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3' end processing of most eukaryotic precursor-mRNAs (pre-mRNAs) is a crucial cotranscriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the four-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the poly(A) polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction, and in vitro biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionarily conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing., (© 2024 Todesca et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA plays a fundamental, ubiquitous role as either substrate or functional component of many large cellular complexes-"molecular machines"-used to maintain and control the readout of genetic information, a functional landscape that we are only beginning to understand. The cellular mechanisms for the spatiotemporal organization of the plethora of RNAs involved in gene expression are particularly poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent mechanistic parameters that govern cellular RNA functions, including those of protein coding messenger RNAs (mRNAs). Progress has been hampered, however, by the scarcity of efficient high-yield methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ T7 RNA polymerase to body label an RNA with a cyanine dye, as well as yeast poly(A) polymerase to strategically place multiple 2'-azido-modifications for subsequent fluorophore labeling either between the body and tail or randomly throughout the tail. Using a combination of biochemical and single-molecule fluorescence microscopy approaches, we demonstrate that both yeast poly(A) polymerase labeling strategies result in fully functional mRNA, whereas protein coding is severely diminished in the case of body labeling., (© 2016 The Protein Society.)

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2-GLD-3, we report here the 2.5 Å resolution structure and biochemical characterization of a GLD-2-RNP-8 core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes., (© 2016 Nakel et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

We present an updated and integrated version of our widely used protein-protein docking and binding affinity benchmarks. The benchmarks consist of non-redundant, high-quality structures of protein-protein complexes along with the unbound structures of their components. Fifty-five new complexes were added to the docking benchmark, 35 of which have experimentally measured binding affinities. These updated docking and affinity benchmarks now contain 230 and 179 entries, respectively. In particular, the number of antibody-antigen complexes has increased significantly, by 67% and 74% in the docking and affinity benchmarks, respectively. We tested previously developed docking and affinity prediction algorithms on the new cases. Considering only the top 10 docking predictions per benchmark case, a prediction accuracy of 38% is achieved on all 55 cases and up to 50% for the 32 rigid-body cases only. Predicted affinity scores are found to correlate with experimental binding energies up to r=0.52 overall and r=0.72 for the rigid complexes., (Copyright © 2015. Published by Elsevier Ltd.)

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Caenorhabditis elegans germ-line development defective (GLD)-2-GLD-3 complex up-regulates the expression of genes required for meiotic progression. GLD-2-GLD-3 acts by extending the short poly(A) tail of germ-line-specific mRNAs, switching them from a dormant state into a translationally active state. GLD-2 is a cytoplasmic noncanonical poly(A) polymerase that lacks the RNA-binding domain typical of the canonical nuclear poly(A)-polymerase Pap1. The activity of C. elegans GLD-2 in vivo and in vitro depends on its association with the multi-K homology (KH) domain-containing protein, GLD-3, a homolog of Bicaudal-C. We have identified a minimal polyadenylation complex that includes the conserved nucleotidyl-transferase core of GLD-2 and the N-terminal domain of GLD-3, and determined its structure at 2.3-Å resolution. The structure shows that the N-terminal domain of GLD-3 does not fold into the predicted KH domain but wraps around the catalytic domain of GLD-2. The picture that emerges from the structural and biochemical data are that GLD-3 activates GLD-2 both indirectly by stabilizing the enzyme and directly by contributing positively charged residues near the RNA-binding cleft. The RNA-binding cleft of GLD-2 has distinct structural features compared with the poly(A)-polymerases Pap1 and Trf4. Consistently, GLD-2 has distinct biochemical properties: It displays unusual specificity in vitro for single-stranded RNAs with at least one adenosine at the 3' end. GLD-2 thus appears to have evolved specialized nucleotidyl-transferase properties that match the 3' end features of dormant cytoplasmic mRNAs.

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 Ca(2+). 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., (© 2013.)

Vaccinia virus poly(A) polymerase (VP55) is the only known polymerase that can translocate independently with respect to single-stranded nucleic acid (ssNA). Previously, its structure has only been solved in the context of the VP39 processivity factor. Here, a crystal structure of unliganded monomeric VP55 has been solved to 2.86 Å resolution, showing the first backbone structural isoforms among either VP55 or its processivity factor (VP39). Backbone differences between the two molecules of VP55 in the asymmetric unit indicated that unliganded monomeric VP55 can undergo a `rocking' motion of the N-terminal domain with respect to the other two domains, which may be `rigidified' upon VP39 docking. This observation is consistent with previously demonstrated experimental molecular dynamics of the monomer during translocation with respect to nucleic acid and with different mechanisms of translocation in the presence and absence of processivity factor VP39. Side-chain conformational changes in the absence of ligand were observed at a key primer contact site and at the catalytic center of VP55. The current structure completes the trio of possible structural forms for VP55 and VP39, namely the VP39 monomer, the VP39-VP55 heterodimer and the VP55 monomer.

