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Nuclease is a type of protein that degrades nucleic acids, which plays an important role in biological processes, including RNA interference efficiency and antiviral immunity. However, no evidence of a link between nuclease and Bombyx mori nucleopolyhedrovirus (BmNPV) infection in silkworm B. mori has been found. In this study, a protein asteroid (BmAst) containing the PIN domain and XPG domain was identified in silkworm B. mori. BmAst gene was highest expressed in hemocytes and fat body of the 5th instar larvae, and high expression in the pupa stage. The transcriptional levels of the BmAst gene in 5th instar larvae were significantly induced by BmNPV or dsRNA. After knocking down BmAst gene expression by specific dsRNA, the proliferation of BmNPV in B. mori was increased significantly, whereas the survival rate of larvae was significantly lower when compared with the control. Our findings indicate that BmAst is involved in silkworm resistance to BmNPV infection.

Emergence of multidrug resistant strains and extremely drug resistant strains of Mycobacterium tuberculosis is due to its ability to form persister cells. The formation of persister cells is assumed to be triggered due to the presence of large number of toxin-antitoxin (TA) systems in its genome. Mtb genome encodes 47 VapBC TA systems. In this work, we aim to biochemically characterize VapC46 toxin of the VapBC46 TA operon from Mycobacterium tuberculosis. Heterologous expression of VapC46 in E. coli is shown to exhibit bacteriostasis and toxicity alters the surface morphology of the E. coli cells. VapC46 is shown to possess ribonuclease activity in a magnesium-dependent manner. Using FRET and pull down assay, VapC46 is shown to interact with VapB46 antitoxin. A model of VapC46 is shown to resemble PIN domain family of proteins and reveals the putative active site required for its ribonuclease activity.

PhoH2 proteins are found in a very diverse range of microorganisms that span bacteria and archaea. These proteins are composed of two domains: an N‐terminal PIN‐domain fused with a C‐terminal PhoH domain. Collectively this fusion functions as an RNA helicase and ribonuclease. In other genomic contexts, PINdomains and PhoHdomains are separate but adjacent suggesting association to achieve similar function. Exclusively among the mycobacteria, PhoH2 proteins are encoded in the genome with an upstream gene, phoAT, which is thought to play the role of an antitoxin (in place of the traditional VapB antitoxin that lies upstream of the 47 other PINdomains in the mycobacterial genome). This review examines PhoH2 proteins as a whole and describes the bioinformatics, biochemical, structural, and biological properties of the two domains that make up PhoH2: PIN and PhoH. We review the transcriptional regulators of phoH2 from two mycobacterial species and speculate on the function of PhoH2 proteins in the context of a Type II toxin–antitoxin system which are thought to play a role in the stress response in bacteria.

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1β mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.

Endonucleolytic removal of 5’-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5’-end in close proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.

Control of gene expression in kinetoplastids depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A) binding protein. MKT1 is recruited to mRNA by sequence-specific RNA-binding proteins, resulting in stabilisation of mRNA. We here show that PBP1, LSM12 and an additional 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and there was evidence for a minor subset of complexes that contained both MKT1 and MKT1L. MKT1 appeared to be associated with many mRNAs, with the exception of those encoding ribosomal proteins. XAC1-containing complexes reproducibly contained RNA-binding proteins that were previously found associated with MKT1. In addition, however, XAC1- or MKT1-containing complexes specifically recruit one of the six translation initiation complexes, EIF4E6-EIF4G5; and yeast 2-hybrid assay results indicated that MKT1 interacts with EIF4G5. The C-terminus of MKT1L resembles MKT1: it contains MKT1 domains and a PIN domain that is probably not active as an endonuclease. MKT1L, however, also has an N-terminal extension with regions of low-complexity. Although MKT1L depletion inhibited cell proliferation, we found no evidence for specific interactions with RNA-binding proteins or mRNA. Deletion of the N-terminal extension, however, enabled MKT1L to interact with EIF4E6. We speculate that MKT1L may either enhance or inhibit the functions of MKT1-containing complexes.

