Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e. a tRNA whose two out of three anticodon nucleotides base-pair with those of the stop codon. Unless programmed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons - PTCs) into coding sequences, where stopping is not desirable. Here, the tRNA’s ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs – tRNATrp and tRNACys, and tRNATyr and tRNAGln, respectively. The readthrough-inducing potential of tRNATrp and tRNATyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNACys in the HEK293T cell line. The tRNACys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNACys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNACys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be utilized in the PTC-associated RNA therapies.

DEAD-box RNA helicases are implicated in most aspects of RNA biology, where these enzymes unwind short RNA duplexes in an ATP-dependent manner. During the central step of the unwinding cycle, the two domains of the helicase core form a distinct closed conformation that destabilizes the RNA duplex, which ultimately leads to duplex melting. Despite the importance of this step for the unwinding process no high resolution structures of this state are available. Here, I employed nuclear magnetic resonance spectroscopy and X-ray crystallography to determine structures of the DEAD-box helicase DbpA in the closed conformation, complexed with substrate duplexes and single-stranded unwinding product. These structures reveal that DbpA initiates duplex unwinding by interacting with up to three base-paired nucleotides and a 5’ single-stranded RNA duplex overhang. These high-resolution snapshots, together with biochemical assays, rationalize the destabilization of the RNA duplex and are integrated into a conclusive model of the unwinding process.

RNA structure regulates bacterial gene expression by several distinct mechanisms via environmental and cellular stimuli, one of which is temperature. While some genome-wide studies have focused on heat shock treatments and the subsequent transcriptomic changes, soil bacteria are less likely to experience such rapid and extreme temperature changes. Though RNA thermometers (RNATs) have been found in 5' untranslated leader regions (5' UTRs) of heat shock and virulence-associated genes, this RNA-controlled mechanism could regulate other genes as well. Using Structure-seq2 and the chemical probe dimethyl sulfate (DMS) at four growth temperatures ranging from 23 to 42 °C, we captured a dynamic response of theBacillus subtilistranscriptome to temperature. Our transcriptome-wide results show RNA structural changes across all four temperatures and reveal non-monotonic reactivity trends with increasing temperature. Then, focusing on subregions likely to contain regulatory RNAs, we examined 5' UTRs to identify large, local reactivity changes. This approach led to the discovery of RNATs that control expression ofglpF(glycerol permease) andglpT(glycerol-3-phosphate permease); expression of both genes increased with increased temperature. Results with mutant RNATs indicate that both genes are controlled at the translational level. Increased import of glycerol at high temperatures could provide thermoprotection to proteins.

Systematic Evolution of Ligands through EXponential enrichment (SELEX) is widely used to identify functional nucleic acids, such as aptamers and ribozymes. Ideally, selective pressure drives enrichment of sequences that display the function of interest (binding, catalysis, etc). However, amplification biases from reverse transcription can overwhelm this enrichment and leave some functional sequences at a disadvantage, with cumulative effects across multiple rounds of selection. Libraries that are designed to include structural scaffolds can improve selection outcomes by sampling sequence space more strategically, but they are also susceptible to such amplification biases, particularly during reverse transcription. Therefore, we tested five reverse transcriptases (RTs) – ImProm-II, Marathon RT (MaRT), TGIRT-III, SuperScript IV (SSIV), and BST 3.0 DNA polymerase (BST) – to determine which enzymes introduced the least bias. We directly compared cDNA yield and processivity for these enzymes on RNA templates with varying degrees of structure under various reaction conditions. In these analyses, BST exhibited excellent processivity, generated large quantities of full length cDNA product, displayed little bias among templates with varying structure and sequence, and performed well on long, highly structured viral RNAs. Additionally, six RNA libraries containing either strong, moderate, or no incorporated structural elements were pooled and competed head-to-head in six rounds of an amplification-only selection without external selective pressure using either SSIV, ImProm-II, or BST during reverse transcription. High-throughput sequencing established that BST maintained the most neutral enrichment values, indicating low inter-library bias over the course of six rounds, relative to SSIV and ImProm-II, and it introduced minimal mutational bias.

