Global view on the metabolism of RNA poly(A) tails in yeast Saccharomyces cerevisiae.

The polyadenosine tail (poly[A]-tail) is a universal modification of eukaryotic messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). In budding yeast, Pap1-synthesized mRNA poly(A) tails enhance export and translation, whereas Trf4/5-mediated polyadenylation of ncRNAs facilitates degradation by the exosome. Using direct RNA sequencing, we decipher the extent of poly(A) tail dynamics in yeast defective in all relevant exonucleases, deadenylases, and poly(A) polymerases. Predominantly ncRNA poly(A) tails are 20-60 adenosines long. Poly(A) tails of newly transcribed mRNAs are 50 adenosine long on average, with an upper limit of 200. Exonucleolysis by Trf5-assisted nuclear exosome and cytoplasmic deadenylases trim the tails to 40 adenosines on average. Surprisingly, PAN2/3 and CCR4-NOT deadenylase complexes have a large pool of non-overlapping substrates mainly defined by expression level. Finally, we demonstrate that mRNA poly(A) tail length strongly responds to growth conditions, such as heat and nutrient deprivation.

Cytoplasmic polyadenylation by TENT5A is required for proper bone formation.

Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

Functions and mechanisms of RNA tailing by metazoan terminal nucleotidyltransferases.

Termini often determine the fate of RNA molecules. In recent years, 3' ends of almost all classes of RNA species have been shown to acquire nontemplated nucleotides that are added by terminal nucleotidyltransferases (TENTs). The best-described role of 3' tailing is the bulk polyadenylation of messenger RNAs in the cell nucleus that is catalyzed by canonical poly(A) polymerases (PAPs). However, many other enzymes that add adenosines, uridines, or even more complex combinations of nucleotides have recently been described. This review focuses on metazoan TENTs, which are either noncanonical PAPs or terminal uridylyltransferases with varying processivity. These enzymes regulate RNA stability and RNA functions and are crucial in early development, gamete production, and somatic tissues. TENTs regulate gene expression at the posttranscriptional level, participate in the maturation of many transcripts, and protect cells against viral invasion and the transposition of repetitive sequences. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.

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.

Controlling the mitochondrial antisense - role of the SUV3-PNPase complex and its co-factor GRSF1 in mitochondrial RNA surveillance.

Transcription of the human mitochondrial genome produces a vast amount of non-coding antisense RNAs. These RNA species can form G-quadraplexes (G4), which affect their decay. We found that the mitochondrial degradosome, a complex of RNA helicase SUPV3L1 (best known as SUV3) and the ribonuclease PNPT1 (also known as PNPase), together with G4-melting protein GRSF1, is a key player in restricting antisense mtRNAs.

Mitochondrial double-stranded RNA triggers antiviral signalling in humans.

Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

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.

The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma.

FAM46C is one of the most frequently mutated genes in multiple myeloma. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FAM46C encodes an active non-canonical poly(A) polymerase which enhances mRNA stability and gene expression. Reintroduction of active FAM46C into multiple myeloma cell lines, but not its catalytically-inactive mutant, leads to broad polyadenylation and stabilization of mRNAs strongly enriched with those encoding endoplasmic reticulum-targeted proteins and induces cell death. Moreover, silencing of FAM46C in multiple myeloma cells expressing WT protein enhance cell proliferation. Finally, using a FAM46C-FLAG knock-in mouse strain, we show that the FAM46C protein is strongly induced during activation of primary splenocytes and that B lymphocytes isolated from newly generated FAM46C KO mice proliferate faster than those isolated from their WT littermates. Concluding, our data clearly indicate that FAM46C works as an onco-suppressor, with the specificity for B-lymphocyte lineage from which multiple myeloma originates. FAM46C is one of the most frequently mutated genes in multiple myeloma (MM), but its molecular function remains unknown. Here the authors show that FAM46C is a poly(A) polymerase and that loss of function of FAM46C drives multiple myeloma through the destabilisation of ER response transcripts.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments.

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a ß-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins.

Perlman syndrome nuclease DIS3L2 controls cytoplasmic non-coding RNAs and provides surveillance pathway for maturing snRNAs.

The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here, we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5' UTR. Importantly, DIS3L2 contributes to surveillance of maturing snRNAs during their cytoplasmic processing. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis.

Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.

MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.

Multiple myeloma-associated hDIS3 mutations cause perturbations in cellular RNA metabolism and suggest hDIS3 PIN domain as a potential drug target.

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.

Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci.

RNA decay is usually mediated by protein complexes and can occur in specific foci such as P-bodies in the cytoplasm of eukaryotes. In human mitochondria nothing is known about the spatial organization of the RNA decay machinery, and the ribonuclease responsible for RNA degradation has not been identified. We demonstrate that silencing of human polynucleotide phosphorylase (PNPase) causes accumulation of RNA decay intermediates and increases the half-life of mitochondrial transcripts. A combination of fluorescence lifetime imaging microscopy with Förster resonance energy transfer and bimolecular fluorescence complementation (BiFC) experiments prove that PNPase and hSuv3 helicase (Suv3, hSuv3p and SUPV3L1) form the RNA-degrading complex in vivo in human mitochondria. This complex, referred to as the degradosome, is formed only in specific foci (named D-foci), which co-localize with mitochondrial RNA and nucleoids. Notably, interaction between PNPase and hSuv3 is essential for efficient mitochondrial RNA degradation. This provides indirect evidence that degradosome-dependent mitochondrial RNA decay takes place in foci.

Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor.

The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a ß-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.

Identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase.

We report here on the identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase. Immunocytochemical experiments confirm that the enzyme indeed localizes to mitochondrial compartment. Inhibition of expression of the enzyme by RNA interference results in significant shortening of the poly(A) tails of the mitochondrial ND3, COX III and ATP 6/8 transcripts, suggesting that the investigated protein represents a bona fide mitochondrial poly(A) polymerase. This is in agreement with our sequencing data which show that poly(A) tails of several mitochondrial messengers are composed almost exclusively of adenosine residues. Moreover, the data presented here indicate that all analyzed mitochondrial transcripts with profoundly shortened poly(A) tails are relatively stable, which in turn argues against the direct role of long poly(A) extensions in the stabilization of human mitochondrial messengers.

Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA.

We characterised the human hSuv3p protein belonging to the family of NTPases/helicases. In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos. Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub-fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix. We expressed and purified recombinant hSuv3p protein from a bacterial expression system. The purified enzyme was capable of hydrolysing ATP with a K(m) of 41.9 micro M and the activity was only modestly stimulated by polynucleotides. hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter. The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution.