Mechanism of inhibition of bacterial RNA helicases by diazo dyes and implications for antimicrobial drug development.

RNA helicases represent attractive drug targets as their activity is linked to several human diseases and impacts microbial infectious processes. While some inhibitors of human RNA helicases demonstrated therapeutic potential as anticancer and antiviral drugs in preclinical trials, chemical inhibition of microbial RNA helicases is less investigated. Here, we address this matter by focusing on the RhlE proteobacterial group of RNA helicases. Having previously shown that the RhlE2 RNA helicase is important for the virulence of the opportunistic pathogen Pseudomonas aeruginosa, we screened a library of 1280 molecules for inhibitors of RhlE2 RNA-dependent ATP hydrolytic activity. The most potent inhibitor is the diazo compound Chicago Sky Blue (CSB). Using hydrogen-deuterium exchange mass spectrometry and biochemical assays, we mapped CSB binding to RhlE2 catalytic core and defined its inhibitory mechanism. Targeting microbial RNA helicases as therapeutic strategy is challenging due to potential side-effects linked to protein conservation across life kingdoms. Interestingly, our structure-activity relationship analysis delineates other diazo dyes closely related to CSB differentially affecting RhlE homologs. Our work could thus be exploited for future drug development studies, which are extremely timely considering the increasing spread of antibiotic resistance among bacterial pathogens.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

RNase J1 and J2 Are Host-Encoded Factors for Plasmid Replication.

Plasmids need to ensure their transmission to both daughter-cells when their host divides, but should at the same time avoid overtaxing their hosts by directing excessive host-resources toward production of plasmid factors. Naturally occurring plasmids have therefore evolved regulatory mechanisms to restrict their copy-number in response to the volume of the cytoplasm. In many plasmid families, copy-number control is mediated by a small plasmid-specified RNA, which is continuously produced and rapidly degraded, to ensure that its concentration is proportional to the current plasmid copy-number. We show here that pSA564 from the RepA_N-family is regulated by a small antisense RNA (RNA1), which, when over-expressed in trans, blocks plasmid replication and cures the bacterial host. The 5' untranslated region (5'UTR) of the plasmid replication initiation gene (repA) potentially forms two mutually exclusive secondary structures, ON and OFF, where the latter both sequesters the repA ribosome binding site and acts as a rho-independent transcriptional terminator. Duplex formation between RNA1 and the 5'UTR shifts the equilibrium to favor the putative OFF-structure, enabling a single small RNA to down-regulate repA expression at both transcriptional and translational levels. We further examine which sequence elements on the antisense RNA and on its 5'UTR target are needed for this regulation. Finally, we identify the host-encoded exoribonucleases RNase J1 and J2 as the enzymes responsible for rapidly degrading the replication-inhibiting section of RNA1. This region accumulates and blocks RepA expression in the absence of either RNase J1 or J2, which are therefore essential host factors for pSA564 replication in Staphylococcus aureus.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences.

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

TRIM5 is an innate immune sensor for the retrovirus capsid lattice.

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys.

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.

The double-stranded RNA binding domain of the vaccinia virus E3L protein inhibits both RNA- and DNA-induced activation of interferon beta.

Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns. This inhibition occurs in both human HeLa and A549 cells, where RIG-I appears to be required for dsDNA-induced IFNbeta activation. Unexpectedly, the two residues most important for dsRNA binding are also critical for this domain's ability to inhibit all three nucleic acid-induced cellular responses.

RIG-I and dsRNA-induced IFNbeta activation.

Except for viruses that initiate RNA synthesis with a protein primer (e.g., picornaviruses), most RNA viruses initiate RNA synthesis with an NTP, and at least some of their viral (ppp)RNAs remain unblocked during the infection. Consistent with this, most viruses require RIG-I to mount an innate immune response, whereas picornaviruses require mda-5. We have examined a SeV infection whose ability to induce interferon depends on the generation of capped dsRNA (without free 5' tri-phosphate ends), and found that this infection as well requires RIG-I and not mda-5. We also provide evidence that RIG-I interacts with poly-I/C in vivo, and that heteropolymeric dsRNA and poly-I/C interact directly with RIG-I in vitro, but in different ways; i.e., poly-I/C has the unique ability to stimulate the helicase ATPase of RIG-I variants which lack the C-terminal regulatory domain.

Activation of the beta interferon promoter by unnatural Sendai virus infection requires RIG-I and is inhibited by viral C proteins.

As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-beta) very poorly, two unnatural SeV infections were used to study virus-induced IFN-beta activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5'-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(+/-), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form double-stranded RNA (dsRNA) with capped 5' ends. We found that (i) virus-induced signaling to IFN-beta depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e., that RIG-I senses both pppRNAs and dsRNA without 5'-triphosphorylated ends, and (ii) it is the viral C protein (as opposed to V) that is primarily responsible for countering RIG-I-dependent signaling to IFN-beta. Nondefective SeV that cannot specifically express C proteins not only cannot prevent the effects of transfected poly(I-C) or (ppp)RNAs on IFN-beta activation but also synergistically enhances these effects. SeV-V(minus) infection, in contrast, behaves mostly like wt SeV and counteracts the effects of transfected poly(I-C) or (ppp)RNAs.

Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin's antifungal activity.

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guanine-binding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo,14 reduced activity in vitro to < or = 2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.

Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: methyl acceptor specificity, inhibition BY S-adenosylmethionine analogs, and structure-guided mutational analysis.

The Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase Ecm1 has been characterized structurally but not biochemically. Here we show that purified Ecm1 is a monomeric protein that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to GTP. The reaction is cofactor-independent and optimal at pH 7.5. Ecm1 also methylates GpppA, GDP, and dGTP but not ATP, CTP, UTP, ITP, or m(7)GTP. The affinity of Ecm1 for the cap dinucleotide GpppA (K 0.1 mm) is higher than that for GTP (K(m) 1 mm) or GDP (K(m) 2.4 mm). Methylation of GTP by Ecm1 in the presence of 5 microm AdoMet is inhibited by the reaction product AdoHcy (IC(50) 4 microm) and by substrate analogs sinefungin (IC(50) 1.5 microm), aza-AdoMet (IC(50) 100 microm), and carbocyclic aza-AdoMet (IC(50) 35 microm). The crystal structure of an Ecm1.aza-AdoMet binary complex reveals that the inhibitor occupies the same site as AdoMet. Structure-function analysis of Ecm1 by alanine scanning and conservative substitutions identified functional groups necessary for methyltransferase activity in vivo. Amino acids Lys-54, Asp-70, Asp-78, and Asp-94, which comprise the AdoMet-binding site, and Phe-141, which contacts the cap guanosine, are essential for cap methyltransferase activity in vitro.

Specificity and mechanism of RNA cap guanine-N2 methyltransferase (Tgs1).

The 2,2,7-trimethylguanosine (TMG) cap structure is characteristic of certain eukaryotic small nuclear and small nucleolar RNAs. Prior studies have suggested that cap trimethylation might be contingent on cis-acting elements in the RNA substrate, protein components of a ribonucleoprotein complex, or intracellular localization of the RNA substrate. However, the enzymatic requirements for TMG cap formation remain obscure because TMG synthesis has not been reconstituted in vitro from defined components. Tgs1 is a conserved eukaryal protein that was initially identified as being required for RNA cap trimethylation in vivo in budding yeast. Here we show that purified recombinant fission yeast Tgs1 catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to m7GTP and m7GDP. Tgs1 also methylates the cap analog m(7)GpppA but is unreactive with GTP, GDP, GpppA, m2,2,7GTP, m2,2,7GDP, ATP, CTP, UTP, and ITP. The products of methyl transfer to m7GTP and m7GDP formed under conditions of excess methyl acceptor are 2,7-dimethyl GTP and 2,7-dimethyl GDP, respectively. Under conditions of limiting methyl acceptor, the initial m2,7GDP product is converted to m2,2,7GDP in the presence of excess AdoMet. We conclude that Tgs1 is guanine-specific, that N7 methylation must precede N2 methylation, that Tgs1 acts via a distributive mechanism, and that the chemical steps of TMG synthesis do not require input from RNA or protein cofactors.

Yeast-like mRNA capping apparatus in Giardia lamblia.

A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the RNA triphosphatase and RNA guanylyltransferase enzymes that catalyze mRNA cap formation. Here we show that the unicellular pathogen Giardia lamblia encodes an mRNA capping apparatus consisting of separate triphosphatase and guanylyltransferase components, which we characterize biochemically. We also show that native Giardia mRNAs have blocked 5'-ends and that 7-methylguanosine caps promote translation of transfected mRNAs in Giardia in vivo. The Giardia triphosphatase belongs to the tunnel family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, microsporidia, and protozoa such as Plasmodium and Trypanosoma. The tunnel enzymes adopt a unique active-site fold and are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants, which comprise part of a bifunctional triphosphataseguanylyltransferase fusion protein. All available evidence now points to the separate tunnel-type triphosphatase and guanylyltransferase as the aboriginal state of the capping apparatus. We identify a putative tunnel-type triphosphatase and a separate guanylyltransferase encoded by the red alga Cyanidioschyzon merolae. These findings place fungi, protozoa, and red algae in a common lineage distinct from that of metazoa and plants.

Arabidopsis C-terminal domain phosphatase-like 1 and 2 are essential Ser-5-specific C-terminal domain phosphatases.

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acid-responsive transcription in Arabidopsis thaliana. Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in Arabidopsis RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity in vitro, includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative double-stranded RNA binding motifs, is essential for the in vivo function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of CPL1 and CPL2 causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.

An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1.

Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes. Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth. Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates. EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro. We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.