The 3' ends of most eukaryotic mRNAs are produced by an endonucleolytic cleavage followed by synthesis of a poly(A) tail. Poly(A) polymerase (PAP), the enzyme that catalyzes the formation of the tail, is subject to tight regulation involving several posttranslational modifications. Here we show that the enzyme poly(ADP-ribose) polymerase 1 (PARP1) modifies PAP and regulates its activity both in vitro and in vivo. PARP1 binds to and modifies PAP by poly(ADP-ribosyl)ation (PARylation) in vitro, which inhibits PAP activity. In vivo we show that PAP is PARylated during heat shock, leading to inhibition of polyadenylation in a PARP1-dependent manner. The observed inhibition reflects reduced RNA binding affinity of PARylated PAP in vitro and decreased PAP association with non-heat shock protein-encoding genes in vivo. Our results provide direct evidence that PARylation can control processing of mRNA precursors, and also identify PARP1 as a regulator of polyadenylation during thermal stress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Megavirus chilensis, a close relative of the Mimivirus giant virus, is also the most complex virus sequenced to date, with a 1.26 Mb double-stranded DNA genome encoding 1120 genes. The two viruses share common regulatory elements such as a peculiar palindrome governing the termination/polyadenylation of viral transcripts. They also share a predicted polyadenylate synthase that presents a higher than average percentage of residue conservation. The Megavirus enzyme Mg561 was overexpressed in Escherichia coli, purified and crystallized. A 2.24 Å resolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF.

Nuclear factors NF90 and NF45 form a complex involved in a variety of cellular processes and are thought to affect gene expression both at the transcriptional and translational level. In addition, this complex affects the replication of several viruses through direct interactions with viral RNA. NF90 and NF45 dimerize through their common 'DZF' domain (domain associated with zinc fingers). NF90 has additional double-stranded RNA-binding domains that likely mediate its association with target RNAs. We present the crystal structure of the NF90/NF45 dimerization complex at 1.9-Å resolution. The DZF domain shows structural similarity to the template-free nucleotidyltransferase family of RNA modifying enzymes. However, both NF90 and NF45 have lost critical catalytic residues during evolution and are therefore not functional enzymes. Residues on NF90 that make up its interface with NF45 are conserved in two related proteins, spermatid perinuclear RNA-binding protein (SPNR) and zinc-finger RNA-binding protein (Zfr). Using a co-immunoprecipitation assay and site-specific mutants, we demonstrate that NF45 is also able to recognize SPNR and Zfr through the same binding interface, revealing that NF45 is able to form a variety of cellular complexes with other DZF-domain proteins.

Addition of poly(A) to the 3' ends of cleaved pre-mRNA is essential for mRNA maturation and is catalyzed by Pap1 in yeast. We have previously shown that a non-viable Pap1 mutant lacking the first 18 amino acids is fully active for polyadenylation of oligoA, but defective for pre-mRNA polyadenylation, suggesting that interactions at the N-terminus are important for enzyme function in the processing complex. We have now identified proteins that interact specifically with this region. Cft1 and Pta1 are subunits of the cleavage/polyadenylation factor, in which Pap1 resides, and Nab6 and Sub1 are nucleic-acid binding proteins with known links to 3' end processing. Our results suggest a novel mechanism for controlling Pap1 activity, and possible models invoking these newly-discovered interactions are discussed., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

PAPD5 is one of the seven members of the family of noncanonical poly(A) polymerases in human cells. PAPD5 was shown to polyadenylate aberrant pre-ribosomal RNAs in vivo, similar to degradation-mediating polyadenylation by the noncanonical poly(A) polymerase Trf4p in yeast. PAPD5 has been reported to be also involved in the uridylation-dependent degradation of histone mRNAs. To test whether PAPD5 indeed catalyzes adenylation as well as uridylation of RNA substrates, we analyzed the in vitro properties of recombinant PAPD5 expressed in mammalian cells as well as in bacteria. Our results show that PAPD5 catalyzes the polyadenylation of different types of RNA substrates in vitro. Interestingly, PAPD5 is active without a protein cofactor, whereas its yeast homolog Trf4p is the catalytic subunit of a bipartite poly(A) polymerase in which a separate RNA-binding subunit is needed for activity. In contrast to the yeast protein, the C terminus of PAPD5 contains a stretch of basic amino acids that is involved in binding the RNA substrate.

