on nearly full-length 16S rRNA gene sequences. A total of 120 genome-sequenced archaea were included in the analysis (see Supporting Information). The phylogenetic tree was generated in ARB release 5.265 using the ‘All-Species Living Tree’ Project (LTP66 ) ARB database release LTPs111 (February 2013). 16S rRNA gene sequences of genome-sequenced strains not included in the LTP database were obtained from NCBI (http://www.ncbi.nlm.nih.gov/), aligned in the SILVA Incremental Aligner (SINA) version v1.2.1167 and added to the LTP tree (maximum-likelihood tree) using the parsimony quick add marked tool of ARB. Sequences of non-genome-sequenced archaea were removed from the tree. Bar, 0.10 nucleotide substitutions per site. Archaea with sequenced genomes were used for 16S rRNA-based phylogenetic analysis.

archaeal RNase J) preferring monophosphorylated 5′ -ends20,21 and by the exosome in 3′ → 5′ direction22 (Figure 3). This review summarizes the current knowledge on the archaeal exosome. A short description of the native exosome in archaea is followed by an overview of the data accumulated by crystallographic and biochemical analyses of reconstituted exosomal complexes. Special attention is given to the interaction of the exosome with RNA. Volume 5, September/October 2014

THE ARCHAEAL EXOSOME IN VIVO The study of the archaeal exosome in vivo was dominated by analyses of protein complexes containing exosomal subunits, complemented by information about the expression levels of exosomal genes from high-throughput studies originally not aiming at the exosome. All studies were performed with wild-type strains because genetic manipulation of archaea is still quite difficult, despite the considerable progress in the development of suitable genetic tools.23 Therefore,

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Archaea with exosome and heteropolymeric RNA tails aCPSF (exo-)

aCPSF (endo-)

Exosome A AG

Archaea without exosome and without RNA tails aCPSF (exo-)

aCPSF (endo-)

GAA

Methanogens Methanopyrus Methanocaldococcus

Pyrococcus, archasoglobus

aCPSF ? (exo-)

Sulfolobus

? aCPSF (endo-)

RNase R

Haloferax

FIGURE 3 | Schematic overview on RNA degradation in different archaeal groups. All archaea with sequenced genomes possess genes for

cleavage and polyadenylation specificity factor (CPSF) homologs with putative exoribonucleolytic 5′ → 3′ and/or endonucleolytic activities,20 but genes for putative 3′ → 5′ exoribonucleases are not present in all of them.15,19,20 Archaeal genera for which exosome, RNase R, and CPSF homologs were characterized as RNases in vitro are indicated. Question marks indicate activities which were not confirmed experimentally in the indicated genera.

there is still no direct evidence for the function of the archaeal exosome in vivo. The conclusions about its dual physiological role as 3′ → 5′ exoribonuclease and polynucleotidylation enzyme are based on enzymatic assays in vitro, comparisons with eukaryotic and bacterial homologs, and comparisons between RNA from archaea with and without exosome.

Composition of the Archaeal Exosome The first experimental evidence for the existence of the archaeal exosome was presented 2 years after its bioinformatics prediction.3,5 The exosome of the hyperthermophilic and acidophilic archaeon S. solfataricus was isolated by coimmunoprecipitation using polyclonal antibodies directed against recombinant SsoRrp41 (Figure 1(a)). In addition to the four predicted subunits of the archaeal exosome, the orthologs of the eukaryotic proteins Rrp41, Rrp42, Rrp4, and Csl4, three other proteins were detected in the complex—a protein annotated as a bacterial-type primase DnaG, and two proteins with chaperone properties, Cdc48 and Cpn.5 In later studies, Cpn was not identified in the complex, and Cdc48 was found to associate with the exosome only after ultracentrifugation in glycerol gradients but was not co-purified directly from cell-free extracts. At present, we do not consider Cdc48 and Cpn proteins as interaction partners of the S. solfataricus exosome. 626

