poly(A) Polymerase Research Articles

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion.

ABSTRACTStar-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3′-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1, and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP–PIPKIα-mediated 3′-end processing as a key anti-invasive mechanism in breast cancer.

Identification of a toxin coding fragment in pBSSB1, a linear plasmid from Salmonella enterica serovar Typhi that can stabilize a multicopy plasmid

Objective: To identify the region conferring stability to pBSSB2 (a linear plasmid, pBSSB1, containing a kanamycin cassette), which is unique to Indonesian isolates of Salmonella enterica serovar Typhi. Methods: The open reading frame (ORF) 009 was identified as a toxin coding gene in the plasmid through introduction of translational termination codons in the ORF. Results: The stability function was located in a fragment that spanned nucleotides 5 766 to 6 828 in the linear plasmid genetic map. Ectopic expression of ORF009 in pBAD18 vector indicated ORF009 codes for a toxin. This fragment could stabilize plasmid pUC18 previously destabilized through mutation of the pcnB (plasmid copy number control) gene that codes for polyA polymerase. Majority of the cells expressing ORF009 were non-viable according to phase contrast microscopy. Conclusions: This study demonstrated that a linear plasmid fragment that carries a gene encoding a toxin possibly conferred stability to the parent plasmid. It was able to stabilize a multicopy plasmid of Escherichia coli.

Distinct regulation of alternative polyadenylation and gene expression by nuclear poly(A) polymerases.

Polyadenylation of nascent RNA by poly(A) polymerase (PAP) is important for 3′ end maturation of almost all eukaryotic mRNAs. Most mammalian genes harbor multiple polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms with distinct functions. How poly(A) polymerases may regulate PAS usage and hence gene expression is poorly understood. Here, we show that the nuclear canonical (PAPα and PAPγ) and non-canonical (Star-PAP) PAPs play diverse roles in PAS selection and gene expression. Deficiencies in the PAPs resulted in perturbations of gene expression, with Star-PAP impacting lowly expressed mRNAs and long-noncoding RNAs to the greatest extent. Importantly, different PASs of a gene are distinctly regulated by different PAPs, leading to widespread relative expression changes of APA isoforms. The location and surrounding sequence motifs of a PAS appear to differentiate its regulation by the PAPs. We show Star-PAP-specific PAS usage regulates the expression of the eukaryotic translation initiation factor EIF4A1, the tumor suppressor gene PTEN and the long non-coding RNA NEAT1. The Star-PAP-mediated APA of PTEN is essential for DNA damage-induced increase of PTEN protein levels. Together, our results reveal a PAS-guided and PAP-mediated paradigm for gene expression in response to cellular signaling cues.

Canonical and noncanonical RNA polyadenylation

Polyadenylation is the non-template addition of adenosine nucleotides at the 3'-end of RNA, which occurs after transcription and generates a poly(A) tail up to 250-300 nucleotides long. In the first section of our review, we consider the classical process of mRNA 3'-terminus formation, which involves the cleavage of the transcript synthesized by RNA polymerase II and the associated poly(A) tail synthesis by canonical polyadenylate polymerase. Nucleotide sequences needed for mRNA cleavage and poly(A) tail synthesis, in particular the AAUAAA polyadenylation signal, as well as numerous proteins and their complexes involved in mRNA cleavage and polyadenylation, is described in detail. The significance of the poly(A) tail for prolonging mRNA lifetime and stimulating their translation is discussed. Data presented in the second section demonstrate that RNA transcribed by RNA polymerase III from certain SINEs (Short Interspersed Elements) can undergo AAUAAA-dependent polyadenylation. The structural and functional features of RNA polymerase III determine the unusual character of polyadenylation of RNAs synthesized by this enzyme. The history of recent developments in this area of study have been described in greater detail, in particular the discovery of AAUAAA-dependent polyadenylation of RNA synthesized by RNA polymerase III, which has not been discussed previously. Data on AAUAAA-independent polyadenylation catalyzed by noncanonical TRAMP poly(A)-polymerases (Trf4 and Trf5) have been presented in the third section. These enzymes promote rapid degradation of RNAs by adding a short poly(A) tail to them. This mechanism enables the recognition, poly(A)-marking, and elimination of incorrectly folded noncoding transcripts (e.g. ribosomal and transfer RNAs).

