Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3'-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3' CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Reproducible and efficient new method of RNA 3'-end labelling by CutA nucleotidyltransferase-mediated CC-tailing.

Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5' or 3' terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5'- or 3'-end of the molecule. An unsurpassed method for 5'-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-32P]ATP. In the case of 3'-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3'-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3'-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5'-32P]pCp ligation to the RNA 3'-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.

The AraC-Type Transcriptional Regulator GliR (PA3027) Activates Genes of Glycerolipid Metabolism in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes a large set of transcriptional regulators (TRs) that modulate and manage cellular metabolism to survive in variable environmental conditions including that of the human body. The AraC family regulators are an abundant group of TRs in bacteria, mostly acting as gene expression activators, controlling diverse cellular functions (e.g., carbon metabolism, stress response, and virulence). The PA3027 protein from P. aeruginosa has been classified in silico as a putative AraC-type TR. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3027 revealed a spectacular increase in the mRNA levels of PA3026-PA3024 (divergent to PA3027), PA3464, and PA3342 genes encoding proteins potentially involved in glycerolipid metabolism. Concomitantly, chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that at least 22 regions are bound by PA3027 in the PAO1161 genome. These encompass promoter regions of PA3026, PA3464, and PA3342, showing the major increase in expression in response to PA3027 excess. In Vitro DNA binding assay confirmed interactions of PA3027 with these regions. Furthermore, promoter-reporter assays in a heterologous host showed the PA3027-dependent activation of the promoter of the PA3026-PA3024 operon. Two motifs representing the preferred binding sites for PA3027, one localized upstream and one overlapping with the -35 promoter sequence, were identified in PA3026p and our data indicate that both motifs are required for full activation of this promoter by PA3027. Overall, the presented data show that PA3027 acts as a transcriptional regulator in P. aeruginosa, activating genes likely engaged in glycerolipid metabolism. The GliR name, from a glycerolipid metabolism regulator, is proposed for PA3027 of P. aeruginosa.

The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459-PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459-PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa.

Elimination of 01/A'-A0 pre-rRNA processing by-product in human cells involves cooperative action of two nuclear exosome-associated nucleases: RRP6 and DIS3.

Pre-rRNA processing generates mature 18S, 5.8S, and 28S/25S rRNAs through multistage removal of surrounding 5'-ETS/3'-ETS and intervening ITS1/ITS2 segments. Endonucleolytic activities release by-products, which need to be eliminated. Here, we investigated the interplay of exosome-associated 3'-5' exonucleases DIS3 and RRP6 in rRNA processing and by-product elimination in human cells. In agreement with previous reports, we observed accumulation of 5.8S and 18S precursors upon dysfunction of these enzymes. However, none of these phenotypes was so pronounced as previously overlooked accumulation of short RNA species derived from 5'-ETS (01/A'-A0), in cells with nonfunctional DIS3. We demonstrate that removal of 01/A'-A0 is independent of the XRN2 5'-3' exonucleolytic activity. Instead, it proceeds rapidly after A0 cleavage and occurs exclusively in the 3'-5' direction in several phases-following initiation by an unknown nuclease, the decay is executed by RRP6 with some contribution of DIS3, whereas the ultimate phase involves predominantly DIS3. Our data shed new light onto the role of human exosome in 5'-ETS removal. Furthermore, although 01/A'-A0 degradation involves the action of two nucleases associated with the exosome ring, similarly to 5.8S 3'-end maturation, it is likely that contrary to the latter process, RRP6 acts prior to or redundantly with DIS3.

Biochemical and structural bioinformatics studies of fungal CutA nucleotidyltransferases explain their unusual specificity toward CTP and increased tendency for cytidine incorporation at the 3'-terminal positions of synthesized tails.

Noncanonical RNA nucleotidyltransferases (NTases), including poly(A), poly(U) polymerases (PAPs/PUPs), and C/U-adding enzymes, modify 3'-ends of different transcripts affecting their functionality and stability. They contain PAP/OAS1 substrate-binding domain (SBD) with inserted NTase domain. Aspergillus nidulans CutA (AnCutA), synthesizes C/U-rich 3'-terminal extensions in vivo. Here, using high-throughput sequencing of the 3'-RACE products for tails generated by CutA proteins in vitro in the presence of all four NTPs, we show that even upon physiological ATP excess synthesized tails indeed contain an unprecedented number of cytidines interrupted by uridines and stretches of adenosines, and that the majority end with two cytidines. Strikingly, processivity assays documented that in the presence of CTP as a sole nucleotide, the enzyme terminates after adding two cytidines only. Comparison of our CutA 3D model to selected noncanonical NTases of known structures revealed substantial differences in the nucleotide recognition motif (NRM) within PAP/OAS1 SBD. We demonstrate that CutA specificity toward CTP can be partially changed to PAP or PUP by rational mutagenesis within NRM and, analogously, Cid1 PUP can be converted into a C/U-adding enzyme. Collectively, we suggest that a short cluster of amino acids within NRM is a determinant of NTases' substrate preference, which may allow us to predict their specificity.