Advances in RNA nanotechnology will depend on the ability to manipulate, probe the structure and engineer the function of RNA with high precision. This article reviews current abilities to incorporate site-specific labels or to conjugate other useful molecules to RNA either directly or indirectly through post-synthetic labeling methodologies that have enabled a broader understanding of RNA structure and function. Readily applicable modifications to RNA can range from isotopic labels and fluorescent or other molecular probes to protein, lipid, glycoside or nucleic acid conjugates that can be introduced using combinations of synthetic chemistry, enzymatic incorporation and various conjugation chemistries. These labels, conjugations and ligations to RNA are quintessential for further investigation and applications of RNA as they enable the visualization, structural elucidation, localization, and biodistribution of modified RNA., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The 3'-ends of almost all eukaryotic mRNAs are formed in a two-step process, an endonucleolytic cleavage followed by polyadenylation (the addition of a poly-adenosine or poly(A) tail). These reactions take place in the pre-mRNA 3' processing complex, a macromolecular machinery that consists of more than 20 proteins. A general framework for how the pre-mRNA 3' processing complex assembles and functions has emerged from extensive studies over the past several decades using biochemical, genetic, computational, and structural approaches. In this article, we review what we have learned about this important cellular machine and discuss the remaining questions and future challenges., (Copyright © 2010 John Wiley & Sons, Ltd.)

Poly(A) polymerase (PAP) synthesizes the polyadenine tail at the 3'-end of messenger RNA. A disulfide cross-linking strategy was implemented to obtain a complex between bovine PAP (bPAP) and a 15-mer oligo(A). All seven endogenous cysteines were mutated to eliminate nonspecific cross-linked complexes. A cysteine residue was introduced at several different positions and A152C was found to achieve maximum specific cross-linking efficiency. The resulting bPAP construct was active and, when mixed with a chemically modified RNA, yielded crystals of a bPAP-RNA complex. The crystals, which belonged to space group P2 and harbored two protein-RNA complexes per asymmetric unit, diffracted X-rays to 2.25 Å resolution.

The messenger RNA precursors (pre-mRNA) 3'-end processing occurs in a two-step co-transcriptional coupled reaction, denoted as cleavage and polyadenylation. Both processes depend on trans-acting factors interacting in a coordinated manner with cis-sequence motifs located at the 3' untranslated region of transcripts. In this paper, we reviewed mechanisms involved in pre-mRNA processing in eukaryotic organisms, including our own findings about sequences and proteins potentially involved in mRNA 3'-end formation in the protozoan parasite Entamoeba histolytica. Interestingly, protein sequence comparisons among E. histolytica, yeast, and human pre-mRNA processing machineries showed that amoeba pre-mRNA 3'-end processing machinery appears to be in an intermediate evolutionary position between mammals and yeast. In addition, the presence of non canonical poly(A) polymerases family recently identified in E. histolytica, adds more complexity to the mRNA 3'-end formation process in this ancient eukaryote.

The fission yeast Cid14 protein belongs to a family of noncanonical poly(A) polymerases which have been implicated in a broad range of biological functions. Here we describe an extensive Cid14 protein-protein interaction network and its biochemical dissection. Cid14 most stably interacts with the zinc-knuckle protein Air1 to form the Cid14-Air1 complex (CAC). Providing a link to ribosomal RNA processing, Cid14 sediments with 60S ribosomal subunits and copurifies with 60S assembly factors. In contrast, no physical link to chromatin has been identified, although gene expression profiling revealed that efficient silencing of a few heterochromatic genes depends on Cid14 and/or Air1.

We report the inhibition of mammalian polyadenylation by the triphosphate derivatives of adenosine analogues, 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-amino-Ado), which are under preclinical and clinical investigations for the treatment of hematological malignancies. The nucleotide substrate specificity of bovine poly(A) polymerase (PAP) towards C8-modified ATP analogues was examined using primer extension assays. Radiolabeled RNA primers were incubated with bovine PAP, and in the absence of ATP, no primer extension was observed with 8-Cl-ATP, whereas 8-amino-ATP resulted in chain termination. The effects of modified ATP analogues on ATP-dependent poly(A)-tail synthesis by bovine PAP also were determined, and incubation with analogue triphosphate resulted in significant reduction of poly(A)-tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was evaluated, and polyadenylation of the primer by bovine PAP with ATP was blocked completely. To explain these experimental observations and probe the possible structural mechanisms, molecular modeling was employed to examine the interactions between PAP and various ATP analogues. Molecular docking demonstrated that C8-modifications of ATP led to increased distance between the 3'-hydroxyl group of the RNA oligonucleotide terminus and the alpha-phosphate of ATP that render the molecules in an unfavorable position for incorporation into RNA. Similarly, C8-substitution with a chlorine or amino group at the 3'-terminal residue of RNA also inhibits further chain elongation by PAP. In conclusion, modified ATP analogues may exert their biological effects through polyadenylation inhibition, and thus may provide an RNA-directed mechanism of action for 8-Cl-Ado and 8-amino-Ado., (2009 Elsevier Inc. All rights reserved.)