The exosome of S. solfataricus was purified by coimmunoprecipitation in many independent experiments using antibodies against Rrp41, Rrp4, Csl4, and DnaG, directly from cell-free extracts or after their fractionation.5–7 These analyses revealed that Rrp41, Rrp42, Rrp4, Csl4, and DnaG are always present in the exosome, and also showed the existence of complexes with different amounts of Rrp4, Csl4, and DnaG in relation to the catalytic hexamer of Rrp41 and Rrp42. Several further proteins like EF1-𝛼 7 and a 16-kDa protein of unknown function6 were co-precipitated with the exosome in different experiments, but their physical and functional association with the complex remains to be verified. The existence of the exosome in vivo was confirmed for two further archaeal species. Global analysis of protein complexes in Methanothermobacter thermautotrophicus revealed orthologs of the proteins Rrp41, Rrp42, Rrp4, and DnaG in a 900-kDa complex together with the splicing endonuclease.24 This finding suggested that the association of DnaG with the exosome may be a general feature of archaea, and that the exosome may have different interaction partners in different species. Csl4 was not found in the complex, but as DnaG interacts exclusively through Csl4 with the S. solfataricus exosome25 (Figure 4(a)), most probably Csl4 was overlooked in the exosome of M. thermautotrophicus. According to our experience

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Structure and function of the archaeal exosome

(a) DnaG Rrp4

Rrp4

Csl4

Rrp4

DnaG

Csl4

DnaG

Csl4

Csl4

Rrp4

Rrp4 Rrp42

DnaG

Rrp42

Rrp41

(b)

Rrp42

Rrp41

Rrp41

(c) KH N Sl Zn-r.

Sl

Rrp4 RNA

N 5′

DnaG

AAA A

Csl4

A A A

RPD RPD

Pi 2– Mg 3′

Rrp41

Rrp42

FIGURE 4 | Schematic representation of the structure of the archaeal exosome and its interaction with RNA. (a) Isoforms of the recombinant exosome. Alternating RPD-containing subunits Rrp41 and Rrp42 (barrels marked with Rrp41 and Rrp42, respectively) are arranged in a hexameric ring. On the top of the ring, three S1-domain-containing subunits (ovals representing Rrp4 or Csl4) are located. DnaG binds to the Csl4-exosome but not to the Rrp4-exosome. Complexes with Rrp4–Csl4–DnaG–caps corresponding to the exosome in vivo can also be reconstituted. Rrp4 and Csl4 are present in different amounts in such complexes. (b) DnaG, an Rrp41, an Rrp42, and an Rrp4 subunit are removed to allow a view into the central channel of the hexameric ring and to the domains of the RNA-binding cap. The nucleotides of an RNA substrate are indicated by gray circles. The first four nucleotides (as numbered from the 3′ -end of the substrate) are in the active site of an Rrp41 subunit. Pi and Mg2+ are needed for catalysis. N, N-terminal domain; S1, S1 domain; KH, KH domain; Zn-r., Zn-ribbon domain. (c) Model of adenine-rich RNA bound to the DnaG-exosome with heteromeric RNA-binding cap, which contains two different proteins with poly(A) preference, Rrp4, and DnaG. We propose that the DnaG and the S1 domain of Rrp4 efficiently and selectively interact with adenine-rich stretches in natural substrates of the archaeal exosome.

it is difficult to stain Csl4, and as it is the shortest polypeptide in the complex and is present in lower amounts than Rrp4 in the soluble fraction, high protein amounts must be loaded to detect Csl4 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses.6,7 The association of DnaG with the archaeal exosome was also shown in a study analyzing the interaction partners of proteins involved or supposed to be involved in replication in Thermococcus kodakarensis.26 His-tagged DnaG was expressed in T. kodakarensis and purified together with the exosome and not with replication proteins. This supports the view that the archaeal DnaG protein is a subunit of the RNA-degrading exosome and participates in processing and/or degradation of RNA. This study also identified other putative interaction partners of the archaeal exosome including an S1-domain-containing, RNA-binding protein TK2227.26