The Canonical Poly (A) Polymerase PAP1 Polyadenylates Non-Coding RNAs and Is Essential for snoRNA Biogenesis in Trypanosoma brucei

The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms that are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases (PAPs) termed PAP1 and PAP2. PAP1 is encoded by one of the only two genes harboring cis-spliced introns in this organism, and its function is currently unknown. In trypanosomes, all mRNAs, and non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the snoRNAs and lncRNAs. Under the silencing of either PAP1 or PAP2, the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both PAP2 and PAP1 and requires the factors essential for the polyadenylation of mRNAs. The dual polyadenylation of the precursor snoRNAs by PAPs may function to recruit the machinery essential for snoRNA processing.

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

194 EFFECT OF SMALL MOLECULE INHIBITORS ON Oct-4, Nanog, AND Sox-2 EXPRESSION IN BOVINE FETAL FIBROBLAST CELLS

Successful reprogramming of somatic cells towards pluripotency requires the epigenetic marks characteristic of the differentiated cell type first be erased in order to inactivate the somatic cell program and activate the embryonic program. However, the majority of cells that undergo reprogramming become trapped in a partially reprogrammed state that is characterised by the down-regulation of somatic cell marker genes, incomplete reactivation of pluripotency genes, maintenance of viral expression, and the inability to form chimeras. Several small molecules, which act on specific signaling pathways or chromatin modifications, have been shown to improve both the kinetics and efficiency of reprogramming. These chemical modifiers aid in overcoming the roadblocks encountered during the reprogramming process by inducing the necessary epigenetic modifications needed to silence the somatic cell genome and reactivate the embryonic stem cell genome. Chemical treatment of cells before reprogramming can remodel the epigenetic landscape to be more like that of embryonic stem cell by removing the repressive epigenetic marks and relaxing chromatin structure to allow the reprogramming factors easier access to target genes. In the present study, we assessed the effect of pretreatment with small molecules on the expression of Oct-4, Nanog, and Sox-2 in bovine fetal fibroblast cells. Chemical treatment consisted of 3 small molecules: PD0325901, a mitogen-activated protein kinase inhibitor; CHIR99021, a glycogen synthase kinase-3 inhibitor; and NuP0178 (NuPotential, Baton Rouge, LA, USA), a G9a histone methyltransferase inhibitor. Cells were seeded at a density of 0.7 × 106 and expanded before being divided evenly into 2 groups. Control cells were cultured in complete culture medium (DMEM with 10% fetal bovine serum), whereas treatment cells were cultured in complete culture medium containing 0.5 mM PD0325901, 3 mM CHIR99021, and 1.8 mM NuP0178. Messenger RNA was isolated from cell cultures using Dynabeads® mRNA DIRECT™ Kit (Dynal Inc., Lake Success, NY, USA) on Day 7 and 14, and the resulting RNA products were transcribed into cDNA using Bio-Rad iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative RT–PCR was performed to measure transcript levels of Oct-4, Nanog, Sox-2, and poly adenylate polymerase in treated and untreated cells at Days 7 and 14. Transcript levels were quantified by relative quantification using the ΔΔCt method and expressed as ratios of target genes (Oct-4, Nanog, Sox-2) to the reference gene (PAP) and normalized against a calibrator consisting of untreated bovine fetal fibroblast cells. No difference in expression levels between untreated and treated cells was detected at either Day 7 or 14. Currently, we are utilising chromatin immunoprecipitation to examine chromatin and DNA methylation patterns around the promoters of Oct-4, Nanog, and Sox-2 to further elucidate the effects of treatment with this combination of small molecule inhibitors.