PAP (polyadenylate polymerase) is the template-independent RNA polymerase responsible for synthesis of the 3' poly(A) tails of mRNA. To investigate the role of proton transfer in the catalytic mechanism of PAP, the pH dependence of the steady-state kinetic parameters of yeast PAP were determined for the forward (adenyl transfer) and reverse (pyrophosphorolysis) reactions. The results indicate that productive formation of an enzyme-RNA-MgATP complex is pH independent over a broad pH range, but that formation of an active enzyme-RNA-MgPPi complex is strongly pH dependent, consistent with the production of a proton on the enzyme in the forward reaction. The pH dependence of the maximum velocity of the forward reaction suggests two protonic species are involved in enzyme catalysis. Optimal enzyme activity requires one species to be protonated and the other deprotonated. The deuterium solvent isotope effect on Vmax is also consistent with proton transfer involved in catalysis of a rate-determining step. Finally, pKa calculations of PAP were performed by the MCCE (multiconformational continuum electrostatic) method. Together, the data support that the protonation of residues Lys215 and Tyr224 exhibit co-operativity that is important for MgATP2- and MgPPi2- binding/dissociation, and suggest these residues function in electrostatic, but not in general acid, catalysis.

Vaccinia virus protein VP55 translocates continuously with respect to single-stranded nucleic acid while extending its 3'end. Here, all key sites of polymerase-primer interaction were identified, demonstrating the wrapping or looping of polyadenylation primer around the polymerase during translocation. Side-chain substitutions at one of the sites indicated its requirement for tail extension beyond approximately 12 nucleotides in length, and conformational changes observed upon oligonucleotide binding suggested allosteric connectivity during translocation. Conformational changes in VP39 upon VP55 binding suggested that, within the VP55-VP39 complex, VP39's mRNA 5' cap binding site closes. The crystallographic structure showed a PAPase catalytic center without side-chain substitutions, possessing two metal ions and with all known reactive and catalytic groups represented, fitting a classical two-metal ion mechanism for phosphoryl transfer.

Translational regulation is heavily employed during developmental processes to control the timely accumulation of proteins independently of gene transcription. In particular, mRNA poly(A) tail metabolism in the cytoplasm is a key determinant for balancing an mRNA's translational output and its decay rate. Noncanonical poly(A) polymerases (PAPs), such as germline development defective-2 (GLD-2), can mediate poly(A) tail extension. Little is known about the regulation and functional complexity of cytoplasmic PAPs. Here we report the discovery of Caenorhabditis elegans GLD-4, a cytoplasmic PAP present in P granules that is orthologous to Trf4/5p from budding yeast. GLD-4 enzymatic activity is enhanced by its interaction with GLS-1, a protein associated with the RNA-binding protein GLD-3. GLD-4 is predominantly expressed in germ cells, and its activity is essential for early meiotic progression of male and female gametes in the absence of GLD-2. For commitment into female meiosis, both PAPs converge on at least one common target mRNA-i.e., gld-1 mRNA-and, as a consequence, counteract the repressive action of two PUF proteins and the putative deadenylase CCR-4. Together our findings suggest that two different cytoplasmic PAPs stabilize and translationally activate several meiotic mRNAs to provide a strong fail-safe mechanism for early meiotic progression.

Regulation of nuclear genome expression in Trypanosoma brucei is critical for this protozoan parasite's successful transition between its vertebrate and invertebrate host environments. The canonical eukaryotic circuits such as modulation of transcription initiation, mRNA splicing and polyadenylation appear to be nearly non-existent in T. brucei suggesting that the transcriptome is primarily defined by mRNA turnover. Our previous work has highlighted sequence similarities between terminal RNA uridylyl transferases (TUTases) and non-canonical poly(A) polymerases, which are widely implicated in regulating nuclear, cytoplasmic and organellar RNA decay throughout the eukaryotic lineage. Here, we have continued characterization of TUTase-like proteins in T. brucei and identified two nuclear non-canonical poly(A) polymerases (ncPAPs). The 82kDa TbncPAP1 is essential for viability of procyclic and bloodstream forms of T. brucei. Similar to Trf4/5 proteins from budding yeast, TbncPAP1 requires protein cofactor(s) to exert poly(A) polymerase activity in vitro. The recombinant 54kDa TbncPAP2 showed a PAP activity as an individual polypeptide. Proteomic analysis of the TbncPAP1 interactions demonstrated its association with Mtr4 RNA helicase and several RNA binding proteins, including a potential ortholog of Air1p/2p proteins, which indicates the presence of a stable TRAMP-like complex in trypanosomes. Our findings suggest that TbncPAP1 may be a "quality control" nuclear PAP involved in targeting aberrant or anti-sense transcripts for degradation by the 3'-exosome. Such mechanisms are likely to play a major role in alleviating promiscuity of the transcriptional machinery.

The Saccharomyces cerevisiae poly(A) polymerases Trf4 and Trf5 are involved in an RNA quality control mechanism, where polyadenylated RNAs are degraded by the nuclear exosome. Although Trf4/5 homologue genes are distributed throughout multicellular organisms, their biological roles remain to be elucidated. We isolated here the two homologues of Trf4/5 in Drosophila melanogaster, named DmTRF4-1 and DmTRF4-2, and investigated their biological function. DmTRF4-1 displayed poly(A) polymerase activity in vitro, whereas DmTRF4-2 did not. Gene knockdown of DmTRF4-1 by RNA interference is lethal in flies, as is the case for the trf4 trf5 double mutants. In contrast, disruption of DmTRF4-2 results in viable flies. Cellular localization analysis suggested that DmTRF4-1 localizes in the nucleolus. Abnormal polyadenylation of snRNAs was observed in transgenic flies overexpressing DmTRF4-1 and was slightly increased by the suppression of DmRrp6, the 3'-5' exonuclease of the nuclear exosome. These results suggest that DmTRF4-1 and DmRrp6 are involved in the polyadenylation-mediated degradation of snRNAs in vivo.

