A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation.

Polynucleotide phosphorylase (PNPase) is an ancient exoribonuclease conserved in the course of evolution and is found in species as diverse as bacteria and humans. Paradoxically, Escherichia coli PNPase can act not only as an RNA degrading enzyme but also by an unknown mechanism as a chaperone for small regulatory RNAs (sRNAs), with pleiotropic consequences for gene regulation. We present structures of the ternary assembly formed by PNPase, the RNA chaperone Hfq, and sRNA and show that this complex boosts sRNA stability in vitro. Comparison of structures for PNPase in RNA carrier and degradation modes reveals how the RNA is rerouted away from the active site through interactions with Hfq and the KH and S1 domains. Together, these data explain how PNPase is repurposed to protect sRNAs from cellular ribonucleases such as RNase E and could aid RNA presentation to facilitate regulatory actions on target genes.

An RNA degradation machine sculpted by Ro autoantigen and noncoding RNA.

Many bacteria contain an ortholog of the Ro autoantigen, a ring-shaped protein that binds noncoding RNAs (ncRNAs) called Y RNAs. In the only studied bacterium, Deinococcus radiodurans, the Ro ortholog Rsr functions in heat-stress-induced ribosomal RNA (rRNA) maturation and starvation-induced rRNA decay. However, the mechanism by which this conserved protein and its associated ncRNAs act has been obscure. We report that Rsr and the exoribonuclease polynucleotide phosphorylase (PNPase) form an RNA degradation machine that is scaffolded by Y RNA. Single-particle electron microscopy, followed by docking of atomic models into the reconstruction, suggests that Rsr channels single-stranded RNA into the PNPase cavity. Biochemical assays reveal that Rsr and Y RNA adapt PNPase for effective degradation of structured RNAs. A Ro ortholog and ncRNA also associate with PNPase in Salmonella Typhimurium. Our studies identify another ribonucleoprotein machine and demonstrate that ncRNA, by tethering a protein cofactor, can alter the substrate specificity of an enzyme.

BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here, we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA sequencing (RNA-seq). We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved non-coding RNAs (ncRNAs) are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the buildup of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

Compartmentalization of RNA Degradosomes in Bacteria Controls Accessibility to Substrates and Ensures Concerted Degradation of mRNA to Nucleotides.

RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication.Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation.

RNA tailing machinery drives amyloidogenic phase transition.

The RNA tailing machinery adds nucleotides to the 3'-end of RNA molecules that are implicated in various biochemical functions, including protein synthesis and RNA stability. Here, we report a role for the RNA tailing machinery as enzymatic modifiers of intracellular amyloidogenesis. A targeted RNA interference screen identified Terminal Nucleotidyl-transferase 4b (TENT4b/Papd5) as an essential participant in the amyloidogenic phase transition of nucleoli into solid-like Amyloid bodies. Full-length-and-mRNA sequencing uncovered starRNA, a class of unusually long untemplated RNA molecules synthesized by TENT4b. StarRNA consists of short rRNA fragments linked to long, linear mixed tails that operate as polyanionic stimulators of amyloidogenesis in cells and in vitro. Ribosomal intergenic spacer noncoding RNA (rIGSRNA) recruit TENT4b in intranucleolar foci to coordinate starRNA synthesis driving their amyloidogenic phase transition. The exoribonuclease RNA Exosome degrades starRNA and functions as a general suppressor of cellular amyloidogenesis. We propose that amyloidogenic phase transition is under tight enzymatic control by the RNA tailing and exosome axis.

RNA degradation in human mitochondria: the journey is not finished.

Mitochondria are vital organelles present in almost all eukaryotic cells. Although most of the mitochondrial proteins are nuclear-encoded, mitochondria contain their own genome, whose proper expression is necessary for mitochondrial function. Transcription of the human mitochondrial genome results in the synthesis of long polycistronic transcripts that are subsequently processed by endonucleases to release individual RNA molecules, including precursors of sense protein-encoding mRNA (mt-mRNA) and a vast amount of antisense noncoding RNAs. Because of mitochondrial DNA (mtDNA) organization, the regulation of individual gene expression at the transcriptional level is limited. Although transcription of most protein-coding mitochondrial genes occurs with the same frequency, steady-state levels of mature transcripts are different. Therefore, post-transcriptional processes are important for regulating mt-mRNA levels. The mitochondrial degradosome is a complex composed of the RNA helicase SUV3 (also known as SUPV3L1) and polynucleotide phosphorylase (PNPase, PNPT1). It is the best-characterized RNA-degrading machinery in human mitochondria, which is primarily responsible for the decay of mitochondrial antisense RNA. The mechanism of mitochondrial sense RNA decay is less understood. This review aims to provide a general picture of mitochondrial genome expression, with a particular focus on mitochondrial RNA (mtRNA) degradation.