International audience; Ribosome biogenesis requires a variety of trans-acting factors in order to produce functional ribosomal subunits. In human cells, the complex formed by the proteins hNob1 and hPno1 is crucial to the site 3 cleavage occurring at the 3'-end of 18S pre-rRNA. However, the properties and activity of this complex are still poorly understood. We present here a detailed characterization of hNob1 organization and its interaction with hPno1. We redefine the boundaries of the endonuclease PIN domain present in hNob1 and we further delineate the precise interacting modules required for complex formation in hNob1 and hPno1. Altogether, our data contributes to a better understanding of the complex biology required during the site 3 cleavage step in ribosome biogenesis.

Lysinibacillus sphaericus C3-41 carries a large low-copy-number plasmid pBsph, which encodes binary toxin proteins. Our previous study found that the transcriptional activator TubX plays an important role in the newly identified type Ⅲ TubRZC replication/partition system in pBsph, and that a vector consisting of tubRZC and tubX is not as stable as pBsph, indicating the presence of other maintenance module(s). In this study, we identified that orf9 and orf10 are necessary for the stability of pBsph by a series of deletion and complementation experiments. Bioinformatics analysis showed that ORF9 contains a PIN domain of VapBC toxin-antitoxin (TA) system, whereas ORF10 share no significant sequence similarity to any of the characterized antitoxins in the database. Further studies revealed that orf9 and orf10 are transcribed as an operon. The overexpression of ORF9 repressed the growth of both Escherichia coli and L. sphaericus, which can be alleviated by overexpression of ORF10. The deletion of orf10 individually or orf9-10 together resulted a decrease on plasmid stability which was restored by the complementation of corresponding gene(s), suggesting that ORF10 plays an important role in plasmid stability. In addition, it was found the plasmid stability is related with the transcription level of tubRZ, and overexpression of TubRZ could neutralize the negative effect on plasmid stability caused by the deletion of orf9-orf10. Moreover, the recombinant vector containing tubRZC, tubX and orf9-10 was more stable than the ones containing only tubRZC and either tubX or orf9-10. The data indicate that the plasmid maintenance system on pBsph includes orf9-orf10 TA system.

MCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3′ untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.

Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.

The PIN (PilT N-terminus) domain is a compact RNA-binding protein domain present in all domains of life. This 120-residue domain consists of a central and parallel β sheet surrounded by α helices, which together organize 4-5 acidic residues in an active site that binds one or more divalent metal ions and in many cases has endoribonuclease activity. In bacteria and archaea, the PIN domain is primarily associated with toxin-antitoxin loci, consisting of a toxin (the PIN domain nuclease) and an antitoxin that inhibits the function of the toxin under normal growth conditions. During nutritional or antibiotic stress, the antitoxin is proteolytically degraded causing activation of the PIN domain toxin leading to a dramatic reprogramming of cellular metabolism to cope with the new situation. In eukaryotes, PIN domains are commonly found as parts of larger proteins and are involved in a range of processes involving RNA cleavage, including ribosomal RNA biogenesis and nonsense-mediated mRNA decay. In this review, we provide a comprehensive overview of the structural characteristics of the PIN domain and compare PIN domains from all domains of life in terms of structure, active site architecture, and activity.

PIN-like domains constitute a widespread superfamily of nucleases, diverse in terms of the reaction mechanism, substrate specificity, biological function and taxonomic distribution. Proteins with PIN-like domains are involved in central cellular processes, such as DNA replication and repair, mRNA degradation, transcription regulation and ncRNA maturation. In this work, we identify and classify the most complete set of PIN-like domains to provide the first comprehensive analysis of sequence–structure–function relationships within the whole PIN domain-like superfamily. Transitive sequence searches using highly sensitive methods for remote homology detection led to the identification of several new families, including representatives of Pfam (DUF1308, DUF4935) and CDD (COG2454), and 23 other families not classified in the public domain databases. Further sequence clustering revealed relationships between individual sequence clusters and showed heterogeneity within some families, suggesting a possible functional divergence. With five structural groups, 70 defined clusters, over 100,000 proteins, and broad biological functions, the PIN domain-like superfamily constitutes one of the largest and most diverse nuclease superfamilies. Detailed analyses of sequences and structures, domain architectures, and genomic contexts allowed us to predict biological function of several new families, including new toxin-antitoxin components, proteins involved in tRNA/rRNA maturation and transcription/translation regulation.