Showing a high sequence similarity, the evolutionary closely related bacterial poly(A) polymerases (PAP) and CCA-adding enzymes catalyze quite different reactions--PAP adds poly(A) tails to RNA 3'-ends, while CCA-adding enzymes synthesize the sequence CCA at the 3'-terminus of tRNAs. Here, two highly conserved structural elements of the corresponding Escherichia coli enzymes were characterized. The first element is a set of amino acids that was identified in CCA-adding enzymes as a template region determining the enzymes' specificity for CTP and ATP. The same element is also present in PAP, where it confers ATP specificity. The second investigated region corresponds to a flexible loop in CCA-adding enzymes and is involved in the incorporation of the terminal A-residue. Although, PAP seems to carry a similar flexible region, the functional relevance of this element in PAP is not known. The presented results show that the template region has an essential function in both enzymes, while the second element is surprisingly dispensable in PAP. The data support the idea that the bacterial PAP descends from CCA-adding enzymes and still carries some of the structural elements required for CCA-addition as an evolutionary relic and is now fixed in a conformation specific for A-addition.

In yeast, the mRNA processing enzyme poly(A) polymerase is tethered to the much larger 3'-end processing complex via Fip1, a 36 kDa protein of unknown structure. We report the 2.6 A crystal structure of yeast poly(A) polymerase in complex with a peptide containing residues 80-105 of Fip1. The Fip1 peptide binds to the outside surface of the C-terminal domain of the polymerase. On the basis of this structure, we designed a mutant of the polymerase (V498Y, C485R) that is lethal to yeast. The mutant is unable to bind Fip1 but retains full polymerase activity. Fip1 is found in all eukaryotes and serves to connect poly(A) polymerase to pre-mRNA processing complexes in yeast, plants, and mammals. However, the Fip1 sequence is highly divergent, and residues on both Pap1 and Fip1 at the observed interaction surface are poorly conserved. Herein we demonstrate using analytical ultracentrifugation, circular dichroism, proteolytic studies, and other techniques that, in the absence of Pap1, Fip1 is largely, if not completely, unfolded. We speculate that flexibility may be important for Fip1's function as a molecular scaffold.

Unlike the heterodimeric poly(A) polymerase (PAP) of vaccinia virus (VACV), the PAP from the Amsacta moorei entomopoxvirus, AMEV, is potentially derived from three subunits: a single large and two small subunits (AMV060 and AMV115). The VACV small subunit serves as a 2'-O-methyltransferase, a processivity factor for mRNA polyadenylation, and a transcription elongation factor. We wished to determine the structure-function relationships of the three putative AMEV PAP subunits. We show that AMV060 is expressed as an early gene persisting throughout infection, whereas AMV115 is expressed late. We demonstrate that AMV060 exhibits 2'-O-methyltransferase activity but the gene is not essential for virus growth. Absence of the AMV060 protein has no effect on the length of the poly(A) tails present in mRNA. No physical association was found between any of the putative AMEV PAP subunits. We therefore propose that mRNA polyadenylation does not require interactions between these three proteins.

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post-transcriptionally and requires extensive U-insertion/deletion mRNA editing. In mitochondrial extracts, 3' adenylation reportedly influences degradation kinetics of synthetic edited and pre-edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A-tails typically found in mitochondrial mRNAs, and decreases the abundance of never-edited and edited transcripts. Pre-edited mRNAs are not destabilized by the lack of 3' adenylation, whereas short A-tails are required and sufficient to maintain the steady-state levels of partially edited, fully edited, and never-edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A-tail in edited molecules. Upon completion of the editing process, the short A-tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3' processing and editing of mitochondrial mRNAs in trypanosomes.

We have recently identified Star-PAP, a nuclear poly(A) polymerase that associates with phosphatidylinositol-4-phosphate 5-kinase Ialpha (PIPKIalpha) and is required for the expression of a specific subset of mRNAs. Star-PAP activity is directly modulated by the PIPKIalpha product phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)), linking nuclear phosphoinositide signaling to gene expression. Here, we show that PI-4,5-P(2)-dependent protein kinase activity is also a part of the Star-PAP protein complex. We identify the PI-4,5-P(2)-sensitive casein kinase Ialpha (CKIalpha) as a protein kinase responsible for this activity and further show that CKIalpha is capable of directly phosphorylating Star-PAP. Both CKIalpha and PIPKIalpha are required for the synthesis of some but not all Star-PAP target mRNA, and like Star-PAP, CKIalpha is associated with these messages in vivo. Taken together, these data indicate that CKIalpha, PIPKIalpha, and Star-PAP function together to modulate the production of specific Star-PAP messages. The Star-PAP complex therefore represents a location where multiple signaling pathways converge to regulate the expression of specific mRNAs.

