Guide RNA acrobatics: the one-for-two shuffle

RNA modifications are crucial for the proper function of the RNAs. The sites of pseudouridines are often specified by dual hairpin guide RNAs, with one or both hairpins identifying a target uridine. In this issue of Genes & Development, Jády and colleagues (pp. 70–83) identify a novel mechanism by which a single guide RNA hairpin can specify two uridines adjacent to each other or separated by 1 nt; i.e., one for two or guide RNA acrobatics.

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

RGS4 RNA Secondary Structure Mediates Staufen2 RNP Assembly in Neurons

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3′-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.

Nonenzymatic loop-closing ligation generates RNA hairpins and is a template-free way to assemble functional RNAs

RNA hairpin loops are the predominant element of secondary structure in functional RNAs. The emergence of primordial functional RNAs, such as ribozymes that fold into complex structures that contain multiple hairpin loops, is generally thought to have been supported by template-directed ligation. However, template inhibition and RNA misfolding problems impede the emergence of function. Here we demonstrate that RNA hairpin loops can be synthesized directly from short RNA duplexes with single-stranded overhangs by nonenzymatic loop-closing ligation chemistry. We show that loop-closing ligation allows full-length functional ribozymes containing a hairpin loop to be assembled free of inhibitory template strands. This approach to the assembly of structurally complex RNAs suggests a plausible pathway for the emergence of functional RNAs before a full-length RNA copying process became available.

Structural basis for the activation of the DEAD-box RNA helicase DbpA by the nascent ribosome

The adenosine triphosphate (ATP)-dependent DEAD-box RNA helicase DbpA from Escherichia coli functions in ribosome biogenesis. DbpA is targeted to the nascent 50S subunit by an ancillary, carboxyl-terminal RNA recognition motif (RRM) that specifically binds to hairpin 92 (HP92) of the 23S ribosomal RNA (rRNA). The interaction between HP92 and the RRM is required for the helicase activity of the RecA-like core domains of DbpA. Here, we elucidate the structural basis by which DbpA activity is endorsed when the enzyme interacts with the maturing ribosome. We used nuclear magnetic resonance (NMR) spectroscopy to show that the RRM and the carboxyl-terminal RecA-like domain tightly interact. This orients HP92 such that this RNA hairpin can form electrostatic interactions with a positively charged patch in the N-terminal RecA-like domain. Consequently, the enzyme can stably adopt the catalytically important, closed conformation. The substrate binding mode in this complex reveals that a region 5′ to helix 90 in the maturing ribosome is specifically targeted by DbpA. Finally, our results indicate that the ribosome maturation defects induced by a dominant negative DbpA mutation are caused by a delayed dissociation of DbpA from the nascent ribosome. Taken together, our findings provide unique insights into the important regulatory mechanism that modulates the activity of DbpA.

Variations in the Abortive HIV-1 RNA Hairpin Do Not Impede Viral Sensing and Innate Immune Responses

The highly conserved trans-acting response element (TAR) present in the RNA genome of human immunodeficiency virus 1 (HIV-1) is a stably folded hairpin structure involved in viral replication. However, TAR is also sensed by viral sensors, leading to antiviral immunity. While high variation in the TAR RNA structure renders the virus replication-incompetent, effects on viral sensing remain unclear. Here, we investigated the role of TAR RNA structure and stability on viral sensing. TAR mutants with deletions in the TAR hairpin that enhanced thermodynamic stability increased antiviral responses. Strikingly, TAR mutants with lower stability due to destabilization of the TAR hairpin also increased antiviral responses without affecting pro-inflammatory responses. Moreover, mutations that affected the TAR RNA sequence also enhanced specific antiviral responses. Our data suggest that mutations in TAR of replication-incompetent viruses can still induce immune responses via viral sensors, hereby underscoring the robustness of HIV-1 RNA sensing mechanisms.