α-Proteobacterial RNA Degradosomes Assemble Liquid-Liquid Phase-Separated RNP Bodies.

Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS condensates that we term bacterial RNP-bodies (BR-bodies), similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related α-proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS condensates, providing an effective strategy for subcellular organization in cells lacking membrane-bound compartments.

Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms.

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

A comprehensive study of the interactions in the B. subtilis degradosome with special emphasis on the role of the small proteins SR1P and SR7P.

Here, we employ coelution experiments and far-western blotting to identify stable interactions between the main components of the B. subtilis degradosome and the small proteins SR1P and SR7P. Our data indicate that B. subtilis has a degradosome comprising at least RNases Y and PnpA, enolase, phosphofructokinase, glycerol-3-phosphate dehydrogenase GapA, and helicase CshA that can be co-purified without cross-linking. All interactions were corroborated by far-western blotting with proteins purified from E. coli. Previously, we discovered that stress-induced SR7P binds enolase to enhance its interaction with and activity of enolase-bound RNase Y (RnY), while SR1P transcribed under gluconeogenic conditions interacts with GapA to stimulate its interaction with and the activity of RnjA (RnjA). We show that SR1P can directly bind RnjA, RnY, and PnpA independently of GapA, whereas SR7P only interacts with enolase. Northern blotting suggests that the degradation of individual RNAs in B. subtilis under gluconeogenic or stress conditions depends on either RnjA or RnY alone or on RnjA-SR1P, RnY-SR1P, or RnY-Eno. In vitro degradation assays with RnY or RnjA substrates corroborate the in vivo role of SR1P. Currently, it is unknown which substrate property is decisive for the utilization of one of the complexes.

PNPASE regulates RNA import into mitochondria.

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3' --> 5' exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE-imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria.

Bacterial RNA Degradosomes: Molecular Machines under Tight Control.

Bacterial RNA degradosomes are multienzyme molecular machines that act as hubs for post-transcriptional regulation of gene expression. The ribonuclease activities of these complexes require tight regulation, as they are usually essential for cell survival while potentially destructive. Recent studies have unveiled a wide variety of regulatory mechanisms including autoregulation, post-translational modifications, and protein compartmentalization. Recently, the subcellular organization of bacterial RNA degradosomes was found to present similarities with eukaryotic messenger ribonucleoprotein (mRNP) granules, membraneless compartments that are also involved in mRNA and protein storage and/or mRNA degradation. In this review, we present the current knowledge on the composition and targets of RNA degradosomes, the most recent developments regarding the regulation of these machineries, and their similarities with the eukaryotic mRNP granules.

Mitochondrial sequencing identifies long noncoding RNA features that promote binding to PNPase.

Extranuclear localization of long noncoding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where polynucleotide phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and cross-linked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. lncRNA sequence and structure were evaluated through supervised [classification and regression trees (CART) and support vector machines (SVM)] machine learning algorithms. In HL-1 cells, quantitative PCR of PNPase CLIP knockout mutants (KH and S1) was performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. One hundred twelve (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most abundant. Most noncoding RNAs binding PNPase were lncRNAs, including Malat1. lncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. The binding of lncRNAs to PNPase was decreased through the knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs binding to PNPase and undergoing import into the mitochondrion.NEW & NOTEWORTHY Long noncoding RNAs (lncRNAs) are relatively novel RNAs with increasingly prominent roles in regulating genetic expression, mainly in the nucleus but more recently in regions such as the mitochondrion. This study explores how lncRNAs interact with polynucleotide phosphorylase (PNPase), a protein that regulates RNA import into the mitochondrion. Machine learning identified several RNA structural features that improved lncRNA binding to PNPase, which may be useful in targeting RNA therapeutics to the mitochondrion.

GDP polyribonucleotidyltransferase domain of vesicular stomatitis virus polymerase regulates leader-promoter escape and polyadenylation-coupled termination during stop-start transcription.

The unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain of the vesicular stomatitis virus (VSV) L protein possesses a dual-functional "priming-capping loop" that governs terminal de novo initiation for leader RNA synthesis and capping of monocistronic mRNAs during the unique stop-start transcription cycle. Here, we investigated the roles of basic amino acid residues on a helix structure directly connected to the priming-capping loop in viral RNA synthesis and identified single point mutations that cause previously unreported defective phenotypes at different steps of stop-start transcription. Mutations of residue R1183 (R1183A and R1183K) dramatically reduced the leader RNA synthesis activity by hampering early elongation, but not terminal de novo initiation or productive elongation, suggesting that the mutations negatively affect escape from the leader promoter. On the other hand, mutations of residue R1178 (R1178A and R1178K) decreased the efficiency of polyadenylation-coupled termination of mRNA synthesis at the gene junctions, but not termination of leader RNA synthesis at the leader-to-N-gene junction, resulting in the generation of larger amounts of aberrant polycistronic mRNAs. In contrast, both the R1183 and R1178 residues are not essential for cap-forming activities. The R1183K mutation was lethal to VSV, whereas the R1178K mutation attenuated VSV and triggered the production of the polycistronic mRNAs in infected cells. These observations suggest that the PRNTase domain plays multiple roles in conducting accurate stop-start transcription beyond its known role in pre-mRNA capping.

Cryo-EM structures of Mycobacterium tuberculosis polynucleotide phosphorylase suggest a potential mechanism for its RNA substrate degradation.

As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.

Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides.

Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3'-5' exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.

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.

Processing of the alaW alaX operon encoding the Ala2 tRNAs in Escherichia coli requires both RNase E and RNase P.

The alaW alaX operon encodes the Ala2 tRNAs, one of the two alanine tRNA isotypes in Escherichia coli. Our previous RNA-seq study showed that alaW alaX dicistronic RNA levels increased significantly in the absence of both RNase P and poly(A) polymerase I (PAP I), suggesting a role of polyadenylation in its stability. In this report, we show that RNase E initiates the processing of the primary alaW alaX precursor RNA by removing the Rho-independent transcription terminator, which appears to be the rate limiting step in the separation and maturation of the Ala2 pre-tRNAs by RNase P. Failure to separate the alaW and alaX pre-tRNAs by RNase P leads to poly(A)-mediated degradation of the dicistronic RNAs by polynucleotide phosphorylase (PNPase) and RNase R. Surprisingly, the thermosensitive RNase E encoded by the rne-1 allele is highly efficient in removing the terminator (>99%) at the nonpermissive temperature suggesting a significant caveat in experiments using this allele. Together, our data present a comprehensive picture of the Ala2 tRNA processing pathway and demonstrate that unprocessed RNase P substrates are degraded via a poly(A) mediated decay pathway.

The association between Hfq and RNase E in long-term nitrogen-starved Escherichia coli.

Under conditions of nutrient adversity, bacteria adjust metabolism to minimize cellular energy usage. This is often achieved by controlling the synthesis and degradation of RNA. In Escherichia coli, RNase E is the central enzyme involved in RNA degradation and serves as a scaffold for the assembly of the multiprotein complex known as the RNA degradosome. The activity of RNase E against specific mRNAs can also be regulated by the action of small RNAs (sRNA). In this case, the ubiquitous bacterial chaperone Hfq bound to sRNAs can interact with the RNA degradosome for the sRNA guided degradation of target RNAs. The RNA degradosome and Hfq have never been visualized together in live bacteria. We now show that in long-term nitrogen starved E. coli, both RNase E and Hfq co-localize in a single, large focus. This subcellular assembly, which we refer to as the H-body, forms by a liquid-liquid phase separation type mechanism and includes components of the RNA degradosome, namely, the helicase RhlB and the exoribonuclease polynucleotide phosphorylase. The results support the existence of a hitherto unreported subcellular compartmentalization of a process(s) associated with RNA management in stressed bacteria.

Multi-scale ensemble properties of the Escherichia coli RNA degradosome.

In organisms from all domains of life, multi-enzyme assemblies play central roles in defining transcript lifetimes and facilitating RNA-mediated regulation of gene expression. An assembly dedicated to such roles, known as the RNA degradosome, is found amongst bacteria from highly diverse lineages. About a fifth of the assembly mass of the degradosome of Escherichia coli and related species is predicted to be intrinsically disordered - a property that has been sustained for over a billion years of bacterial molecular history and stands in marked contrast to the high degree of sequence variation of that same region. Here, we characterize the conformational dynamics of the degradosome using a hybrid structural biology approach that combines solution scattering with ad hoc ensemble modelling, cryo-electron microscopy, and other biophysical methods. The E. coli degradosome can form punctate bodies in vivo that may facilitate its functional activities, and based on our results, we propose an electrostatic switch model to account for the propensity of the degradosome to undergo programmable puncta formation.

Impact of pseudouridylation, substrate fold, and degradosome organization on the endonuclease activity of RNase E.

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multicomponent RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5' nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognized uracil is isomerized to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric autoregulation. The catalytic power of the enzyme is boosted when it is assembled into the multienzyme RNA degradosome, most likely as a consequence of substrate capture and presentation. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.