131 EFFECT OF PROLONGED PROGESTERONE EXPOSURE OF BEEF COWS ON THE EXPRESSION OF OOCYTE DEVELOPMENTAL COMPETENCE-ASSOCIATED GENES

Progesterone is a key hormone in reproduction and plays a crucial regulatory role during the oestrous cycle and pregnancy. The effect of progesterone on the developmental competence of bovine oocytes is yet to be fully understood. Molecular tools might provide further information on the role of progesterone on expression of developmentally important genes. The objective of this study was to evaluate the effect of the prolonged exposure of beef cows (days = 32) to different progesterone levels on the expression of genes associated with oocyte developmental competence. Fifteen cows were used in the experiment and were divided in 3 treatment groups (group n = 5). Treatment 1 consisted of no progesterone supplementation, treatment 2 consisted of supplementation by insertion of one controlled internal drug release device (CIDR) per animal, and treatment 3 consisted of supplementation with the insertion of 2 CIDR per animal. All cows were subjected to follicle ablation 4 days before initiation of the experiment. At Day 0, prostaglandin F2α (PGF2α, 25 mg of dinoprost tromethamine, i.m., Lutalyze®) was administered and CIDR devices were inserted in cows designated to treatments 2 and 3. Cumulus-oocyte complexes were collected through ovum pickup where follicles >2 mm were aspirated. Samples evaluated were obtained at Day 0, 8, 16, 24, and 32. The oocyte developmental competence-associated genes METAP2, FOXM1, HMGA1 were analysed in the study. Polyadenylate polymerase was used as normalization gene. Relative gene expression was calculated utilising a comparative CT method (2−ΔΔCT) where data were calculated as fold increase (>1) or decrease (<1) from a calibrator included in each PCR reaction. Mean values of the fold increase/decrease were compared between treatments and were analysed by repeated-measures ANOVA. No statistical difference was detected between treatments at Day 0, 8, 16, 24, or 32 when analysing independent relative expression of oocyte developmental competence-associated genes METAP2, FOXM1, or HMGA1 (P > 0.05). Similarly, no significant differences between treatments was detected in relative expression of METAP2, FOXM1, or HMGA1 across time (P > 0.05). Prolonged exposure of progesterone at different levels does not affect the expression of METAP2, FOXM1, and HMGA1 genes in bovine cumulus-oocyte complexes as indicated by the results of this experiment.

Antagonistic control of flowering time by functionally specialized poly(A) polymerases in Arabidopsis thaliana.

Polyadenylation is a critical 3'-end processing step during maturation of pre-mRNAs, and the length of the poly(A) tail affects mRNA stability, nuclear export and translation efficiency. The Arabidopsis thaliana genome encodes three canonical nuclear poly(A) polymerase (PAPS) isoforms fulfilling specialized functions, as reflected by their different mutant phenotypes. While PAPS1 affects several processes, such as the immune response, organ growth and male gametophyte development, the roles of PAPS2 and PAPS4 are largely unknown. Here we demonstrate that PAPS2 and PAPS4 promote flowering in a partially redundant manner. The enzymes act antagonistically to PAPS1, which delays the transition to flowering. The opposite flowering-time phenotypes in paps1 and paps2 paps4 mutants are at least partly due to decreased or increased FLC activity, respectively. In contrast to paps2 paps4 mutants, plants with increased PAPS4 activity flower earlier than the wild-type, concomitant with reduced FLC expression. Double mutant analyses suggest that PAPS2 and PAPS4 act independently of the autonomous pathway components FCA, FY and CstF64. The direct polyadenylation targets of the three PAPS isoforms that mediate their effects on flowering time do not include FLC sense mRNA and remain to be identified. Thus, our results uncover a role for canonical PAPS isoforms in flowering-time control, raising the possibility that modulating the balance of the isoform activities could be used to fine tune the transition to flowering.

Structure of mitochondrial poly(A) RNA polymerase reveals the structural basis for dimerization, ATP selectivity and the SPAX4 disease phenotype.