Heterogeneity and Subcellular Localization Virtually all analyses of the native archaeal exosome in vivo were performed with S. solfataricus. When Volume 5, September/October 2014

cell-free extract was separated in glycerol6 or sucrose gradients27 containing 150 mM NaCl, the exosome was found in fractions corresponding to 250–270 kDa and in 30S–50S fractions. At high salt (0.5 M or 1 M NaCl), most of the exosomes were found in very-high-molecular-weight fractions together with the membrane,7,27 suggesting membrane localization via hydrophobic interactions. Immunofluorescence microscopy confirmed the localization of Rrp4 and DnaG at the periphery of S. solfataricus cells.27 Important components of the RNA-degrading machinery in the bacteria Escherichia coli and Bacillus subtilis also show specific subcellular localization at the membrane.28–31 The subcellular localization of the exosome probably serves to regulate its availability at different locations in the prokaryotic archaeal cell. Exosomes purified from different fractions of sucrose gradients show differences in the relative amounts of Rrp4, Csl4, and DnaG in relation to the catalytic Rrp41–Rrp42 ring.7 Thus, heterogeneous exosomal complexes are present in the cell. The relative amounts of Rrp4 were higher in the 250–270 kDa

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fraction of the exosome, whereas the 30S–50S fraction contained higher relative amount of Csl4 and DnaG, suggesting that DnaG, which interacts with the exosome via Csl4, is important for the sedimentation properties of the complex. Depletion experiments to separate DnaG–Csl4-containing complexes from those containing Rrp4 failed: Rrp4, DnaG, and Csl4 were always present in the exosome albeit in different stoichiometric amounts. It seems that complexes with different sedimentation properties interact with different proteins: EF1-𝛼 was co-purified only with the lighter complex of S. solfataricus.7 They may also interact with different substrates, but it is still not clear whether the sedimentation of the exosome in the 30S–50S fraction is due to interaction with ribosomal RNA.5

Function as an RNA-Tailing Enzyme in Archaea The structural similarities between the archaeal exosome and bacterial PNPase suggested that they may have similar functions in the cell. In E. coli, PNPase has two functions: using inorganic phosphate, it degrades RNA in 3′ → 5′ direction, while using ribonucleoside 5′ -diphosphates (rNDPs), it synthesizes adenine-rich, heteropolymeric tails at the 3′ -end of various cellular RNAs.32 The RNA tails are important for loading of 3′ → 5′ exoribonucleases (RNase II; RNase R, PNPase itself) and facilitate RNA degradation. While in addition to PNPase, E. coli has a canonical poly(A) polymerase synthesizing homopolymeric poly(A) tails, PNPase is the only RNA-tailing enzyme in cyanobacteria and some organelles which lack poly(A) polymerase.33,34 Archaea also do not have any candidate gene encoding poly(A) polymerase and the exosome was proposed to be its only RNA-tailing enzyme.15 Comparison of RNA from selected archaea with and without exosome revealed that while all the analyzed species having exosome also have adenine-rich, heteropolymeric tails, all those without exosome do not have any posttranscriptional modification at the 3′ -end of their RNAs.15,17 Indeed, exosome-less archaea are the first-described living organisms without posttranscriptionally added RNA tails.15,34 The proposed dual function of the archaeal exosome was supported by biochemical analysis in vitro.35

Regulation of the Archaeal Exosome How the dual function of the exosome is regulated in the archaeal cell is an unresolved question. Like in the case of PNPase, it was proposed that the local environment determines the direction of the reaction 628

(phosphorolysis or RNA synthesis).35 Rrp41, Rrp42, and Rrp4 are encoded in an operon and are most probably co-transcribed, whereas Csl4 and DnaG belong to different operons and might be differently regulated.3,5 Nothing is known, however, about differential expression of these operons under different environmental conditions. Interestingly, several genome-wide studies have shown that the expression of exosomal genes changes under stress. The mRNA levels of rrp41, rrp42, and rrp4 were decreased under heat stress in S. solfataricus,36 whereas mRNA and protein levels were increased under cold stress in Methanolobus psychrophylus37 and Methanococcoides burtonii38 . The upregulation of the exosome under cold stress parallels the upregulation of bacterial 3′ → 5′ exoribonucleases like PNPase39 and RNase R40 under similar conditions, and probably helps in the degradation of RNA secondary structures arising or stabilized at low temperatures.

