Fantastic ribonucleoprotein complexes and how to study them. Architectural principles of regulatory assemblies in bacteria.

The fate of a bacterial RNA transcript is controlled by three key players: ribonucleases, RNA binding proteins (RBPs) and small regulatory RNAs (sRNAs). Together these form an information-rich regulatory network that exercises post-transcriptional control of gene expression. A key RBP in bacteria is Hfq, which is best studied as a facilitator for sRNA-mRNA pairing. In Pseudomonas aeruginosa, however, Hfq is known to modulate translation of more than 100 genes in cooperation with an effector protein, Crc, by sequestering the ribosome binding site of the corresponding mRNA molecules. In this thesis, Cryo-EM studies on target RNAs such as amiE and rbsB, reveal how Hfq presents multiple A-rich motifs in the mRNA 5ʹ-end to Crc and triggers assembly of higher order complexes. The structures suggest that secondary structure elements and the sequence of the target RNA determine the quaternary structure of the Hfq-Crc translation-repression assembly, introducing a degree of structural diversity that is accommodated by polymorphic interaction surfaces on both Hfq and Crc. In Escherichia coli, the homolog of the same RBP (Hfq) can modulate sRNA lifetime by repurposing the conserved ribonuclease PNPase (polynucleotide phosphorylase) into an RNA chaperone. Cryo-EM structures presented here show how the flexible KH-S1 portal of PNPase generally guides the 3ʹ-end of the sRNA sponge 3ʹETSleuZ to the catalytic core for digestion but reroutes the sRNA when bound to Hfq. In particular, an A-rich motif in the 5ʹ-end of 3ʹETSleuZ is presented to the KH and S1 domains by the Hfq distal side, while the 3ʹ-end is sequestered on the proximal side. Gel shift- and activity assays suggest that these repurposed ‘RNA carrier’ complexes protect sRNAs from other ribonucleases such as RNase E and RNase III, and potentially lend their KH-S1 portal as an interaction platform to promote sRNA/RNA target pairing. A fraction of PNPase in the cell associates with the endoribonuclease RNase E to form the E. coli ‘RNA degradosome’, a multi-enzyme ribonuclease assembly responsible for the bulk RNA turnover in the cell. Other partner enzymes include the DEAD-box helicase RhlB and Enolase. The core of the assembly, RNase E, has a long C-terminal scaffold domain that has a conserved natively unstructured character. The subcellular localization of the RNA degradosome, anchored to the cell membrane, provides a spatial component to post-transcriptional gene regulation. In this work, holistic experimental strategies to study this molecular machine are developed. Reproducible reconstitution of a truncated version of the RNA degradosome was achieved with a mix of detergents. Using small angle X-ray solution scattering (SAXS) combined with conformational ensemble computation indicates that the truncated degradosome is a highly flexible system with extended scaffold domains but can adopt more compact conformations when 9S rRNA is bound. Complementary cryo-EM studies of the catalytic core and subsequent 3D variability analyses reveal three modes of molecular motion of the RNase E protomers, adding to the overall flexibility of the RNA degradosome. Lastly, cryo-electron tomography studies of the truncated degradosome tethered to lipid vesicles provide the first visualization of the degradosome in its native environment and hint towards a degree of structural order in its components. Subsequent sub-tomogram averaging approaches to investigate putative membrane-bound RNA surveillance complexes set a foundation for future structural studies

Stabilization of Hfq-mediated translational repression by the co-repressor Crc in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery

Architectural principles for Hfq/Crc-mediated regulation of gene expression.

In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen Pseudomonas aeruginosa inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts as part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.BFL, XYP and TD are supported by the Welcome Trust (200873/Z/16/Z). TD is also supported by an AstraZeneca Studentship. UB and ES are supported by the Austrian Science Fund (FWF) (www.fwf.ac.at/en) [P28711-B22]

Radiation Hardness of dSiPM Sensors in a Proton Therapy Radiation Environment

In vivo verification of dose delivery in proton therapy by means of positron emission tomography (PET) or prompt gamma imaging is mostly based on fast scintillation detectors. The digital silicon photomultiplier (dSiPM) allows excellent scintillation detector timing properties and is thus being considered for such verification methods. We present here the results of the first investigation of radiation damage to dSiPM sensors in a proton therapy radiation environment. Radiation hardness experiments were performed at the AGOR cyclotron facility at the KVI-Center for Advanced Radiation Technology, University of Groningen. A 150-MeV proton beam was fully stopped in a water target. In the first experiment, bare dSiPM sensors were placed at 25 cm from the Bragg peak, perpendicular to the beam direction, a geometry typical for an in situ implementation of a PET or prompt gamma imaging device. In the second experiment, dSiPM-based PET detectors containing lutetium yttrium orthosilicate scintillator crystal arrays were placed at 2 and 4 m from the Bragg peak, perpendicular to the beam direction; resembling an in-room PET implementation. Furthermore, the experimental setup was simulated with a Geant4-based Monte Carlo code in order to determine the angular and energy distributions of the neutrons and to determine the 1-MeV equivalent neutron fluences delivered to the dSiPM sensors. A noticeable increase in dark count rate (DCR) after an irradiation with about 108 1-MeV equivalent neutrons/cm2 agrees with observations by others for analog SiPMs, indicating that the radiation damage occurs in the single photon avalanche diodes and not in the electronics integrated on the sensor chip. It was found that in the in situ location, the DCR becomes too large for successful operation after the equivalent of a few weeks of use in a proton therapy treatment room (about 5× 103 protons). For PET detectors in an in-room setup, detector performance was unchanged even after an irradiation equivalent to three years of use in a treatment room (3× 1015 protons)

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.ERC, BBSR

Multi‐scale ensemble properties of the Escherichia coli RNA degradosome

Abstract: 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

Dip-a-Dee-Doo-Dah: Bacteriophage-Mediated Rescoring of a Harmoniously Orchestrated RNA Metabolism.

status: publishe

RNA lifetime control, from stereochemistry to gene expression.

Through the activities of various multi-component assemblies, protein-coding transcripts can be chaperoned toward protein synthesis or nudged into a funnel of rapid destruction. The capacity of these machine-like assemblies to tune RNA lifetime underpins the harmony of gene expression in all cells. Some of the molecular machines that mediate transcript turnover also contribute to on-the-fly surveillance of aberrant mRNAs and non-coding RNAs. How these dynamic assemblies distinguish functional RNAs from those that must be degraded is an intriguing puzzle for understanding the regulation of gene expression and dysfunction associated with disease. Recent data illuminate what the machines look like, and how they find, recognise and operate on transcripts to sculpt the dynamic regulatory landscape. This review captures current structural and mechanistic insights into the key enzymes and their effector assemblies that contribute to the fate-determining decision points for RNA in post-transcriptional control of genetic information.Welcome Trus

A flexible framework for multi-particle refinement in cryo-electron tomography

International audienceCryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M . We illustrate the power of working within this framework by applying it to EMPIAR-10164 , a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164 . The guide is hosted on https://teamtomo.org/ , a collaborative online platform we establish for sharing knowledge about cryo-ET