SKI complex: A multifaceted cytoplasmic RNA exosome cofactor in mRNA metabolism with links to disease, developmental processes, and antiviral responses.

RNA stability and quality control are integral parts of gene expression regulation. A key factor shaping eukaryotic transcriptomes, mainly via 3'-5' exoribonucleolytic trimming or degradation of diverse transcripts in nuclear and cytoplasmic compartments, is the RNA exosome. Precise exosome targeting to various RNA molecules requires strict collaboration with specialized auxiliary factors, which facilitate interactions with its substrates. The predominant class of cytoplasmic RNA targeted by the exosome are protein-coding transcripts, which are carefully scrutinized for errors during translation. Normal, functional mRNAs are turned over following protein synthesis by the exosome or by Xrn1 5'-3'-exonuclease, acting in concert with Dcp1/2 decapping complex. In turn, aberrant transcripts are eliminated by dedicated surveillance pathways, triggered whenever ribosome translocation is impaired. Cytoplasmic 3'-5' mRNA decay and surveillance are dependent on the tight cooperation between the exosome and its evolutionary conserved co-factor-the SKI (superkiller) complex (SKIc). Here, we summarize recent findings from structural, biochemical, and functional studies of SKIc roles in controlling cytoplasmic RNA metabolism, including links to various cellular processes. Mechanism of SKIc action is illuminated by presentation of its spatial structure and details of its interactions with exosome and ribosome. Furthermore, contribution of SKIc and exosome to various mRNA decay pathways, usually converging on recycling of ribosomal subunits, is delineated. A crucial physiological role of SKIc is emphasized by describing association between its dysfunction and devastating human disease-a trichohepatoenteric syndrome (THES). Eventually, we discuss SKIc functions in the regulation of antiviral defense systems, cell signaling and developmental transitions, emerging from interdisciplinary investigations. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

An Adenosine Triphosphate- Dependent 5'-3' DNA Helicase From sk1-Like Lactococcus lactis F13 Phage.

Here, we describe functional characterization of an early gene (gp46) product of a virulent Lactococcus lactis sk1-like phage, vB_Llc_bIBBF13 (abbr. F13). The GP46 F13 protein carries a catalytically active RecA-like domain belonging to the P-loop NTPase superfamily. It also retains features characteristic for ATPases forming oligomers. In order to elucidate its detailed molecular function, we cloned and overexpressed the gp46 gene in Escherichia coli. Purified GP46 F13 protein binds to DNA and exhibits DNA unwinding activity on branched substrates in the presence of adenosine triphosphate (ATP). Size exclusion chromatography with multi-angle light scattering (SEC-MALS) experiments demonstrate that GP46 F13 forms oligomers, and further pull-down assays show that GP46 F13 interacts with host proteins involved in replication (i.e., DnaK, DnaJ, topoisomerase I, and single-strand binding protein). Taking together the localization of the gene and the obtained results, GP46 F13 is the first protein encoded in the early-expressed gene region with helicase activity that has been identified among lytic L. lactis phages up to date.

Immunoglobulin expression and the humoral immune response is regulated by the non-canonical poly(A) polymerase TENT5C.

TENT5C is a non-canonical cytoplasmic poly(A) polymerase highly expressed by activated B cells to suppress their proliferation. Here we measure the global distribution of poly(A) tail lengths in responsive B cells using a Nanopore direct RNA-sequencing approach, showing that TENT5C polyadenylates immunoglobulin mRNAs regulating their half-life and consequently steady-state levels. TENT5C is upregulated in differentiating plasma cells by innate signaling. Compared with wild-type, Tent5c-/- mice produce fewer antibodies and have diminished T-cell-independent immune response despite having more CD138high plasma cells as a consequence of accelerated differentiation. B cells from Tent5c-/- mice also have impaired capacity of the secretory pathway, with reduced ER volume and unfolded protein response. Importantly, these functions of TENT5C are dependent on its enzymatic activity as catalytic mutation knock-in mice display the same defect as Tent5c-/-. These findings define the role of the TENT5C enzyme in the humoral immune response.