STRUCTURAL STUDIES ON THE ARCHAEAL EXOSOME The Hexameric Ring and Its Catalytic Mechanism The crystal structures of reconstituted exosomes of several archaeal species are available. Structures of the minimal catalytic subunit of the exosome, the Rrp41–Rp42 hexamer of S. solfataricus,22,41–43 Archaeoglobus fulgidus,44 Pyrococcus abyssi,45 and M. thermoautotrophicus,46 were resolved, in some cases with bound RNA substrates or cofactors. These studies revealed that the catalytic core of the archaeal exosome is composed of three Rrp41–Rrp42 dimers arranged in a hexameric ring containing three active sites in the Rrp41 subunits, although both Rrp41 and Rrp42 contain RNase PH-like domains (RPDs). Rrp41 alone is not capable of RNA phosphorolysis because the active site is located near the surface interacting with Rrp42, and the last four nucleotides (nt) at the 3′ -end of an RNA substrate are bound in a cleft of an Rrp41–Rrp42 dimer by ionic interactions with conserved arginine residues from both proteins.22,41,43,45 Single-stranded region of 10 nt at the 3′ -end of an RNA molecule is needed to span the distance from the active site near the bottom of the Rrp41–Rrp42 hexamer to a narrow neck of 8–10 Å near the top of the hexamer22,44,45,47 (Figure 4(b)). Thus, the Rrp41–Rrp42 hexamer represents a catalytic chamber accessible only for single-stranded RNA explaining the single-strand specificity of the exosome. Binding of RNA to the exosome at the active site and at the narrow neck is important for the processive degradation of RNA.6,48

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Structure and function of the archaeal exosome

It must be pointed out that the structure of the archaeal Rrp41–Rrp42 hexamer is very similar to the catalytic ring of the bacterial PNPase.22,49 This ring is built of the six RPDs of the homotrimeric PNPase and exhibits phosphorolytic 3′ → 5′ activity similarly to the hexameric ring of the archaeal exosome. The eukaryotic exosome also contains a ring of six proteins, each with an RPD.50 Although the eukaryotic hexamer is catalytically inactive,51 its structure is very similar to those of the archaeal exosome and bacterial PNPase.50 Like PNPase, the archaeal exosome needs inorganic phosphate (Pi) or rNDPs and Mg2+ for phosphorolysis or RNA synthesis in vitro, respectively.35,52 In the early crystal structures, a phosphate-mimicking ion was used to visualize the phosphate-binding sites in the Rrp41–Rrp42 hexamer,44 and a direct attack of a conserved aspartate D182 in the Rrp41 of S. solfataricus leading to 3′ -end cleavage of the RNA substrate was proposed.41 This model did not explain the essentiality of divalent metal ions for catalysis.35 The recent model of metal-assisted phosphorolysis of RNA by the archaeal exosome (Figure 4(b)) is based on two studies, in which phosphate ions were detected in the active sites of archaeal exosomes,43,46 and on the detection of Mn2+ at the catalytic sites of E. coli PNPase.52 In the crystal structures of the archaeal exosome, the Pi closely approaches the phosphate of the 3′ -end nucleotide of the RNA, in a suitable position to perform a nucleophilic attack.43,46 In PNPase, the divalent cation is coordinated by highly conserved aspartate residues corresponding to D182 and D188 in Rrp41 of S. solfataricus. It was proposed that in both PNPase and in the archaeal exosome, the active site contains a conserved Mg2+ binding site, and that the divalent cation lowers the energy of the transition state and facilitates the phosphorolytic reaction.43,52 In the recent model of the active site of the archaeal exosome, the Mg2+ ion is positioned between the Pi and the 3′ -phosphate of the RNA substrate, and facilitates the nucleophilic attack of Pi on the phosphate of the 3′ -end nucleotide by stabilization of the transition state.43 After phosphorolysis, the rNDP product is actively released in concert with the entry of a Pi through a conserved side channel of the hexamer. This is followed by a translocation of the RNA substrate and the most 3′ -end nucleotide is positioned at the so-called N1-binding site.45 It can be assumed that during RNA synthesis, the steps described above are followed in the opposite direction: rNDPs are used as substrates and Pi is released. Volume 5, September/October 2014

