CCR4-NOT is a large protein complex present both in cytoplasm and the nucleus of eukaryotic cells. Although it is involved in a variety of distinct processes related to expression of genetic information such as poly(A) tail shortening, transcription regulation, nuclear export and protein degradation, there is only fragmentary information available on some of its nine subunits. Here we show a comprehensive structural characterization of the native CCR4-NOT complex from Schizosaccharomyces pombe. Our cryo-EM 3D reconstruction of the complex, combined with techniques such as immunomicroscopy, RNA-nanogold labelling, docking of the available high-resolution structures and models of different subunits and domains, allow us to propose its full molecular architecture. We locate all functionally defined domains endowed with deadenylating and ubiquitinating activities, the nucleus-specific RNA-interacting subunit Mmi1, as well as surfaces responsible for protein-protein interactions. This information provides insight into cooperation of the different CCR4-NOT complex functions.

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.

Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.

Although interactions between microorganisms involved in biogas production are largely uncharted, it is commonly accepted that methanogenic Archaea are essential for the process. Methanogens thrive in various environments, but the most extensively studied communities come from biogas plants. In this study, we employed a metagenomic analysis of deeply sequenced methanogenic communities, which allowed for comparison of taxonomic and functional diversity as well as identification of microorganisms directly involved in various stages of methanogenesis pathways.

Biochemical, physiological and genomic comparisons of two Pseudomonas strains, assigned previously to the Pseudomonas jessenii subgroup, which are efficient SDS-degraders were carried out. A GO enrichment analysis showed that the genomes of SDS-degraders encode more genes connected with bacterial cell wall biosynthesis and alkanesulfonate monooxygenase activity than their closest relatives from the P. jessenii subgroup. A transcriptomic analysis of the most promising strain exposed to detergent suggests that although SDS can be later utilized as a carbon source, in early stages it influences cell envelope integrity, causing a global stress response followed by cell wall modification and induction of repair mechanisms. Genomes of the analyzed strains from P. jessenii group encode multiple putative sulfatases and their enzymatic activity was experimentally verified, which led to the identification of three novel enzymes exhibiting activity toward SDS. Two of the novel alkylsulfatases showed their highest activity at pH 8.0 and the temperature of 60°C or 70°C. One of the enzymes retained its activity even after 1 h of incubation at 60°C. Ions like K+ and Mg2+ enhanced enzymatic activity of both proteins, whereas Cu2+ or EDTA had inhibitory effects.

Prokaryotic Ligase D is a conserved DNA repair apparatus processing DNA double-strand breaks in stationary phase. An orthologous Ligase C (LigC) complex also co-exists in many bacterial species but its function is unknown. Here we show that the LigC complex interacts with core BER enzymes in vivo and demonstrate that together these factors constitute an excision repair apparatus capable of repairing damaged bases and abasic sites. The polymerase component, which contains a conserved C-terminal structural loop, preferentially binds to and fills-in short gapped DNA intermediates with RNA and LigC ligates the resulting nicks to complete repair. Components of the LigC complex, like LigD, are expressed upon entry into stationary phase and cells lacking either of these pathways exhibit increased sensitivity to oxidising genotoxins. Together, these findings establish that the LigC complex is directly involved in an excision repair pathway(s) that repairs DNA damage with ribonucleotides during stationary phase.

The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. β-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the β-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level.

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.

Plasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions.

The exosome complex is a major eukaryotic exoribonuclease that requires the SKI complex for its activity in the cytoplasm. In yeast, the Ski7 protein links both complexes, whereas a functional equivalent of the Ski7 has remained unknown in the human genome. Proteomic analysis revealed that a previously uncharacterized short splicing isoform of HBS1L (HBS1LV3) is the long-sought factor linking the exosome and SKI complexes in humans. In contrast, the canonical HBS1L variant, HBS1LV1, which acts as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. Interestingly, both HBS1LV1 and HBS1LV3 interact with the SKI complex and HBS1LV1 seems to antagonize SKI/exosome supercomplex formation. HBS1LV3 contains a unique C-terminal region of unknown structure, with a conserved RxxxFxxxL motif responsible for exosome binding and may interact with the exosome core subunit RRP43 in a way that resembles the association between Rrp6 RNase and Rrp43 in yeast. HBS1LV3 or the SKI complex helicase (SKI2W) depletion similarly affected the transcriptome, deregulating multiple genes. Furthermore, half-lives of representative upregulated mRNAs were increased, supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway, essential for transcriptome homeostasis in the cytoplasm.

Innate immunity is the first line of host defense against pathogens. Here, through global transcriptome and proteome analyses, we uncover that newly described cytoplasmic poly(A) polymerase TENT-5 (terminal nucleotidyltransferase 5) enhances the expression of secreted innate immunity effector proteins in Caenorhabditis elegans. Direct RNA sequencing revealed that multiple mRNAs with signal peptide-encoding sequences have shorter poly(A) tails in tent-5-deficient worms. Those mRNAs are translated at the endoplasmic reticulum where a fraction of TENT-5 is present, implying that they represent its direct substrates. Loss of tent-5 makes worms more susceptible to bacterial infection. Notably, the role of TENT-5 in innate immunity is evolutionarily conserved. Its orthologs, TENT5A and TENT5C, are expressed in macrophages and induced during their activation. Analysis of macrophages devoid of TENT5A/C revealed their role in the regulation of secreted proteins involved in defense response. In summary, our study reveals cytoplasmic polyadenylation to be a previously unknown component of the posttranscriptional regulation of innate immunity in animals.

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.

The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.

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.

Sphingopyxis inhabit diverse environmental niches, including marine, freshwater, oceans, soil and anthropogenic sites. The genus includes 20 phylogenetically distinct, valid species, but only a few with a sequenced genome. In this work, we analyzed the nearly complete genome of the newly described species, Sphingopyxislindanitolerans, and compared it to the other available Sphingopyxis genomes. The genome included 4.3 Mbp in total and consists of a circular chromosome, and two putative plasmids. Among the identified set of lin genes responsible for γ-hexachlorocyclohexane pesticide degradation, we discovered a gene coding for a new isoform of the LinA protein. The significant potential of this species in the remediation of contaminated soil is also correlated with the fact that its genome encodes a higher number of enzymes potentially involved in aromatic compound degradation than for most other Sphingopyxis strains. Additional analysis of 44 Sphingopyxis representatives provides insights into the pangenome of Sphingopyxis and revealed a core of 734 protein clusters and between four and 1667 unique proteins per genome.

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.

Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased ∼2.5-fold, covering ∼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of ∼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome.

The GC skew in vertebrate mitochondrial genomes results in synthesis of RNAs that are prone to form G-quadruplexes (G4s). Such RNAs, although mostly non-coding, are transcribed at high rates and are degraded by an unknown mechanism. Here we describe a dedicated mechanism of degradation of G4-containing RNAs, which is based on cooperation between mitochondrial degradosome and quasi-RNA recognition motif (qRRM) protein GRSF1. This cooperation prevents accumulation of G4-containing transcripts in human mitochondria. In vitro reconstitution experiments show that GRSF1 promotes G4 melting that facilitates degradosome-mediated decay. Among degradosome and GRSF1 regulated transcripts we identified one that undergoes post-transcriptional modification. We show that GRSF1 proteins form a distinct qRRM group found only in vertebrates. The appearance of GRSF1 coincided with changes in the mitochondrial genome, which allows the emergence of G4-containing RNAs. We propose that GRSF1 appearance is an evolutionary adaptation enabling control of G4 RNA.

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 β-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.

Processive exoribonucleases are executors of RNA decay. In humans, their physical but not functional interactions were thoughtfully investigated. Here we have screened cells deficient in DIS3, XRN2, EXOSC10, DIS3L, and DIS3L2 with a custom siRNA library and determined their genetic interactions (GIs) with diverse pathways of RNA metabolism. We uncovered a complex network of positive interactions that buffer alterations in RNA degradation and reveal reciprocal cooperation with genes involved in transcription, RNA export, and splicing. Further, we evaluated the functional distinctness of nuclear DIS3 and cytoplasmic DIS3L using a library of all known genes associated with RNA metabolism. Our analysis revealed that DIS3 mutation suppresses RNA splicing deficiency, while DIS3L GIs disclose the interplay of cytoplasmic RNA degradation with nuclear RNA processing. Finally, genome-wide DIS3 GI map uncovered relations with genes not directly involved in RNA metabolism, like microtubule organization or regulation of telomerase activity.