Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The polypyrimidine tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.
Triphosphate tunnel metalloenzymes (TTMs) are a newly recognized superfamily of phosphotransferases defined by a unique active site residing within an eight-stranded  barrel. The prototypical members are the eukaryal metal-dependent RNA triphosphatases, which catalyze the initial step in mRNA capping. Little is known about the activities and substrate specificities of the scores of TTM homologs present in bacterial and archaeal proteomes, nearly all of which are annotated as adenylate cyclases. Here we have conducted a biochemical and structure-function analysis of a TTM protein (CthTTM) from the bacterium Clostridium thermocellum. CthTTM is a metal-dependent tripolyphosphatase and nucleoside triphosphatase; it is not an adenylate cyclase. We have identified 11 conserved amino acids in the tunnel that are critical for tripolyphosphatase and ATPase activity. The most salient findings are that (i) CthTTM is 150-fold more active in cleaving tripolyphosphate than ATP and (ii) the substrate specificity of CthTTM can be transformed by a single mutation (K8A) that abolishes tripolyphosphatase activity while strongly stimulating ATP hydrolysis. Our results underscore the plasticity of CthTTM substrate choice and suggest how novel specificities within the TTM superfamily might evolve through changes in the residues that line the tunnel walls.
Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2,3-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His 98 ), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys 74 ). We find that Rv0805 has a previously unappreciated 2,3-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2,3-cAMP than 3,5-cAMP. Changing His 98 to alanine or asparagine suppressed the 2,3-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2,3-cNMP phosphodiesterase by introducing histidine in lieu of Cys 74 . YfcE-C74H cleaved the P-O2 bond of 2,3-cAMP to yield 3-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2 or P-O3 to yield a mixture of 3-AMP and 2-AMP products, with a bias toward 3-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3 bond of 2,3-cAMP to generate 2-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.The binuclear metallophosphoesterases comprise a vast enzyme superfamily distributed widely among taxa. A prototypal member is bacteriophage phosphatase (-Pase), 2 which has been characterized structurally and biochemically (1-7).-Pase uses Mn 2ϩ to catalyze phosphoester hydrolysis with a variety of substrates, including phosphopeptides, phosphoproteins, nucleoside 2Ј,3Ј-cyclic phosphates, and "generic" organic phosphomonoesters and diesters such as p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate. Although the physiological substrate(s) and biological function of -Pase remain obscure, other well studied members of the binuclear metallophosphoesterase superfamily play key physiological roles in cellular pathways of signal transduction (e.g. the phosphoprotein phosphatase calcineurin), DNA repair (e.g. the DNA nuclease Mre11), or RNA processing (e.g. the RNA debranching enzyme Dbr1) (8 -10).The signature feature of the metallophosphoesterase superfamily is an active site composed of two metal ions (typically manganese, iron or zinc) coordinated with octahedral geometry by a cage of histidine, aspartate, and asparagine side chains (Fig. 1). The metals directly coordinate the scissile phosphate ...
Most human genes generate multiple protein isoforms through alternative pre-mRNA splicing, but the mechanisms controlling alternative splicing choices by RNA binding proteins are not well understood. These proteins can have multiple paralogs expressed in different cell types and exhibiting different splicing activities on target exons. We examined the paralogous polypyrimidine tract binding proteins PTBP1 and PTBP2 to understand how PTBP1 can exhibit greater splicing repression activity on certain exons. Using both an in vivo coexpression assay and an in vitro splicing assay, we show that PTBP1 is more repressive than PTBP2 per unit protein on a target exon. Constructing chimeras of PTBP1 and 2 to determine amino acid features that contribute to their differential activity, we find that multiple segments of PTBP1 increase the repressive activity of PTBP2. Notably, when either RRM1 of PTBP2 or the linker peptide separating RRM2 and RRM3 are replaced with the equivalent PTBP1 sequences, the resulting chimeras are highly active for splicing repression. These segments are distinct from the known region of interaction for the PTBP1 cofactors Raver1 and Matrin3 in RRM2. We find that RRM2 of PTBP1 also increases the repression activity of an otherwise PTBP2 sequence, and that this is potentially explained by stronger binding by Raver1. These results indicate that multiple features over the length of the two proteins affect their ability to repress an exon.
Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni2+/Mn2+-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn2+-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and kcat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.
