Concurrent Session 2: RNA Structure and Regulation 14:00 - 16:00 Wednesday, 27th May, 2020 Talk Session Chair Ming Lei

74 The first crystal structure of the human and the chimp CPEB3 ribozyme: a snapshot of the catalytic core reveals an unexpected fold of the HDV-like ribozymes

Anna Ilaria Przytula-Mally1, Sylvain Engilberge2, Silke Johannsen1, Eva Freisinger1, Vincent Olieric2, Roland K.O. Sigel1 1University of Zurich, Zurich, Switzerland. 2Paul Scherrer Institut, Villigen, Switzerland

Abstract

Cytoplasmic polyadenylation element binding (CPEB) proteins are involved in many vital processes including cell division, synaptic plasticity, learning and memory. A highly conserved, short mammalian ribozyme was found within an intron of the CPEB3 protein. This CPEB3 ribozyme is the third ribozyme confirmed in humans, besides the ribosome and the spliceosome, and belongs to the broad family of the hepatitis delta virus (HDV)- like ribozyme. All members of the HDV-like family have several features in common: (i) a nested double- pseudoknot structure, (ii) 5’-end self-cleavage activity, and (iii) a conserved cytosine in the catalytic core. However, their self-cleavage rates are highly divergent and it is assumed that the rates directly depend on the overall stabilization of the catalytic core. Since the first crystal structure of the cleaved HDV ribozyme in 1998 followed by structures of uncleaved, mutant-inhibited and ion-complexed forms, no three-dimensional structure of any other ribozyme of this family was published. Here we present the first crystal structures of the cleaved human and chimp CPEB3 ribozyme in complex with the U1A spliceosomal protein. Theses sequences differ by only a single nucleotide but show a difference in cleavage rate by around ~1 order of magnitude. Our crystallographic data disclosed two highly similar structures with the proposed sophisticated double-pseudoknot fold. However, only the four helical regions P1, P2, P3 and P4 are present, whereas P1.1 consisting of only one Watson-Crick base pair and a U-U wobble is not formed. Instead, we observed an alternative interaction in which two copies of RNA base pair within the L3 region with each other. The dimer formation was also consistent with SEC-SAXS experiments and suggesting the highly dynamic behavior of the catalytic core. This is well in accordance with our NMR data of the cleaved wild-type human and chimp ribozyme in which P1.1 formation occurs only in the presence of at least 5 mM Mg2+ ions.

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RNA Structure, Folding & Modeling 259 Competition between ligand binding and transcription factor NusA modulates riboswitch-mediated regulation of transcription

Adrien Chauvier, Pujan Ajmera, Nils Walter University of Michigan, Ann Arbor, Michigan, USA

Abstract

Transcriptional pausing by RNA polymerase (RNAP) is a main feature in all organisms and is a prerequisite for transcription termination. In bacteria, the essential transcription factor NusA regulates both pausing and termination, thus acting as a key player for genetic regulation. Riboswitches are genetic elements frequently found in the 5’ untranslated regions of mRNAs, where they respond to cellular metabolites to regulate gene expression either at the level of transcription or translation. The fluoride riboswitch from Bacillus cereus regulates gene expression by a transcriptional mechanism in which the binding of its ligand, the negative ion fluoride, causes a conformational change in the RNA structure that prevents the formation of a terminator hairpin. Previous studies have deciphered the structure of the riboswitch alone and in its transcriptional elongation complex (EC), but little is known about the impact of the transcriptional machinery on riboswitch folding and vice versa. Using in vitro transcription assays, we found that fluoride and NusA each modulate the termination efficiency in dependence of the riboswitch while competing with each other. Interestingly, we found that a downstream transcriptional pause of RNAP is modulated by the presence of fluoride, rendering the EC insensitive to NusA in the ligand-bound conformation. Developing single molecule fluorescence assays of the EC using fluorescently labeled transcriptional factor, we found that NusA transiently binds to the EC, fine-tuning the transcription rate similar to an anti-lock braking system. Moreover, analysis of the binding kinetics reveals that the presence of fluoride prevents the recruitment of NusA to the EC upon formation of an RNA long-range interaction, defining a novel class of RNAP pausing behavior. Finally, using single molecule transcription assays in real-time we found that fluoride and NusA compete for binding the EC, thus finely modulating the transcription rate and final output of transcription. This work provides unprecedented insights into the powerful mechanisms by which RNA structure can modulate the function of RNA polymerase and vice versa.