Unfolding of RNA via Translocation Through a Nanopore

RNA unfolding and refolding are important biological phenomena, which occur during the transfer of genetic information from DNA to RNA to proteins. During these processes, RNA is found in single stranded, secondary and tertiary structures, including secondary conformations like hairpins and pseudoknots. Understanding the diverse conformations of RNA and how these influence the dynamics of unfolding and refolding is crucial to gain insight to fundamental biological processes. In this work, we employ coarse-grained Langevin dynamics simulations of a simple model of different RNA hairpins passing through a geometric nanopore to find the influence of structural changes on the translocation dynamics. The threshold voltage of unfolding depends on the length of the hairpin attached to the tail. The lag time to unfold is longer for smaller applied voltages and for the architectures containing a longer hairpin attached to the tail. Chain translocation dynamics for different architectures are largely collapsed by the threshold. A distinct signature for the base unfolding time was observed for the bases around the unpaired bases in all the RNA hairpin models. These results can motivate future technologies or experiments that use translocation to predict secondary structures of polynucleotides.

Specific Interaction of DDX6 with an RNA Hairpin in the 3´-UTR of the Dengue Genome Mediates G1 Phase Arrest

The extent to which viral genomic RNAs interact with host factors and contribute to host response and disease pathogenesis is not well known. Here, we report that the human RNA helicase DDX6 specifically binds to the viral most conserved RNA hairpin in the A3 element in the dengue 3´-UTR, with nanomolar affinities. DDX6 CLIP confirmed the interaction in HuH-7 cells infected by dengue virus serotype 2. This interaction requires three conserved residues, Lys 307 , Lys 367 , and Arg 369 , as well as the unstructured extension in the C-terminal domain of DDX6. Interestingly, alanine substitution of these three basic residues resulted in RNA-independent ATPase activity, suggesting a mechanism by which RNA-binding and ATPase activities are coupled in DEAD-box helicases. Furthermore, we applied a cross-omics gene enrichment approach to suggest that DDX6 is functionally related to cell cycle regulation and viral pathogenicity. Indeed, infected cells exhibited cell cycle arrest in G1 phase and a decrease in the early S phase. Exogeneous expression of intact DDX6, but not A3-binding-deficient mutants, alleviated these effects by rescue of the DNA pre-initiation complex expression. Disruption of the DDX6-binding site was found in dengue and Zika live-attenuated vaccine strains. Our results suggested that dengue virus has evolved an RNA aptamer against DDX6 to alter host cell states, and defined DDX6 as a new regulator of G1/S transition. Importance Dengue virus (DENV) is transmitted by mosquitoes to humans, infecting 390 million individuals per year globally. About 20% of infected patients shows a spectrum of clinical manifestation, ranging from a mild flu-like syndrome, dengue fever (DF), to life-threatening severe dengue (SD) diseases including dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). There is currently no specific treatment for dengue diseases and the molecular mechanism underlying dengue pathogenesis remains poorly understood. In this study, we combined biochemical, bioinformatics, high-content analysis, and RNA sequencing approaches to characterize a highly conserved interface of the RNA genome of DENV with a human factor named DDX6 in infected cells. The significance of our research is in identifying the mechanism for a viral strategy to alter host cell fates, which conceivably allows us to generate a model for live-attenuated vaccine and the design of new therapeutic reagent for dengue diseases.

RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures

Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5′UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.

CCG•CGG interruptions in high penetrance SCA8 families increase RAN translation and protein toxicity

Spinocerebellar ataxia type 8 (SCA8), a dominantly inherited neurodegenerative disorder caused by a CTG•CAG expansion, is unusual because most individuals that carry the mutation do not develop ataxia. To understand the variable penetrance of SCA8 we studied the molecular differences between highly penetrant families and more common sporadic cases (82%) using a large cohort of SCA8 families (N=77). We show that repeat expansion mutations from individuals with two or more affected family members have CCG•CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG•CGG interruptions correlates with age at onset. At the molecular level, CCG•CGG interruptions increase RNA hairpin stability and steady state levels of SCA8 RAN polyAla and polySer proteins. Additionally, the CCG•CGG interruptions, which encode arginine interruptions in the polyGln frame increase the toxicity of the resulting proteins. In summary, CCG•CGG interruptions increase polyAla and polySer RAN protein levels, polyGln protein toxicity and disease penetrance and provide novel insight into the molecular differences between SCA8 families with high vs. low disease penetrance.