FAM46B is a prokaryotic-like cytoplasmic poly(A) polymerase essential in human embryonic stem cells.

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.

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines.

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.

Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity.

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.

Updates to the Integrated Protein-Protein Interaction Benchmarks: Docking Benchmark Version 5 and Affinity Benchmark Version 2.

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.

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.

Structural basis for the activation of the C. elegans noncanonical cytoplasmic poly(A)-polymerase GLD-2 by GLD-3.

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.

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, ß 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.

Domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid.

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.

Preliminary crystallographic analysis of a polyadenylate synthase from Megavirus.

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 Šresolution MAD data set was recorded from a single crystal on the ID29 beamline at the ESRF.

PARP1 represses PAP and inhibits polyadenylation during heat shock.

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.

NF45 dimerizes with NF90, Zfr and SPNR via a conserved domain that has a nucleotidyltransferase fold.

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.

Novel interactions at the essential N-terminus of poly(A) polymerase that could regulate poly(A) addition in Saccharomyces cerevisiae.

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.

Pre-mRNA 3'-end processing complex assembly and function.

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.

PAPD5, a noncanonical poly(A) polymerase with an unusual RNA-binding motif.

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.

RNA labeling, conjugation and ligation.

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.

Purification, crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine poly(A) polymerase and a chemically modified 15-mer oligo(A) RNA.

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 Šresolution.

Recent insights in pre-mRNA 3'-end processing signals and proteins in the protozoan parasite Entamoeba histolytica.

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.

Proteomic and functional analysis of the noncanonical poly(A) polymerase Cid14.

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.

Chain termination and inhibition of mammalian poly(A) polymerase by modified ATP analogues.

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.