The Archaeal Nine Subunit Exosome Crystal structures of the two recombinant isoforms of the A. fulgidus exosome with homotrimeric RNA-binding caps build of Rrp4 or Csl4 are available,44 together with several crystallographic studies of the Rrp4-exosome of S. solfataricus.42,43,47 Furthermore, small-angle X-ray scattering (SAXS) analyses of recombinant Pyroccoccus53 and Archaeoglobus48 complexes in solution contributed to our understanding of the structure of the archaeal nine-subunit exosome. In summary, these analyses revealed that the RNA-binding cap is a flexible structure interacting mainly via the N-terminal domains of Rrp4 and Csl4 with the top of the Rrp41–Rrp42 hexamer. Each of the Rrp4 and Csl4 proteins harbors two RNA-binding domains: Rrp4 contains an S1 (ribosomal protein S1 homology) and a KH (protein K homology) domain, whereas Csl4 contains an S1 and a Zn-ribbon domain.3 The S1 domains are oriented to the middle and build an S1-pore above the central opening of the hexameric ring.44 The KH and Zn-ribbon domain are located more at the periphery but have not identical locations in the two different isoforms. The arrangement of Rrp4 and Csl4 in the two isoforms of the A. fulgidus exosome suggested that heteromeric RNA-binding caps are possible.44 Exosomes with such mixed caps were reconstituted in vitro25,44 and were detected in vivo7 (Figure 4(a)). The binding of either Rrp4 or Csl4 changes the shape of the central channel of the Rrp41–Rrp42 hexamer and may allosterically influence its activity.44 Rrp4 and Csl4 are bound to the hexameric ring through hydrophobic interactions mainly by their N-terminal domains, and the RNA-binding domains are quite flexible. In a crystal structure of the Rrp4-exosome of S. solfataricus, Lu et al. found that each of the Rrp4 subunits possess distinct thermal and conformational characteristics, while the Rrp41–Rrp42 hexamer was rigid.42 Earlier SAXS analyses of the Rrp4-exosome from Pyrococcus suggested that the KH and S1 domains are like moving arms in solution.53 Furthermore, recent SAXS analyses of the A. fulgidus exosome with bound RNA suggested that RNA is bound by the flexible KH domain of Rrp4 at the periphery of the exosome and also interacts with the S1 domain.48 Based on these data, it is assumed that the flexible RNA-binding domains of the cap recruit RNA substrates, which then enter the catalytic chamber. This is in agreement with the faster degradation of RNA by nine-subunit exosomes containing the RNA-binding cap when compared with the RNA degradation only by the hexameric ring.6,48,53 The flexibility of the KH- and S1-domaincontaining cap of the archaeal exosome underlines

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its structural and functional similarity to the bacterial PNPase. Each monomer of the homotrimeric PNPase contains an S1 and a KH domain. The three S1 and KH domains of the trimer are arranged on the top of a hexameric ring formed by six RNase PH-like domains. The S1 and KH domains of PNPase are highly flexible and were usually disordered in the crystal structures of bacterial PNPases.49,52,54 Recently, the crystal structure of the Caulobacter crescentus PNPase with resolved S1 and KH domains was published, with and without bound RNA.55 The most exciting finding was that the three KH domains interact with RNA in an asymmetric manner like three hands holding a rope. Asymmetry was also observed in the catalytic hexameric ring containing three active sites. A speculative model was proposed, according to which the KH domains capture the substrate and thread it through the S1-pore into the catalytic hexameric chamber. It was proposed that a mechanical ratcheting is responsible for the movement of the substrate down to the catalytic site during phosphorolysis or in the opposite direction during RNA tailing. The proposed ratcheting of the RPD hexamer implies that after each catalytic step the substrate is bound by one of the three active sites and does not remain at one of the active sites during the processive degradation of RNA.55 This model can also be applied to the archaeal exosome, which has very similar architecture and catalytic mechanism. A demand for flexibility and movements of the hexameric core during catalysis fits well with the observed failure of RNA processing by rigidified, cross-linked archaeal Rrp41–Rrp42 hexamer.48

BIOCHEMICAL CHARACTERIZATION OF THE ARCHAEAL EXOSOME Enzymatic assays in vitro were performed with reconstituted archaeal exosomes and exosomes isolated by coimmunoprecipitation directly from lysed archaeal cells. The results are compatible with the above-described models and highlight the interdependence of the subunits in the complex as well as their individual functions.