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Presenting author email [email protected]

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Ribozymes & Riboswitches 127 Structural insights into multivalent and multimodal tRNA-mRNA interactions from the T-box riboswitches

Krishna Suddala1, Shuang Li1, Zhaoming Su2, Jean Lehmann3, Vasiliki Stamatopoulou4, Nikoleta Giarimoglou4, Frances Henderson1, Lixin Fan5, Yun-Xing Wang5, Constantinos Stathopoulos4, Wah Chiu2, Jinwei Zhang1 1NIDDK, NIH, Bethesda, MD, USA. 2Stanford University, Stanford, CA, USA. 3Université Paris-Sud 11, Paris, France. 4University of Patras, Patras, Greece. 5NCI, NIH, Frederick, MD, USA

Abstract

Higher-order, multivalent RNA-RNA interactions play key roles in biological processes from long noncoding RNA and viral genome organization to RNA condensation and granule formation. Yet, little is known about the nature of these interactions due to lack of structurally tractable systems. The T-box riboswitch-tRNA interactions provide a unique vantage point to understand how discrete RNA domains recognize each other with multivalent affinity and exquisite specificity.

The T-box riboswitches are widespread bacterial noncoding RNAs that directly bind specific tRNAs, sense their aminoacylation states, and switch conformations to control amino-acid metabolism. Despite their prevalence in numerous human pathogens and importance to nutritional homeostasis, it remains unclear how the T-box mRNA recognizes its tRNA ligand, senses aminoacylation, and controls the transcription or translation of downstream genes.

Here, we present two co-crystal structures (2.7 Å and 2.9 Å) and a 4.9 Å cryo-EM structure of T-box-tRNA complexes. These three structures and functional analyses define the core mechanisms of tRNA recognition, amino acid sensing, and conformational switching by the T-boxes. Specifically, T-boxes form a U-shaped molecular vise that clamps the tRNA and captures its 3′-end using an elaborate “discriminator” structure. Numerous tertiary contacts, especially those emanating from strings of single-stranded purines in Stems II and III regions, act in concert to reinforce codon-anticodon and stacking interactions. These coordinated contacts enable the T-box discriminator to sense tRNA aminoacylation using a bipartite steric sieve, and to couple this readout to a conformational switch mediated by tRNA-T-box stacking.

The T-box paradigm reveals that noncoding RNAs can interact through multiple coordinated contacts, concatenation of stacked helices, and mutually induced fit. These insights inform investigations into other complex RNA structures and assemblies, development of T-box-targeted antimicrobials, and design and engineering of novel RNA sensors, regulators, and interfaces.

1. Van Treeck & Parker, Cell 2018. 2. Grundy & Henkin, Cell 1993. 3. Zhang & Ferré-D'Amaré, Nature 2013. 4. Grigg & Ke, Structure 2013. 5. Zhang & Ferré-D'Amaré, Mol Cell 2014. 6. Li, Su et al., NSMB 2019. 7. Suddala & Zhang, NSMB 2019. 8. Battaglia et al., NSMB 2019. 9. Weaver & Serganov, NSMB 2019.

Supported in part by the intramural research program of NIDDK, NIH.

Presenting author email [email protected] Topic category

Ribozymes & Riboswitches 233 Single-molecule and ensemble analysis of protein-induced frameshifting

Neva Caliskan1,2, Lukas Pekarek1, Anuja Kibe1 1Helmholtz Institute for RNA-based Infection Research, Würzburg, Bayern, Germany. 2Faculty of Medicine, University of Würzburg, Würzburg, Bayern, Germany

Abstract

The coding region of many genes contains sequence elements that constitute roadblocks during mRNA translation. These roadblocks present problems, but also opportunities for the cell to regulate gene expression and increase the genetic repertoire by so-called programmed ribosome frameshifting (PRF). Efficient frameshifting is mediated by stimulatory-RNA structures and slippery sequences embedded in the mRNA. There are also numerous cellular factors and small RNAs involved in the regulation frameshifting, acting as riboswitches. However, how these interactions work at the molecular level remains elusive. Since, interactions between RNA and interacting partners are mostly short-lived, or weak; it is difficult to decipher the underlying physical principles and precise control mechanisms. In this study, we chose the encephalomyocarditis virus (EMCV) 2A protein as a model to study the molecular details of protein-induced frameshifting using ensemble and single-molecule analysis tools. In order to gain an in-depth insight into the dynamics of cardiovirus frameshifting riboswitch, we employed state of the art optical tweezers assisted with high-resolution imaging and microfluidics. We probed how interactions of 2A protein alter the cardiovirus RNA structure in single- nucleotide resolution, using the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. We further characterized binding kinetics of mutant RNA variants using the highly sensitive microscale thermophoresis tool and with that looked into the interplay of the 2A protein with its frameshifting-RNA target. We found that immediate upstream bases of the predicted hairpin are crucial for high-affinity interactions with the protein. Our results demonstrate that the binding of the 2A-protein stabilizes the frameshift RNA structure and several combinations of the hairpins and pseudoknots can effectively stimulate PRF, rather than a static RNA. We anticipate our study to be a starting point for more sophisticated analyses of the kinetics of frameshifting riboswitches and their interplay by RNA binding factors.