The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis , a founding member of the NYN (Nedd4‐BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo . Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo . Critically, we also demonstrate translation‐dependent Rae1 cleavage of this substrate in a purified translation assay in vitro . Multiple lines of evidence converge to suggest that Rae1 is an A‐site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A‐site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo .

Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminus (PIN) domain containing conserved amino acids considered essential for activity. The vapBC ( v irulence a ssociated p rotein) TA system is the largest subfamily in this class, and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 A resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi Δ vapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC- 1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivo . IMPORTANCE: Herein the crystal structure of the VapBC-1 complex from nontypeable Haemophilus influenzae (NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in E. coli , but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues ex vivo . Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to the in cis wild-type toxin under the same conditions.

The monocyte chemoattractant protein-induced protein (MCPIP) family consists of 4 members (MCPIP1–4) encoded by theZC3h12A-Dgenes, which are located at different loci. The common features of MCPIP proteins are the zinc finger domain, consisting of three cysteines and one histidine (CCCH), and the N-terminal domain of the PilT protein (PilT-N-terminal domain (PIN domain)). All family members act as endonucleases controlling the half-life of mRNA and microRNA (miRNA). The best-studied member of this family is MCPIP1 (also known as Regnase-1).In this review, we discuss the current knowledge on the role of MCPIP1 in cancer-related processes. Because the characteristics of MCPIP1 as a fundamental negative regulator of immune processes have been comprehensively described in numerous studies, we focus on the function of MCPIP1 in modulating apoptosis, angiogenesis and metastasis.

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms.

The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg(2+) -dependent ribonuclease and has been shown to target initiator tRNA(fMet) in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892-899. © 2016 Wiley Periodicals, Inc.

DIS3 (defective in sister chromatid joining) is the catalytic subunit of the exosome, a protein complex involved in the 3'-5' degradation of RNAs. DIS3 is a highly conserved exoribonuclease, also known as Rrp44. Global sequencing studies have identified DIS3 as being mutated in a range of cancers, with a considerable incidence in multiple myeloma. In this work, we have identified two protein-coding isoforms of DIS3. Both isoforms are functionally relevant and result from alternative splicing. They differ from each other in the size of their N-terminal PIN (PilT N-terminal) domain, which has been shown to have endoribonuclease activity and tether DIS3 to the exosome. Isoform 1 encodes a full-length PIN domain, whereas the PIN domain of isoform 2 is shorter and is missing a segment with conserved amino acids. We have carried out biochemical activity assays on both isoforms of full-length DIS3 and the isolated PIN domains. We find that isoform 2, despite missing part of the PIN domain, has greater endonuclease activity compared with isoform 1. Examination of the available structural information allows us to provide a hypothesis to explain this altered behaviour. Our results also show that multiple myeloma patient cells and all cancer cell lines tested have higher levels of isoform 1 compared with isoform 2, whereas acute myeloid leukaemia and chronic myelomonocytic leukaemia patient cells and samples from healthy donors have similar levels of isoforms 1 and 2. Taken together, our data indicate that significant changes in the ratios of the two isoforms could be symptomatic of haematological cancers.