Acinetobacter baumannii is a nosocomial pathogen capable of causing serious infections associated with high rates of morbidity and mortality. Due to its antimicrobial drug resistance profile, A. baumannii is categorized as an urgent priority pathogen by the Centers for Disease Control and Prevention in the United States and a priority group 1 critical microorganism by the World Health Organization. Understanding how A. baumannii adapts to different host environments may provide critical insights into strategically targeting this pathogen with novel antimicrobial and biological therapeutics. Exposure to human fluids was previously shown to alter the gene expression profile of a highly drug-susceptible A. baumannii strain A118 leading to persistence and survival of this pathogen. Herein, we explore the impact of human pleural fluid (HPF) and human serum albumin (HSA) on the gene expression profile of a highly multi-drug-resistant strain of A. baumannii AB5075. Differential expression was observed for ~30 genes, whose products are involved in quorum sensing, quorum quenching, iron acquisition, fatty acid metabolism, biofilm formation, secretion systems, and type IV pilus formation. Phenotypic and further transcriptomic analysis using quantitative RT-PCR confirmed RNA-seq data and demonstrated a distinctive role of HSA as the molecule involved in A. baumannii’s response.
Clostridium thermocellum polynucleotide kinase/phosphatase (Pnkp) 2 is a multifunctional enzyme that catalyzes the phosphorylation of 5Ј-OH termini of DNA or RNA polynucleotides and the dephosphorylation of 3Ј-phosphate and 2Ј-phosphate ribonucleotides (1). CthPnkp also catalyzes an autoadenylylation reaction via a polynucleotide ligase-type mechanism. These characteristics are consistent with a role in end-healing during RNA or DNA repair. The 870-amino acid CthPnkp polypeptide is composed of 3 catalytic domains, which are an N-terminal module that resembles the polynucleotide kinase domain of bacteriophage T4 Pnkp, a central metal-dependent phosphoesterase module, and a C-terminal module that resembles the nucleotidyltransferase domain of polynucleotide ligases (1). Homologs of CthPnkp are found in the proteomes of other bacterial genera, including Kinetococcus, Nostoc, Streptomyces, Fusobacterium, Bacillus, Helicobacter, Deinococcus, and Thermobifida.CthPnkp and its relatives differ from other RNA/DNA repair enzymes insofar as the 3Ј end-modification component belongs to the binuclear metallophosphoesterase superfamily (2). The metallophosphoesterase superfamily embraces cellular and viral proteins from all domains of life (3-5). Although possessed of a common active site fold and two-metal mechanism, individual family members differ with respect to their biological functions, metal ligands, and substrate specificities (6 -14). The biochemical properties of CthPnkp resemble in many respects those of bacteriophage phosphatase. Like phosphatase (15), CthPnkp is specifically dependent on either nickel or manganese for its activity (1). The crystal structure of phosphatase showed that the active site contains two manganese ions coordinated directly or via water by aspartate, histidine, and asparagine side chains plus a sulfate ion coordinated by the two metals, an arginine and a histidine, in a position that is proposed to mimic the product complex of the enzyme with phosphate (7). The metal and phosphate ligands in phosphatase are strictly conserved in CthPnkp (Fig. 1).A mutational analysis of the phosphatase domain of CthPnkp, guided by its similarity to phosphatase, pinpointed 11 residues required for Ni 2ϩ -dependent hydrolysis of 2Ј-AMP and 3Ј-AMP (2). The ensemble of essential side chains includes the CthPnkp counterparts (Asp-187, His-189, Asp-233, Arg-237, Asn-263, His-264, His-323, His-376, and Asp-392) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of phosphatase (Fig. 1). However, three residues (Asp-236, His-264, and Arg-237) required for activity with 2Ј-AMP or 3Ј-AMP were dispensable for Ni 2ϩ -dependent hydrolysis of the non-nucleotide substrate p-nitrophenyl phosphate. Whereas Arg-237 coordinates two of the phosphate oxygens and is presumed to stabilize the transition state,
Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5¢-OH termini of DNA or RNA polynucleotides and Ni 2+ /Mn 2+ -dependent dephosphorylation of 2¢,3¢ cyclic phosphate, 2¢-phosphate, and 3¢-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an Nterminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallophosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni 2+ -dependent phosphatase activity with 2¢-AMP and 3¢-AMP. Eight of the 11 CthPnkp side chains were also required for Ni 2+ -dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage l phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2¢-AMP or 3¢-AMP were dispensable for Ni 2+ -dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).
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