Structure and mechanism of mRNA cap (guanine-N7) methyltransferase.

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.

Ambisense sendai viruses are inherently unstable but are useful to study viral RNA synthesis.

Ambisense Sendai virus (SeV) was prepared in order to study the control of viral RNA synthesis. In these studies, we found that the relative ratios of genomes/antigenomes formed during infection are largely determined by the relative strengths of the replication promoters, independent of the presence of a functional mRNA start site. We also found that the ability of the viral polymerase (vRdRP) to respond to an mRNA editing site requires prior (re)initiation at an mRNA start site, similar to the acquisition of vRdRP processivity in the absence of nascent chain coassembly. During these studies, the inherent instability of ambisense SeV upon passage in embryonated chicken eggs was noted and was found to be associated with a point mutation in the ambisense mRNA (ambi-mRNA) start site that severely limited its expression. Since the interferon (IFN)-induced antiviral state is mediated in part via double-stranded RNA (dsRNA), the efficiency of the ambi-mRNA poly(A)/stop site was examined. This site was found to operate in a manner similar to that of other SeV mRNA poly(A)/stop sites, i.e., at approximately 95% efficiency. This modest level of vRdRP read-through is apparently tolerable for natural SeV because the potential to form dsRNA during infection remains limited. However, when mRNAs are expressed from ambisense SeV antigenomes, vRdRP read-through of the ambi-mRNA poly(A)/stop site creates a capped transcript that can potentially extend the entire length of the antigenome, since there are no further poly(A)/stop sites here. In support of this hypothesis, loss of ambi-mRNA expression during passage of ambisense SeV stocks in eggs is also characterized by conversion of virus that grows poorly in IFN-sensitive cultures and is relatively IFN sensitive to virus that grows well even in IFN-pretreated cells that restrict vesicular stomatitis virus replication, i.e., the wild-type SeV phenotype. The selection of mutants unable to express ambi-mRNA on passage in chicken eggs is presumably due to increased levels of dsRNA during infection. How natural ambisense viruses may deal with this dilemma is discussed.

Characterization of the CTD phosphatase Fcp1 from fission yeast. Preferential dephosphorylation of serine 2 versus serine 5.

The C-terminal domain (CTD) of RNA polymerase II undergoes extensive phosphorylation and dephosphorylation at positions Ser2 and Ser5 during the transcription cycle. A single CTD phosphatase, Fcp1, has been identified in yeast and metazoans. Here we conducted a biochemical characterization of Fcp1 from the fission yeast Schizosaccharomyces pombe. The 723-amino acid Fcp1 protein was expressed at high levels in bacteria. Recombinant Fcp1 catalyzed the metal-dependent hydrolysis of para-nitrophenyl phosphate with a pH optimum of 5.5 (kcat = 2 s(-1); K(m) = 19 mm). Deletion analysis showed that 139- and 143-amino acid segments could be deleted from the N and C termini of Fcp1, respectively, without affecting phosphatase activity. A segment containing amino acids 487-580, deletion of which abolished activity, embraces a BRCT domain present in all known Fcp1 orthologs. Mutations of residues Asp170 and Asp172 abrogated Fcp1 phosphatase activity; the essential aspartates are located within a 170DXDXT172 motif that defines a superfamily of metal-dependent phosphotransferases. We exploited defined synthetic CTD phosphopeptide substrates to show for the first time that: (i) Fcp1 CTD phosphatase activity is not confined to native polymerase II and (ii) Fcp1 displays an inherent preference for a particular CTD phosphorylation array. Using equivalent concentrations (25 microm) of CTD peptides of identical amino acid sequence and phosphoserine content, which differed only in the positions of phosphoserine within the heptad, we found that Fcp1 was 10-fold more active in dephosphorylating Ser2-PO4 than Ser5-PO4.

Characterization of the mRNA capping apparatus of the microsporidian parasite Encephalitozoon cuniculi.

A scheme of eukaryotic phylogeny has been suggested based on the structure and physical linkage of the enzymes that catalyze mRNA cap formation. Here we show that the intracellular parasite Encephalitozoon cuniculi encodes a complete mRNA capping apparatus consisting of separate triphosphatase (EcCet1), guanylyltransferase (EcCeg1), and methyltransferase (Ecm1) enzymes, which we characterize biochemically and genetically. The triphosphatase EcCet1 belongs to a metal-dependent phosphohydrolase family that includes the triphosphatase components of the capping apparatus of fungi, DNA viruses, and the malaria parasite Plasmodium falciparum. These enzymes are structurally and mechanistically unrelated to the metal-independent cysteine phosphatase-type RNA triphosphatases found in metazoans and plants. Our findings support the proposed evolutionary connection between microsporidia and fungi, and they place fungi and protozoa in a common lineage distinct from that of metazoans and plants. RNA triphosphatase presents an attractive target for antiprotozoal/antifungal drug development.