Egg activation is the process that modifies mature, arrested oocytes so that embryo development can proceed. One key aspect of egg activation is the cytoplasmic polyadenylation of certain maternal mRNAs to permit or enhance their translation. wispy (wisp) maternal-effect mutations in Drosophila block development during the egg-to-embryo transition. We show here that the wisp gene encodes a member of the GLD-2 family of cytoplasmic poly(A) polymerases (PAPs). The WISP protein is required for poly(A) tail elongation of bicoid, Toll, and torso mRNAs upon egg activation. In Drosophila, WISP and Smaug (SMG) have previously been reported to be required to trigger the destabilization of maternal mRNAs during egg activation. SMG is the major regulator of this activity. We report here that SMG is still translated in activated eggs from wisp mutant mothers, indicating that WISP does not regulate mRNA stability by controlling the translation of smg mRNA. We have also analyzed in detail the very early developmental arrest associated with wisp mutations. Pronuclear migration does not occur in activated eggs laid by wisp mutant females. Finally, we find that WISP function is also needed during oogenesis to regulate the poly(A) tail length of dmos during oocyte maturation and to maintain a high level of active (phospho-) mitogen-activated protein kinases (MAPKs).

Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by which they directly regulate nuclear functions remains unknown. PIPKIs regulate cellular functions through interactions with protein partners, often PtdIns4,5P2 effectors, that target PIPKIs to discrete subcellular compartments, resulting in the spatial and temporal generation of PtdIns4,5P2 required for the regulation of specific signalling pathways. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIalpha. Here we show that PIPKIalpha co-localizes at nuclear speckles and interacts with a newly identified non-canonical poly(A) polymerase, which we have termed Star-PAP (nuclear speckle targeted PIPKIalpha regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIalpha function together in a complex to control the expression of select mRNAs, including the transcript encoding the key cytoprotective enzyme haem oxygenase-1 (refs 8, 9) and other oxidative stress response genes by regulating the 3'-end formation of their mRNAs. Taken together, the data demonstrate a model by which phosphoinositide signalling works in tandem with complement pathways to regulate the activity of Star-PAP and the subsequent biosynthesis of its target mRNA. The results reveal a mechanism for the integration of nuclear phosphoinositide signals and a method for regulating gene expression.

Poly(A) polymerase (PAP), which adds poly(A) tails to the 3' end of mRNA, can be phosphorylated at several sites in the C-terminal domain. Phosphorylation often mediates regulation by extracellular stimuli, suggesting PAP may be regulated by such stimuli. In this study, we found that phosphorylation of PAP was increased upon growth stimulation and that the mitogen-activated protein kinase ERK was responsible for the increase in phosphorylation. We identified serine 537 of PAP as a unique phosphorylation site by ERK. PAP phosphorylation of serine 537 by ERK increased its nonspecific polyadenylation activity in vitro. This PAP activity was also activated by stimulation of ERK with phorbol-12-myristate-13-acetate in vivo. These data suggest that ERK is a novel regulatory kinase for PAP and further, that PAP activity could be regulated by extracellular stimuli through an ERK-dependent signaling pathway(s).

Fission yeast Cid14, a component of the TRAMP (Cid14/Trf4-Air1-Mtr4 polyadenylation) complex, polyadenylates nuclear RNA and stimulates degradation by the exosome for RNA quality control. Here, we analyze patterns of global gene expression in cells lacking the Cid14 or the Dis3/Rpr44 subunit of the nuclear exosome. We found that transcripts from many genes induced during meiosis, including key regulators, accumulated in the absence of Cid14 or Dis3. Moreover, our data suggest that additional substrates include transcripts involved in heterochromatin assembly. Mutant cells lacking Cid14 and/or Dis3 accumulate transcripts corresponding to naturally silenced repeat elements within heterochromatic domains, reflecting defects in centromeric gene silencing and derepression of subtelomeric gene expression. We also uncover roles for Cid14 and Dis3 in maintaining the genomic integrity of ribosomal DNA. Our data indicate that polyadenylation-assisted nuclear RNA turnover functions in eliminating a variety of RNA targets to control diverse processes, such as heterochromatic gene silencing, meiotic differentiation, and maintenance of genomic integrity.