Catalysis by Rrp41–Rrp42 Dimers Without a Ring Rrp41 alone cannot degrade or synthesize RNA, but the hexameric Rrp41–Rr42 complex is active6,22,44,53 and interactions of the substrate with the catalytic site and the neck of the hexamer are crucial for activity.41,45 There are three catalytic centers in the hexamer, and it is still an open question whether only one of them is used during the processive degradation 630

of RNA. But is the hexameric ring really necessary for RNA degradation? Hartung et al. prepared mutated Rrp41–Rrp42 dimers which cannot assemble into a ring, showed that they are active, and concluded that ring formation in the cell is not necessary for high activity, but is probably important for controlled RNA degradation.48

Recruitment, Loading, and Degradation of Substrates In agreement with the RNA-binding properties of Rrp4 and Csl4 and their role in substrate recruitment, reconstituted nine-subunit exosomes containing these proteins degrade RNA faster than the Rrp41–Rrp42 hexamer.6,44,48,53,56 The traditional model for substrate loading proposes that the 3′ -end of RNA is threaded through the narrow neck of the hexamer into the catalytic chamber till it reaches one of the three active sites.47 According to an alternative model, the substrate is recruited by the RNA-binding cap and is loaded laterally by a breathing of the hexameric ring.48 This model is supported by the failure of a cross-linked hexameric ring to degrade RNA. The dramatic reduction of catalytic efficiency of the rigid exosome was either due to difficulties in loading the substrate and/or due to missing flexibility preventing the ratcheting, which may be necessary for the processing of the substrate.48,55 The model of lateral loading does not easily explain the single-strand specificity of the exosome, which is ensured if RNA is threaded through the neck of the catalytic chamber. In any case, a stable interaction between the exosome and the substrate resulting in processive degradation includes binding of RNA to the active site and to the neck in the hexamer, and additional interactions with the RNA-binding cap.45,47,48 The kinetics of degradation of RNA by different isoforms of the exosome was studied using reconstituted complexes of A. fulgidus. Careful quantitative measurements with mathematical modeling of the parameters were performed with poly(A) substrates of 30 and 7 nt showing that poly(A) substrates of different length are processed with different kinetics by the two isoforms of the exosome.48 The start of the reaction was faster with the Rrp4-exosome, but the degradation of substrates between 30 and 24 nt was slower than by the Csl4 exosome. This was explained by stronger binding of the poly(A) substrate by the Rrp4-exosome.48 The stronger binding in turn may reflect a poly(A) specificity of the A. fulgidus Rrp4 similar to that described for S. solfataricus.57 Generally, while substrates longer than 10 nt are degraded processively, shorter substrates are degraded distributively by the archaeal exosome.6,48 Such short

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substrates do not interact simultaneously with the neck of the ring and one of the active sites, and are probably released in the central channel after each catalytic step. RNA is shortened to final products of 4 to 3 nt, as the binding of shorter substrates to the active site is too weak.35,48 So far, an enzyme degrading RNA oligonucleotides to mononucleotides is not known in archaea. In bacteria, specialized and essential oligoribonucleases are responsible for these last steps of RNA degradation.58,59 It is important to note that some structured RNAs like tRNAs were easily polyadenylated in vitro by the Rrp41–Rrp42 hexamer (thus the 3′ -end reached the active site under the given experimental condition), but for their degradation Rrp4 or Csl4 was needed.35 Thus, multiple RNA-binding domains in the cap of the exosome serve not only for substrate recruitment but most probably lead to unwinding of structured substrate in an ATP-independent manner. Furthermore, the multiple RNA-binding domains probably ensure the efficient interaction of the archaeal exosome with longer RNA molecules, which more closely resemble natural substrates of the exosome that often used short poly(A) RNAs.57