The chromosome of Mycobacterium tuberculosis (Mtb) contains a large number of Type II toxin-antitoxin (TA) systems. The majority of these belong to the VapBC TA family, characterised by the VapC protein consisting of a PIN domain with four conserved acidic residues, and proposed ribonuclease activity. Characterisation of five VapC (VapC1, 19, 27, 29 and 39) proteins from various regions of the Mtb chromosome using a combination of pentaprobe RNA sequences and mass spectrometry revealed a shared ribonuclease sequence-specificity with a preference for UAGG sequences. The TA complex VapBC29 is auto-regulatory and interacts with inverted repeat sequences in the vapBC29 promoter, whereas complexes VapBC1 and VapBC27 display no auto-regulatory properties. The difference in regulation could be due to the different properties of the VapB proteins, all of which belong to different VapB protein families. Regulation of the vapBC29 operon is specific, no cross-talk among Type II TA systems was observed. VapC29 is bacteriostatic when expressed in Mycobacterium smegmatis, whereas VapC1 and VapC27 displayed no toxicity upon expression in M. smegmatis. The shared sequence specificity of the five VapC proteins characterised is intriguing, we propose that the differences observed in regulation and toxicity is the key to understanding the role of these TA systems in the growth and persistence of Mtb.

The precursor ribosomal RNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs. Utp24 is a PIN domain endonuclease in the early 90S precursor of small ribosomal subunit and is proposed to cleave at sites A1 and A2 of pre-rRNA. Here we determine the crystal structure of Utp24 from Schizosaccharomyces pombe at 2.1 angstrom resolution. Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a Zn-finger motif. Functional analysis in Saccharomyces cerevisiae shows that depletion of Utp24 disturbs the assembly of 90S and abolishes cleavage at sites A0, A1 and A2. The 90S assembled with inactivated Utp24 is arrested at a post-A0-cleavage state and contains enriched nuclear exosome for degradation of 5' ETS. Despite of high sequence conservation, Utp24 from other organisms is unable to form an active 90S in S. cerevisiae, suggesting that Utp24 needs to be precisely positioned in 90S. Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.

Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation - related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPβ) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3'UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in WTMCPIP1 than in D141NMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.

The mammalian Zc3h12a/MCPIP1/Regnase-1, an extensively studied regulator of inflammatory response, is the founding member of a ribonuclease family, which includes proteins related by the presence of the so-called Zc3h12a-like NYN domain. Recently, several related proteins have been described in Caenorhabditis elegans, allowing comparative evaluation of molecular functions and biological roles of these ribonucleases. We discuss the structural features of these proteins, which endow some members with ribonuclease (RNase) activity while others with auxiliary or RNA-independent functions. We also consider their RNA specificity and highlight a common role for these proteins in cellular defense, which is remarkable considering the evolutionary distance and fundamental differences in cellular defense mechanisms between mammals and nematodes.

Synechococcus is an abundant marine cyanobacterium that significantly contributes to primary production. Lytic phages are thought to have a major impact on cyanobacterial population dynamics and evolution. Previously, an investigation of the transcriptional response of three Synechococcus strains to infection by the T4-like cyanomyovirus, Syn9, revealed that while the transcript levels of the vast majority of host genes declined soon after infection, those for some genes increased or remained stable. In order to assess the role of two such host-response genes during infection, we inactivated them in Synechococcus sp. strain WH8102. One gene, SYNW1659, encodes a domain of unknown function (DUF3387) that is associated with restriction enzymes. The second gene, SYNW1946, encodes a PIN-PhoH protein, of which the PIN domain is common in bacterial toxin-antitoxin systems. Neither of the inactivation mutations impacted host growth or the length of the Syn9 lytic cycle. However, the DUF3387 mutant supported significantly lower phage DNA replication and yield of phage progeny than the wild-type, suggesting that the product of this host gene aids phage production. The PIN-PhoH mutant, on the other hand, allowed for significantly higher Syn9 genomic DNA replication and progeny production, suggesting that this host gene plays a role in restraining the infection process. Our findings indicate that host-response genes play a functional role during infection and suggest that some function in an attempt at defense against the phage, while others are exploited by the phage for improved infection.

Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis determined to 2.1Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB2C2) and two heterotrimers (VapBC2) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB2C2 and VapBC2 emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin-antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15's toxicity.

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4 ) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex.

The importance of monocyte chemotactic protein-1-induced protein 1 (MCPIP1) in the negative regulation of inflammatory reactions has already been extensively studied. However, its role in cancer is not yet established. We studied MCPIP1 gene expression in primary human neuroblastomas and several neuroblastoma cell lines. Our results showed a lack of MCPIP1 expression in primary neuroblastoma tumors. Moreover, it was found that the low expression of the protein measured in human neuroblastoma cell lines might be important for neuroblastoma survival, since enforced MCPIP1 gene expression in human neuroblastoma BE(2)-C cells caused a significant decrease in neuroblastoma cell viability and proliferation.

The PilT N terminus (PIN) domains with about 130 amino acids in length comprise a very large protein family present in all three kingdoms of life (Arcus et al. 2011). In the Pfam database, the PIN-domain family (PF01850) currently contains 8,807 members from bacteria, archaea, and eukaryotes. The biological functions of PIN-domains are diverse in various species. Most PIN domains have ribonuclease activity involved in different biological process. In eukaryotes, PIN domains are involved in nonsense mediated mRNA decay (NMD), RNA interference (RNAi), ribosomal RNA processing, and RNA degradation in immune response regulation (Bleichert et al. 2006; Xu et al. 2012). In prokaryotes, the majority of PIN domain proteins are the toxic components of VapBC-type toxinantitoxin systems for stress response, while the toxic activity comes from their nuclease activity (Arcus et al. 2011; Blower et al. 2011). Recently, PIN domains were found in Chp1 of RITS (the RNA-induced initiation of transcriptional gene silencing) complex (Schalch et al. 2011) and Rrp44 in the yeast exosome (Makino et al. 2013). PIN-domains have poor sequence conservation but a conserved three-dimensional structure (Arcus et al. 2011). As is shown by the determined structures of many PINdomain proteins using X-ray crystallography, PIN-domains from various organisms have a 3-layer a/b/a sandwich structure which contains a 5-stranded parallel b-sheet with the order 32145 (Arcus et al. 2011). The revealed structural fold of PIN-domains has significant similarity with the Rossmann fold, a nucleotide-binding module existing in many dehydrogenases, kinases, and flavodoxins (Rossmann et al. 1974). Besides the conserved core structure, PINdomain proteins often contain structural decorations and variations of loop and secondary structure elements, such as different a-helix orientation, different length of b-stand, and additional a-helix or b-stand (Takeshita et al. 2007; Bunker et al. 2008). Despite the poor sequence conservation, PIN domains contain a highly conserved active site constituted by several acidic residues for metal binding and ribonuclease activity (Arcus et al. 2011). Protein SSO1118 with the full length of 111 residues from hyperthermophilic archaeon Sulfolobus solfataricus P2 was annotated as a hypothetical protein conserved in Sulfolobale (Fig. 1a). Our previous sequence analysis and NMR chemical shift assignment studies suggested that SSO1118 is a novel putative PIN domain protein (Xuan et al. 2011). Most archaeal PIN domains are from VapC gene of VapBC toxinantitoxin pair whose genes are in an operon in genome, but the gene of SSO1118 is alone in the genome of S. solfataricus P2. Blast search in PDB does not give significant hit. This indicates that SSO1118 has no significant homology with structure-known proteins. In the present study, the solution structure of SSO1118 was determined by NMR J. Xuan (&) X. Song Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Beijing 100083, China e-mail: jsxuan@sas.ustb.edu.cn

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.