RNA-specific nucleotidyl transferases (rNTrs) are a diverse family of template-independent polymerases that add ribonucleotides to the 3'-ends of RNA molecules. All rNTrs share a related active-site architecture first described for DNA polymerase beta and a catalytic mechanism conserved among DNA and RNA polymerases. The best known examples are the nuclear poly(A) polymerases involved in the 3'-end processing of eukaryotic messenger RNA precursors and the ubiquitous CCA-adding enzymes that complete the 3'-ends of tRNA molecules. In recent years, a growing number of new enzymes have been added to the list that now includes the "noncanonical" poly(A) polymerases involved in RNA quality control or in the readenylation of dormant messenger RNAs in the cytoplasm. Other members of the group are terminal uridylyl transferases adding single or multiple UMP residues in RNA-editing reactions or upon the maturation of small RNAs and poly(U) polymerases, the substrates of which are still not known. 2'-5'Oligo(A) synthetases differ from the other rNTrs by synthesizing oligonucleotides with 2'-5'-phosphodiester bonds de novo.

We report the 1.8 A structure of yeast poly(A) polymerase (PAP) trapped in complex with ATP and a five residue poly(A) by mutation of the catalytically required aspartic acid 154 to alanine. The enzyme has undergone significant domain movement and reveals a closed conformation with extensive interactions between the substrates and all three polymerase domains. Both substrates and 31 buried water molecules are enclosed within a central cavity that is open at both ends. Four PAP mutants were subjected to detailed kinetic analysis, and studies of the adenylyltransfer (forward), pyrophosphorolysis (reverse), and nucleotidyltransfer reaction utilizing CTP for the mutants are presented. The results support a model in which binding of both poly(A) and the correct nucleotide, MgATP, induces a conformational change, resulting in formation of a stable, closed enzyme state. Thermodynamic considerations of the data are discussed as they pertain to domain closure, substrate specificity, and catalytic strategies utilized by PAP.

Polyadenylate polymerase (PAP) catalyzes the synthesis of poly(A) tails on the 3'-end of pre-mRNA. PAP is composed of three domains: an N-terminal nucleotide-binding domain (homologous to the palm domain of DNA and RNA polymerases), a middle domain (containing other conserved, catalytically important residues), and a unique C-terminal domain (involved in protein-protein interactions required for 3'-end formation). Previous X-ray crystallographic studies have shown that the domains are arranged in a V-shape such that they form a central cleft with the active site located at the base of the cleft at the interface between the N-terminal and middle domains. In the previous studies, the nucleotides were bound directly to the N-terminal domain and exhibited a conspicuous lack of adenine-specific interactions that would constitute nucleotide recognition. Furthermore, it was postulated that base-specific contacts with residues in the middle domain could occur either as a result of a change in the conformation of the nucleotide or domain movement. To address these issues and to better characterize the structural basis of substrate recognition and catalysis, we report two new crystal structures of yeast PAP. A comparison of these structures reveals that the N-terminal and C-terminal domains of PAP move independently as rigid bodies along two well defined axes of rotation. Modeling of the nucleotide into the most closed state allows us to deduce specific nucleotide interactions involving residues in the middle domain (K215, Y224 and N226) that are proposed to be involved in substrate binding and specificity. To further investigate the nature of PAP domain flexibility, 2-aminopurine labeled molecular probes were employed in steady state fluorescence and acrylamide quenching experiments. The results suggest that the closed domain conformation is stabilized upon recognition of the correct subtrate, MgATP, in an enzyme-substrate ternary complex. The implications of these results on the enzyme mechanism of PAP and the possible role for domain motion in an induced fit mechanism are discussed.

CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.

In Escherichia coli, RNA polyadenylation is catalyzed mainly by poly(A) polymerase I (PAP I). Here we demonstrate that a PAP I variant with a C-terminal His tag (PAP I-His) can be phosphorylated both in vivo and in an artificial in vitro system. The in vivo phosphorylation of PAP I-His impairs activity of this enzyme. Previous studies, performed by others, indicated that phosphorylation of His-tagged proteins usually reflects such a modification of their native counterparts in bacterial cells. Therefore, our results suggest that phosphorylation and dephosphorylation of PAP I may be important regulatory processes in the control of activity of this enzyme.

Polyadenylation of mRNAs in poxviruses, crucial for virion maturation, is carried out by a poly(A) polymerase heterodimer composed of a catalytic component, VP55, and a processivity factor, VP39. The ATP-gamma-S bound and unbound crystal structures of the vaccinia polymerase reveal an unusual architecture for VP55 that comprises of N-terminal, central or catalytic, and C-terminal domains with different topologies and that differs from many polymerases, including the eukaryotic poly(A) polymerases. Residues in the active site of VP55, located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition. VP55's concave surface docks the globular VP39. A model for RNA primer binding that involves all three VP55 domains and VP39 is proposed. The model supports biochemical evidence that VP39 functions as a processivity factor by partially enclosing the RNA primer at the heterodimer interface.

microRNAs (miRNAs) and small interfering RNAs (siRNAs) in plants bear a methyl group on the ribose of the 3' terminal nucleotide. We showed previously that the methylation of miRNAs and siRNAs requires the protein HEN1 in vivo and that purified HEN1 protein methylates miRNA/miRNA* duplexes in vitro. In this study, we show that HEN1 methylates both miRNA/miRNA* and siRNA/siRNA* duplexes in vitro with a preference for 21-24 nt RNA duplexes with 2 nt overhangs. We also demonstrate that HEN1 deposits the methyl group on to the 2' OH of the 3' terminal nucleotide. Among various modifications that can occur on the ribose of the terminal nucleotide, such as 2'-deoxy, 3'-deoxy, 2'-O-methyl and 3'-O-methyl, only 2'-O-methyl on a small RNA inhibits the activity of yeast poly(A) polymerase (PAP). These findings indicate that HEN1 specifically methylates miRNAs and siRNAs and implicate the importance of the 2'-O-methyl group in the biology of RNA silencing.