Presenting author email [email protected]

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Ribozymes & Riboswitches 179 Strand threading in subgenomic flavivirus RNAs generates exoribonuclease-resistance mechanically

Meng Zhao, Michael Woodside University of Alberta, Edmonton, Alberta, Canada

Abstract

Recently, a new class of non-coding RNA from flaviviruses was discovered that resists digestion of the invading viral RNA by host exoribonucleases. These exoribunuclease-resistant RNAs (xrRNAs) contain knotted structures (which we term “ring-knots”) featuring a single strand threaded through a ring closed by secondary and tertiary interactions. The ring-knots in xrRNAs are proposed to act as roadblocks halting digestion via their mechanical resistance to unfolding, thereby generating sub-genomic RNAs that interact with various host proteins to increase viral pathogenicity. The hypothesis that resistance to mechanical unfolding by RNases is the key factor conferring exoribonuclease resistance (XR), however, has not been tested. We used optical tweezers to apply force to an xrRNA from Zika virus, mimicking the forces applied during the helicase activity of RNases, to measure its mechanical stability and determine the sequence of steps leading to ring-knot formation. We found that this xrRNA was the most mechanically stable compact RNA structure observed to date, unfolding at forces well above other tertiary structures and in the same range as the duplex overstretching transition. The ring-knot formed by a hierarchical pathway, with the 5′-end threading into the cleft in a Mg2+-coordinated three-helix junction before closure of the ring by pseudoknot interactions. Both the threading and pseudoknot were required to generate extreme force resistance. XR levels correlated directly with ring-knot formation when comparing the wild-type xrRNA to a mutant with lower XR, confirming that the knot acts as a mechanical roadblock for exoribonucleases. This work reveals how folding and function are related in an important new class of RNA fold.

Presenting author email [email protected]

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RNA Structure, Folding & Modeling 312 RNA structure-mediated RNP assembly in Staufen granules

Flora Lee1,2, Anob Chakrabarti1,3, Reem Abouward1, Sandra Fernández-Moya4, Janina Ehses4, Michael Kiebler4, Nicholas Luscombe1,3, Jernej Ule1,2 1The Francis Crick Institute, London, United Kingdom. 2UCL Queen Square Institute of Neurology, London, United Kingdom. 3UCL Genetics Institute, London, United Kingdom. 4BioMedical Center, Division of Cell Biology, Ludwig Maximilians University, Munich, Germany

Abstract

The structure of RNA molecules is crucial in mediating post-transcriptional regulation through an interplay with RNA binding proteins (RBPs). In particular, Staufen proteins contain multiple domains that bind to double- stranded RNA regions, formed predominantly through base-pairing complementarity. However, how RNA- structure links to the various regulatory functions of Staufen proteins remains poorly understood. To investigate this question in a physiological context, we determined an atlas of RNA secondary structures that are bound by STAU1 & STAU2 in neurons using hiCLIP (RNA hybrid individual nucleotide resolution crosslinking and immunoprecipitation). We identified hundreds of thousands of stable RNA secondary structures which are enriched in synaptically localised transcripts. Notably, we observed patterns of dense RNA-RNA contacts that reflect the formation of “Staufen-Associated Domains” (STADs) on long 3’UTRs. Comparative analysis revealed variations in Staufen-bound RNA structures across neuronal differentiation of mouse and human stem cells, and in vivo across multiple ages of rat cortex, suggesting that the ensemble of 3’UTR conformations is regulated throughout development. Based on integration of our hiCLIP data with various iCLIP datasets, we found a positional relationship between conserved STADs and the binding of ELAVL1-4 and other sequence- specific RBPs. Our evidence points towards a working model where RNA-structure dependent compaction of long 3’UTRs organises multi-protein RNP assembly in Staufen-containing granules to mediate neuronal RNA localisation.

Presenting author email [email protected]

Topic category

RNA Structure, Folding & Modeling