The major human pathogen Mycobacterium tuberculosis can survive in the host organism for decades without causing symptoms. A large cohort of Toxin-Antitoxin (TA) modules contribute to this persistence. Of these, 48 TA modules belong to the vapBC (virulence associated protein) gene family. VapC toxins are PIN domain endonucleases that, in enterobacteria, inhibit translation by site-specific cleavage of initiator tRNA. In contrast, VapC20 of M. tuberculosis inhibits translation by site-specific cleavage of the universally conserved Sarcin-Ricin loop (SRL) in 23S rRNA. Here we identify the cellular targets of 12 VapCs from M. tuberculosis by applying UV-crosslinking and deep sequencing. Remarkably, these VapCs are all endoribonucleases that cleave RNAs essential for decoding at the ribosomal A-site. Eleven VapCs cleave specific tRNAs while one exhibits SRL cleavage activity. These findings suggest that multiple vapBC modules contribute to the survival of M. tuberculosis in its human host by reducing the level of translation.

Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.

VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 A resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg2+ ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg2+ ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 A closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

The generalized inflammatory response leads to activation of hundreds of genes transcribed in an established sequence in specialized cells. Transcriptome analysis of human monocyte-derived cells stimulated with IL-1beta or with monocyte chemotactic protein-1 (MCP-1) has led to the identification of a new inflammation-related gene ZC3H12A encoding a chain of 599 amino acids corresponding to a 66-kDa protein. The protein, given a provisional name of MCPIP1 (monocyte chemotactic protein-induced protein-1), is expressed in several human and murine tissues such as bone marrow, spleen, heart and placenta. In in vivo studies, mice with inactivated MCPIP1-encoding gene showed growth retardation, lymphadenopathy, splenomegaly and enhanced inflammatory symptoms. Principal molecular features of MCPIP1 include a single zinc finger motif, an RNase-like PIN domain and ubiquitin-binding domain. Reports from independent laboratories suggest that MCPIP1 may function also as a deubiquitinase. Although MCPIP1 is regarded by some authors as a new transcription factor or cell differentiation factor modulating angiogenesis or adipogenesis, its principal function appears to be downregulation of inflammatory responses through at least two independent mechanisms: increased degradation of cytokine mRNAs and inhibition of LPS- and IL-1-induced NF-kappaB signaling pathway. The interference with NF-kappaB activation is highly complex and includes TRAF6 and TANK interaction with the ubiquitin-associated (UBA) domain of MCPIP1. Purified MCPIP1 protein was reported to degrade specific mRNA and cleave K48- and K63-linked polyubiquitin chains. Although some structural features and the mechanism of action of MCPIP1 are not fully explained yet, its importance in the regulation of inflammatory reactions has been firmly established.

RNA interference (RNAi) is critical for the assembly of heterochromatin at Schizosaccharomyces pombe centromeres. Central to this process is the RNA-induced initiation of transcriptional gene silencing (RITS) complex, which physically anchors small noncoding RNAs to chromatin. RITS includes Ago1, the chromodomain protein Chp1, and Tas3, which forms a bridge between Chp1 and Ago1. Chp1 is a large protein with no recognizable domains, apart from its chromodomain. Here we describe how the structured C-terminal half of Chp1 binds the Tas3 N-terminal domain, revealing the tight association of Chp1 and Tas3. The structure also shows a PIN domain at the C-terminal tip of Chp1 that controls subtelomeric transcripts through a post-transcriptional mechanism. We suggest that the Chp1-Tas3 complex provides a solid and versatile platform to recruit both RNAi-dependent and RNAi-independent gene-silencing pathways for locus-specific regulation of heterochromatin.

Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin—antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA fMet in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translation in vivo and in vitro. Translation-reactions could be reactivated by the addition of VapB and extra charged tRNA fMet . Similarly, ectopic production of tRNA fMet counteracted VapC in vivo. Thus, tRNA fMet is the only cellular target of VapC. Depletion of tRNA fMet by vapC induction was bacteriostatic and stimulated ectopic translation initiation at elongator codons. Moreover, addition of chloramphenicol to cells carrying vapBC induced VapC activity. Thus, by cleavage of tRNA fMet , VapC simultaneously may regulate global cellular translation and reprogram translation initiation.