The physiological significance and metabolism of oligoadenylated and polyadenylated human mitochondrial mRNAs are not known to date. After study of eight mitochondrial transcripts (ND1, ND2, ND3, ND5, CO1, CO2, ATP6/8 and Cyt. b) we found a direct correlation between the half-lives of mitochondrial mRNAs and their steady-state levels. Investigation of the mt-mRNA decay after thiamphenicol treatment indicated that three transcripts (ND2, ND3 and Cyt. b) are significantly stabilized after inhibition of mitochondrial translation. Careful analysis one of them, ND3, showed that inaccurate processing of the H-strand RNA precursor may occasionally occur between the ND3 and tRNA(Arg) locus leading to synthesis of ND3 mRNAs lacking the STOP codon. However, analysis of the oligo(A) fraction observed in case of the ND3 indicates that partially polyadenylated mRNAs are linked rather to the transcription process than to the translation-dependent deadenylation. Analysis of ND3 mRNA turnover in cells with siRNA-mediated knock-down of the mitochondrial poly(A) polymerase shows that strongly decreased polyadenylation does not markedly affect the decay of this transcript. We present a model where oligoadenylated mitochondrial transcripts are precursors of molecules containing full length poly(A) tails.

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively.

Cytoplasmic polyadenylation is important in the control of mRNA stability and translation, and for early animal development and synaptic plasticity. Here, we focus on vertebrate poly(A) polymerases that are members of the recently described GLD2 family. We identify and characterize two closely related GLD2 proteins in Xenopus oocytes, and show that they possess PAP activity in vivo and in vitro and that they bind known polyadenylation factors and mRNAs known to receive poly(A) during development. We propose that at least two distinct polyadenylation complexes exist in Xenopus oocytes, one of which contains GLD2; the other, maskin and Pumilio. GLD2 protein interacts with the polyadenylation factor, CPEB, in a conserved manner. mRNAs that encode GLD2 in mammals are expressed in many tissues. In the brain, mouse, and human GLD2 mRNAs are abundant in anatomical regions necessary for long-term cognitive and emotional learning. In the hippocampus, mouse GLD2 mRNA colocalizes with CPEB1 and Pumilio1 mRNAs, both of which are likely involved in synaptic plasticity. We suggest that mammalian GLD2 poly(A) polymerases are important in synaptic translation, and in polyadenylation throughout the soma.

Polyadenylate polymerase (PAP) catalyzes the synthesis of 3'-polyadenylate tails onto mRNA. A comprehensive steady-state kinetic analysis of PAP was conducted which included initial velocity studies of the forward and reverse reactions, inhibition studies, and the use of alternative substrates. The reaction (A(n) + ATP <--> A(n+1) + PP(i)) is adequately described by a rapid equilibrium random mechanism. Several thermodynamic parameters for the reaction were determined or calculated, including the overall equilibrium constant (K(eq) = 84) and the apparent equilibrium constant of the internal step (K(int) = 4) which involves the rate-determining interconversion of central complexes. A large (100-fold) difference in Vmax accounts for nucleotide specificity (ATP vs CTP), despite an only 3-fold difference in Km. Comparison of the sulfur elemental effect on Vmax for ATP and CTP suggests that the chemical step is rate-determining for both reactions. Comparison of the sulfur elemental effect on Vmax/Km revealed differences in the mechanism by which either nucleotide is incorporated. Consistent with these data, an induced fit mechanism for nucleotide specificity is proposed whereby PAP couples a uniform binding mechanism, which selects for ATP, with a ground-state destabilization mechanism, which serves to accelerate the velocity for the correct substrate.

Over the past 15 years considerable progress has been made in understanding the molecular mechanisms of mRNA decay in both prokaryotes and eukaryotes. Interestingly, unlike other important biological reactions such as DNA replication and repair, many features of mRNA decay differ between prokaryotes or eukaryotes. Even when a particular enzyme like poly(A) polymerase has been conserved, polyadenylation of mRNAs in prokaryotes appears to serve a very different function than it does in eukaryotes. Furthermore, while mRNA degrading multiprotein complexes have been identified in both prokaryotes and eukaryotes, their composition and biochemical mechanisms are significantly different. Accordingly, this review seeks to provide a concise comparison of our current knowledge regarding the pathways of mRNA decay in two model organisms, the prokaryote Escherichia coli and the eukaryote Saccharomyces cerevisiae.

The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. 32P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)