Differential Role for Rrp4 and Csl4 in the Archaeal Exosome The RNA-binding cap of the archaeal nine-subunit exosome contains both highly conserved subunits Rrp4 and Csl4, suggesting that they may be important for the interaction with different substrates and other proteins.7,44 In vitro assays with different isoforms of the exosome revealed that Csl4 does not change the substrate specificity of the catalytic hexamer, while Rrp4 confers strong poly(A) specificity.57 As the KH domain of Rrp4 is dispensable for this specificity,7 it most probably resides in the S1 domain of the protein such as in the PNPase of chloroplasts.60 Furthermore, it turned out that Csl4 is needed for the interaction of DnaG with the exosome, whereas Rrp4 does not take part in this interaction.25 As DnaG cannot bind to Csl4 alone or to the Rrpr1–Rrp42 hexamer but strongly binds the Csl4 isoform of the exosome, either surfaces of both Csl4 and the hexamer are needed for interaction with DnaG, or in the nine-subunit exosome, Csl4 adopts a conformation necessary for this interaction.25

DnaG, an Additional Poly(A) Binding Subunit of the Exosome The recent reconstitution of DnaG-containing exosomes of S. solfataricus enabled the in vitro analysis of the function of DnaG in RNA degradation.25 Interestingly DnaG modulates the substrate specificity of Volume 5, September/October 2014

the Csl4-exosome: it positively influences the degradation of adenine-rich transcripts corresponding to native RNA tails by the Csl4-exosome and negatively influences the degradation of an adenine-poor synthetic RNA. Furthermore, DnaG shows strong poly(A) binding preference and confers such a preference to the Csl4-exosome. Finally, the presence of DnaG in recombinant exosomes containing both Rrp4 and Csl4 speeds up the degradation of poly(A) RNA in competition assays with heteropolymeric RNA.25 The fact that two different proteins (Rrp4 and DnaG) in the RNA-binding cap of the archaeal exosome contribute to its poly(A) preference is intriguing, as homopolymeric poly(A) tails are not present in archaea.15 At least in the case of S. solfataricus, this poly(A) preference probably serves the efficient interaction with RNAs containing short poly(A) stretches, which are common in this species with average GC-content of 37%61 (Figure 4(c)). However, selective recruitment of adenine-rich substrates by the exosome must not be restricted to Sulfolobus because poly(A) RNA is also bound better by the Rrp4-exosome than by the Csl4 exosome of P. abyssi.56 The different kinetics of poly(A) degradation by the two isoforms of the A. fulgidus exosome can also be explained by the assumption that poly(A) is bound stronger by the Rrp4-exosome than by the Csl4 exosome.48 But why the archaeal exosome should prefer adenine-rich substrates? Most probably, because this helps the exosome to efficiently select the RNA molecules, which are tagged for degradation. In prokaryotes, RNA is tagged for degradation by the posttranscriptional synthesis of poly(A) or adenine-rich tails, which serve as single-stranded, loading paths for 3′ → 5′ exoribonucleases.59,62 The heteropolymeric adenine-rich tails in archaea are synthesized by the exosome.15,17 Rrp4 and DnaG probably help the exosome to remain bound to or to find the tagged substrates for the subsequent degradation process (Figure 4(c)).

Sequence Preferences of the Rrp41–Rrp42 Hexamer Not only the RNA-binding cap but also the hexameric ring of the S. solfataricus exosome seems to exhibit preference for certain RNA sequences or rNDPs. In presence of 10 mM of each rNDP, the hexamer synthesizes very long poly(A) and poly(G) tails of more than 700 nt, but the length of the synthesized poly(U) and poly(C) tails does not exceed 20 nt.35 Thus, the nucleotide distribution in the RNA tails in vivo (45% A; 32% G, 18% U, and 5% C)57 may reflect the efficiency of use of the individual rNDPs by the exosome