Polyadenylation, performed by poly(A) polymerases (PAPs), is a ubiquitous post-transcriptional modification that plays key roles in multiple aspects of RNA metabolism. Although cytoplasmic and nuclear PAPs have been studied extensively, the mechanism by which mitochondrial PAP (mtPAP) selects adenosine triphosphate over other nucleotides is unknown. Furthermore, mtPAP is unique because it acts as a dimer. However, mtPAP's dimerization requirement remains enigmatic. Here, we show the structural basis for mtPAP's nucleotide selectivity, dimerization and catalysis. Our structures reveal an intricate dimerization interface that features an RNA-recognition module formed through strand complementation. Further, we propose the structural basis for the N478D mutation that drastically reduces the length of poly(A) tails on mitochondrial mRNAs in patients with spastic ataxia 4 (SPAX4), a severe and progressive neurodegenerative disease.

Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3′-end processing.

Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs.

Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3′-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.

MRNA maturation in giant viruses: variation on a theme.

Giant viruses from the Mimiviridae family replicate entirely in their host cytoplasm where their genes are transcribed by a viral transcription apparatus. mRNA polyadenylation uniquely occurs at hairpin-forming palindromic sequences terminating viral transcripts. Here we show that a conserved gene cluster both encode the enzyme responsible for the hairpin cleavage and the viral polyA polymerases (vPAP). Unexpectedly, the vPAPs are homodimeric and uniquely self-processive. The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication. A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5′-GpppA 2′O methyltransferase activity but is also able to internally methylate the mRNAs’ polyA tails. These findings elucidate how the arm wrestling between hosts and their viruses to access the translation machinery is taking place in Mimiviridae.

Analysis of RNA processing reactions using cell free systems: 3' end cleavage of pre-mRNA substrates in vitro.

The 3' end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3' end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5' cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3' end processing of HIV-1 mRNAs.

Arabidopsis poly(A) polymerase PAPS1 limits founder‐cell recruitment to organ primordia and suppresses the salicylic acid‐independent immune response downstream of EDS1/PAD4

Polyadenylation of pre-mRNAs by poly(A) polymerase (PAPS) is a critical process in eukaryotic gene expression. As found in vertebrates, plant genomes encode several isoforms of canonical nuclear PAPS enzymes. In Arabidopsis thaliana these isoforms are functionally specialized, with PAPS1 affecting both organ growth and immune response, at least in part by the preferential polyadenylation of subsets of pre-mRNAs. Here, we demonstrate that the opposite effects of PAPS1 on leaf and flower growth reflect the different identities of these organs, and identify a role for PAPS1 in the elusive connection between organ identity and growth patterns. The overgrowth of paps1 mutant petals is due to increased recruitment of founder cells into early organ primordia, and suggests that PAPS1 activity plays unique roles in influencing organ growth. By contrast, the leaf phenotype of paps1 mutants is dominated by a constitutive immune response that leads to increased resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis and reflects activation of the salicylic acid-independent signalling pathway downstream of ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1)/PHYTOALEXIN DEFICIENT4 (PAD4). These findings provide an insight into the developmental and physiological basis of the functional specialization amongst plant PAPS isoforms.

Protein-Protein Interaction between Poly(A) Polymerase and Cyclophilin A in Chemotactic Cells

Poly(A) polymerase (PAP) play an essential role for maturation of mRNA by adding the adenylate residues at the 3' end. PAP functions are regulated through protein-protein interaction at its C-terminal region. In this study, cyclophilin A (CypA), a member of the peptidyl-prolyl cis-trans isomerase family, was identified as a partner protein interacting with the C-terminal region PAP. The interaction between PAP and CypA was inhibited by the immunosuppressive drug cyclosporine A. Deletion analysis revealed that the N-terminal 56 residues of CypA are sufficient for the interaction with PAP. Interestingly, we observed that PAP and CypA colocalize in the nucleus during SDF-1-induced chemotaxis, implying that CypA could be involved in the regulation of polyadenylation by PAP in the chemotactic cells.

