The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC co-evolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and re-activates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.

Double-stranded RNA (dsRNA) has aroused widespread interest due to its effects on immunity and applications based on RNAi. However, in vitro preparation of dsRNA is costly and laborious. In this study, we developed a novel and interesting method designated as pfRCT (promoter free Rolling-Circle Transcription) for direct, facile, and efficient dsRNA preparation. This method generates equal amounts of sense strands and antisense strands simultaneously from a single circular dsDNA template. To initiate transcription by T7 RNA polymerase without direction preference, a 9-15-bp bubble (mismatched duplex with strong sequence symmetry) is introduced to the template. During RCT, all the necessary reagents, including the template, NTPs, RNA polymerase, RNase H, and Helpers, are present in one pot; and the just transcribed RNA is immediately truncated by RNase H to monomers with the desired size. The ends of dsRNA product can also be simply sealed by T4 RNA ligase 1 after pfRCT. This new approach is expected to promote the applications of dsRNA.

Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well-established; its role in RNA localization to the ER remains however unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding alpha subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SRP receptor (SR) knockout mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SRP receptor expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER..

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity towards RNA containing secondary structures, and broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA-mediated interference is alleviated by genetic perturbations that elicit precocious lncRNA 3'-processing and transcription termination, such as: (i) the inositol pyrophosphate pyrophosphatase-defective asp1-H397A allele, which results in elevated levels of IP8; and (ii) absence of the 14-3-3 protein Rad24. Combining rad24∆ with asp1-H397A causes a severe synthetic growth defect. A forward genetic screen for SRA (Suppressor of Rad24 Asp1-H397A) mutations identified a novel missense allele (Tyr86Asp) of Pla1, the essential poly(A) polymerase subunit of the fission yeast CPF (cleavage and polyadenylation factor) complex. The pla1-Y86D allele was viable but slow-growing in an otherwise wild-type background. Tyr86 is a conserved active site constituent that contacts the RNA primer 3' nucleotide and the incoming ATP. The Y86D mutation elicits a severe catalytic defect in RNA-primed poly(A) synthesis in vitro and in binding to an RNA primer. Yet, analyses of specific mRNAs indicate that poly(A) tails in pla1-Y86D cells are not different in size than those in wild-type cells, suggesting that other RNA interactors within CPF compensate for the defects of isolated Pla1-Y86D. Transcriptome profiling of pla1-Y86D cells revealed the accumulation of multiple RNAs that are normally rapidly degraded by the nuclear exosome under the direction of the MTREC complex, with which Pla1 associates. We suggest that Pla1-Y86D is deficient in the hyperadenylation of MTREC targets that precedes their decay by the exosome.

It is known that mRNAs and the machinery that translates them are not uniformly distributed throughout the cytoplasm. As a result, the expression of some genes is localized to particular parts of the cell and this makes it possible to carry out important activities, such as growth and signaling, in three-dimensional space. However, the functions of localized gene expression are not fully understood and the underlying mechanisms that enable localized expression have not been determined in many cases. One consideration that could help in addressing these challenges is the role of quality control (QC) mechanisms that monitor translating ribosomes. On a global level, QC pathways are critical for detecting aberrant translation events, such as a ribosome that stalls while translating, and responding by activating stress pathways and resolving problematic ribosomes and mRNAs at the molecular level. However, it is unclear how these pathways, even when uniformly active throughout the cell, affect local translation. Importantly, some QC pathways have themselves been reported to be enriched in the proximity of particular organelles but the extent of such localized activity remains largely unknown. Here we describe the major QC pathways and review studies that have begun to explore their roles in localized translation. Given the limited data in this area, we also pose broad questions about the possibilities and limitations for how QC pathways could facilitate localized gene expression in the cell with the goal of offering ideas for future experimentation.

The conserved family of RNA-binding proteins (RBP), IGF2BPs play an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs, IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells and its expression correlates with poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we introduced highly-sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified protocol "IR-PAR-CLIP". Third, we systematically compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.

The identification of catalytic RNAs is typically achieved through primarily experimental means. However, only a small fraction of sequence space can be analyzed even with high-throughput techniques. Methods to extrapolate from a limited data set to predict additional ribozyme sequences, particularly in a human-interpretable fashion, could be useful both for designing new functional RNAs and for generating greater understanding about a ribozyme fitness landscape. Using information theory, we express the effects of epistasis (i.e., deviations from additivity) on a ribozyme. This representation was incorporated into a simple model of the epistatic fitness landscape, which identified potentially exploitable combinations of mutations. We used this model to theoretically predict mutants of high activity for a self-aminoacylating ribozyme, identifying potentially active triple and quadruple mutants beyond the experimental data set of single and double mutants. The predictions were validated experimentally, with nine out of nine sequences being accurately predicted to have high activity. This set of sequences included mutants that form a previously unknown evolutionary 'bridge' between two ribozyme families that share a common motif. Individual steps in the method could be examined, understood, and guided by a human, combining interpretability and performance in a simple model to predict ribozyme sequences by extrapolation.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events, so cells need to carefully control SAM levels. MAT2A encodes the only SAM synthetase expressed in the majority of human cells and its 3' UTR has six conserved regulatory hairpins (hp1-6) that can be methylated by the N6-methyladenosine methyltransferase METTL16. Hp1 begins 8 nucleotides (nt) from the stop codon while hp2-6 are clustered further downstream (~800 nt). These hairpins have been proposed to regulate MAT2A mRNA levels in response to intracellular SAM levels by regulating intron detention of the last intron of MAT2A and by modulating stability of the fully spliced mRNA. However, a dissection of these proposed activities has not been previously reported. Using a modular reporter system, we show that hp1 functions only when the detained intron is included in the reporter and when that intron has a suboptimal polypyrimidine tract. In contrast, the hp2-6 cluster modulates mRNA stability independent of the detained intron. Taken with previously published reports, these data support a two-tiered model for MAT2A posttranscriptional regulation by METTL16 through its interactions with hp1 and hp2-6. In the upstream tier, hp1 and METTL16 control MAT2A intron detention while the second tier involves METTL16-dependent methylation of hp2-6 to control MAT2A mRNA stability. Thus, cells employ a similar set of molecular factors to achieve considerable complexity in the posttranscriptional regulation of SAM homeostasis.