Understanding the frequency and structural context of discrete noncovalent interactions between nucleotides is of pivotal significance in establishing the rules that govern RNA structure and dynamics. Although T-shaped contacts (i.e., perpendicular stacking contacts) between aromatic amino acids and nucleobases at the nucleic acid–protein interface have recently garnered attention, the analogous contacts within the nucleic acid structures have not been discussed. In this work, we have developed an automated method for identifying and unambiguously classifying T-shaped interactions between nucleobases. Using this method, we identified a total of 3261 instances of T-shaped (perpendicular stacking) contacts between two nucleobases in an array of RNA structures from an up-to-date dataset of <= 3.5 Å resolution crystal structures deposited in the Protein Data Bank.

RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to a RBP. When the RBP binds to a mRNA, ADAR catalyzes A to G modifications in the target mRNA that can be easily identified in standard RNA-sequencing. In STAMP, the concept is the same except the ADARcd is replaced by the RNA editing enzyme APOBEC. Here we compared TRIBE and STAMP side-by-side in human and Drosophila cells. The goal is to learn the pros and cons of each method so that researchers can choose the method best suited to their RBP and system. In human cells, TRIBE and STAMP were performed using the RBP TDP-43. Although they both identified TDP-43 target mRNAs, combining the two methods more successfully identified high confidence targets. In Drosophila cells, RBP-APOBEC fusions generated only low numbers of editing sites, comparable to the level of control editing. This was true for two different RBPs, Hrp48 and Thor (Drosophila EIF4E-BP), indicating that STAMP does not work well in Drosophila.

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.

The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and Trinucleotide Repeat Containing Six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRbase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The miRgeneDB resource offers closest alignment with experimental results. By contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously-expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA dependence, and physical association with AGO when forming hypotheses related to their function.

The interactions of iron regulatory proteins (IRPs) with mRNAs containing an iron-responsive element (IRE) maintain cellular iron homeostasis and coordinate it with metabolism and possibly cellular behavior. The mRNA encoding transferrin receptor-1 (TFRC, TfR1), which is a major means of iron importation, has five IREs within its 3’ UTR, and IRP interactions help maintain cytosolic iron through protection of the TfR1 mRNA from degradation. An IRE within the 3’ UTR of a mRNA splice variant encoding human cell division cycle 14A (CDC14A) has the potential to coordinate the cellular iron status with cellular behavior through a similar IRP-mediated mechanism. However, the stability of the CDC14A splice variant was reported earlier to be unaffected by the cellular iron status, which suggested that the IRE is not functional. We labeled newly synthesized mRNA in HEK293 cells with 5-ethynyl uridine and found that the stability of the CDC14A variant is responsive to iron deprivation, but there are two major differences from the regulation of TfR1 mRNA stability. First, the decay of the CDC14A mRNA does not utilize the Roquin-mediated reaction that acts on the TfR1 mRNA indicating that there is flexibility in the degradative machinery antagonized by the IRE-IRP interactions. Second, the stabilization of the CDC14A mRNA is delayed relative to the TfR1 mRNA and does not occur until IRP binding activity has been induced. The result is consistent with a hierarchy of IRP interactions in which the maintenance of cellular iron through the stabilization of the TfR1 mRNA is initially prioritized.

The SRP9/SRP14 heterodimer is a central component of signal recognition particle (SRP) RNA (7SL) processing and Alu retrotransposition. In this study, we sought to establish the role of nuclear SRP9/SRP14 in the transcriptional regulation of 7SL and BC200 RNA. 7SL and BC200 RNA steady state levels, rate of decay, and transcriptional activity were evaluated under SRP9/SRP14 knockdown conditions. Immunofluorescent imaging, and subcellular fractionation of MCF-7 cells, revealed a distinct nuclear localization for SRP9/SRP14. The relationship between this localization and transcriptional activity at 7SL and BC200 genes was also examined. These findings demonstrate a novel nuclear function of SRP9/SRP14 establishing that this heterodimer transcriptionally regulates 7SL and BC200 RNA expression. We describe a model in which SRP9/SRP14 co-transcriptionally regulate 7SL and BC200 RNA expression. Our model is also a plausible pathway for regulating Alu RNA transcription and is consistent with the hypothesized roles of SRP9/SRP14 transporting 7SL RNA into the nucleolus for post-transcriptional processing, and trafficking of Alu RNA for retrotransposition.