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and not necessarily their intracellular concentration. Weaker binding of pyrimidines compared with purines to the active site of the S. solfataricus hexamer may also explain the very low efficiency of degradation and tailing of synthetic poly(U)20 and GGG(CA)15 transcripts by all isoforms of the exosome. On the other hand, a G(GU)16 transcript is degraded and prolonged with a similar efficiency like the poly(A) RNA,57 which is often used as a standard single-stranded substrate in assays with the archaeal exosome.6,22,44,56 If we assume that the processing of certain substrates is strongly delayed due to their high U and (especially) C content, it is tempting to speculate that the 3′ -end sequences of RNAs with different stabilities may reflect the coevolution of the exosome and its substrates. In line with this speculation, the 3′ -end of the stable 16S rRNA has the sequence 5′ -UCACCUC-3′ , and a 170-nt transcript corresponding to the 3′ -portion of 16S rRNA was neither degraded nor polyadenylated in vitro by any of the nine-subunit isoforms of the archaeal exosome.35

CONCLUSION The nine-subunit archaeal exosome containing Rrp41, Rrp42, Rrp4, and Csl4 is a conserved protein complex with structural similarities to the catalytically inactive core of the essential eukaryotic exosome and to the bacterial PNPase, with which it also shares similar catalytic mechanism and cellular functions. The structure and the catalytic mechanism of the Rrp41–Rrp42 hexamer of the archaeal exosome are well understood. It was also shown that the RNA-binding proteins Rrp4 and Csl4 form a heteromeric caps with different stoichiometries on the top of the ring, and that these two proteins have different roles in the most intensely studied archaeal exosome, the one of

S. solfataricus. In this exosome, Rrp4 shows poly(A) specificity for which its KH domain is dispensable, and Csl4 enables the strong binding of the archaeal DnaG, which also exhibits poly(A) specificity. Both Rrp4 and DnaG enhance the interaction of the S. solfataricus exosome with adenine-rich substrates in vitro. It is still an open question whether Rrp4 and DnaG in exosomes of other archaeal species also selectively recruit adenine-rich RNA substrates. As the eukaryotic exosome is activated by addition of poly(A) tails to its substrates,63,64 it will also be interesting to know whether proteins in the RNA-binding cap of the eukaryotic exosome confer poly(A) specificity. Although structural studies suggest sequenceindependent binding of RNA to the active site of several archaeal exosomes, in vitro assays show that the hexameric ring of the S. solfataricus exosome exhibits a preference for purines and seems to be inhibited by high pyrimidine content at the 3′ -end of a substrate. The basis for this substrate preference is not clear. So far, most of the in vitro assays were performed with artificial RNA substrates and the in vitro processing of an obvious substrate candidate like the 3′ -portion of 16S rRNA failed. It can be expected that some of the (potential) interaction partners of the exosome are necessary for the processing of such natural substrates and for the regulation of the activities of the exosome in vivo. For direct analysis of the role of the archaeal exosome in vivo, mutants or manipulated strains with changed expression of specific exosomal genes are needed. The analysis of such strains will also shed light on the mechanisms for subcellular localization of the archaeal exosome. Last but not least, as the archaeal DnaG is an integral part of the exosome, structural studies on DnaG-containing exosomes are necessary for better understanding of the mechanisms of RNA processing and degradation in the third domain of life.

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poly(A) polymerase complex involved in RNA quality control. PLoS Biol 2005, 3:e189. 64. LaCava J, Houseley J, Saveanu C, Petfalski E, Thompson E, Jacquier A, Tollervey D. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 2005, 121:713–724. 65. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar , Buchner A, Lai T, Steppi S, Jobb G, et al. ARB: a software environment for sequence data. Nucleic Acids Res 2004, 32:1363–1371. 66. Yarza P, Richter M, Peplies J, Euzeby J, Amann R, Schleifer KH, Ludwig W, Glöckner FO, Rosselló-Móra R. The all-species living tree project: a 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl Microbiol 2008, 31:241–250. doi: 10.1016/j.syapm.2008.07.001. 67. Pruesse E, Peplies J, Glöckner FO. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 2012, 28:1823–1829. doi: 10.1093/bioinformatics/bts252.

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