The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

Crystal Structure of Human Poly(A) Polymerase Gamma Reveals a Conserved Catalytic Core for Canonical Poly(A) Polymerases

In eukaryotes, the poly(A) tail added at the 3′ end of an mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing or post-translational modifications. The complexity of PAP isoforms suggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs are grouped into three clades termed α, β and γ, which originated from two gene duplication events. To date, all the available PAP structures are from the PAPα clade. Here, we present the crystal structure of the first representative of the PAPγ clade, human PAPγ bound to cordycepin triphosphate (3′dATP) and Ca2+. The structure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservation reveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are more divergent.

ISOLATION AND CHARACTERIZATION OF A CERVIDPOXVIRUS FROM A GOITERED GAZELLE (GAZELLA SUBGUTTUROSA) FROM A ZOOLOGIC PARK IN MINNESOTA

Deerpox virus (DPV) is the sole member of the newly ratified Cervidpoxvirus genus in the subfamily Chordopoxvirinae. Presented here is the first diagnostic report of isolation of DPV from a goitered gazelle (Gazella subgutturosa). A tissue homogenate was submitted by a zoologic park to the Minnesota Veterinary Diagnostic Laboratory at the University of Minnesota for poxvirus diagnostic investigation and then referred to Plum Island Foreign Animal Disease Diagnostic Laboratory for confirmation. Poxviral infection was confirmed using electron microscopy. The virus was cultured in vero cells and subjected to further diagnoses for characterization. Polymerase chain reaction targeting the major envelope (B2L) protein and RNA polymerase of parapoxviruses, and the poly-A polymerase gene of capripoxviruses, were all negative. Degenerative pan-poxvirus primers that target the DNA polymerase (DNApol) and DNA topoisomerase (DNAtopo) genes, however, successfully amplified poxviral DNA fragments. Amplification of the DNApol and DNAtopo genes yielded fragments of 543 and 344 base pairs, respectively. DNA sequence and phylogenetic analysis of each gene fragment from the gazelle isolate showed >97% identity in BLAST searches with two DPV virus strains (W848-83 and W-1170-84) isolated from North American mule deer (Odocoileus hemionus) in 1983-1984. Neighbor-joining trees indicate that the isolate is a member of the Cervidpoxvirus genus and shows a more-distant relationship to other ruminant poxviruses, namely the Capripoxvirus genus consisting of lumpy skin disease, sheeppox, and goatpox viruses. This report documents the premiere finding of DPV, a recently characterized virus, in gazelles and demonstrates the need for broadened investigation when diagnosing poxvirus infections in ruminants.

Target specificity among canonical nuclear poly(A) polymerases in plants modulates organ growth and pathogen response

Polyadenylation of pre-mRNAs is critical for efficient nuclear export, stability, and translation of the mature mRNAs, and thus for gene expression. The bulk of pre-mRNAs are processed by canonical nuclear poly(A) polymerase (PAPS). Both vertebrate and higher-plant genomes encode more than one isoform of this enzyme, and these are coexpressed in different tissues. However, in neither case is it known whether the isoforms fulfill different functions or polyadenylate distinct subsets of pre-mRNAs. Here we show that the three canonical nuclear PAPS isoforms in Arabidopsis are functionally specialized owing to their evolutionarily divergent C-terminal domains. A strong loss-of-function mutation in PAPS1 causes a male gametophytic defect, whereas a weak allele leads to reduced leaf growth that results in part from a constitutive pathogen response. By contrast, plants lacking both PAPS2 and PAPS4 function are viable with wild-type leaf growth. Polyadenylation of SMALL AUXIN UP RNA (SAUR) mRNAs depends specifically on PAPS1 function. The resulting reduction in SAUR activity in paps1 mutants contributes to their reduced leaf growth, providing a causal link between polyadenylation of specific pre-mRNAs by a particular PAPS isoform and plant growth. This suggests the existence of an additional layer of regulation in plant and possibly vertebrate gene expression, whereby the relative activities of canonical nuclear PAPS isoforms control de novo synthesized poly(A) tail length and hence expression of specific subsets of mRNAs.