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Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a cytidine-adding enzyme that is likely to be part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing. Ribonucleotidyl transferases tethered to an RNA reporter add untemplated RNA tails. TRAID-seq identifies the activities of 22 enzymes, including the addition of poly(UG).

The microbiome plays a central role in biochemical cycling and nutrient turnover of most ecosystems. Because it can comprise myriad microbial prokaryotes, eukaryotes and viruses, microbiome characterization requires high-throughput sequencing to attain an accurate identification and quantification of such co-existing microbial populations. Short-read next-generation-sequencing (srNGS) revolutionized the study of microbiomes and remains the most widely used approach, yet read lengths spanning only a few of the nine hypervariable regions of the 16S rRNA gene limit phylogenetic resolution leading to misclassification or failure to classify in a high percentage of cases. Here we evaluate a synthetic long-read (SLR) NGS approach for full-length 16S rRNA gene sequencing that is high-throughput, highly accurate and low-cost. The sequencing approach is amenable to highly multiplexed sequencing and provides microbiome sequence data that surpasses existing short and long-read modalities in terms of accuracy and phylogenetic resolution. We validated this commercially-available technology, termed LoopSeq, by characterizing the microbial composition of well-established mock microbiome communities and diverse real-world samples. SLR sequencing revealed differences in aquatic community complexity associated with environmental gradients, resolved species-level community composition of uterine lavage from subjects with histories of misconception and accurately detected strain differences, multiple copies of the 16S rRNA in a single strain’s genome, as well as low-level contamination in soil cyanobacterial cultures. This approach has implications for widespread adoption of high-resolution, accurate long-read microbiome sequencing as it is generated on popular short read sequencing platforms without the need for additional infrastructure.

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid(TNA) extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities., Graphical abstract Unlabelled Image

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet, little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities – Puf3p, Puf4p, and Puf5p of S. cerevisiae. Using RNA Tagging, we identified a “super-network” comprised of four sub-networks: Puf3p, Puf4p, and Puf5p sub-networks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, are determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: the remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution.

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy inS. cerevisiaeto identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase,C. elegansMUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2′-O-methylation of C32 and N34 of tRNAPhe, tRNATrp, and tRNALeu(UAA) to form Cm32 and Nm34, and trm7-Δ mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNAPhe suppresses the growth defect of trm7-Δ mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNAPhe. We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 2′-O-methylation of C32, and with Rtt10 (named Trm734) for 2′-O-methylation of N34 of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNAPhe, in which formation of Cm32 and Gm34 drives modification of m1G37 (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-Δ mutants is due to the lack of both Cm32 and Nm34, and the accompanying loss of yW, because trm732-Δ trm734-Δ mutants phenocopy trm7-Δ mutants, whereas each single mutant is healthy; nonetheless, TRM732 and TRM734 each have distinct roles, since mutations in these genes have different genetic interactions with trm1-Δ mutants, which lack m2,2G26 in their tRNAs. We speculate that 2′-O-methylation of the anticodon loop may be important throughout eukaryotes because of the widespread conservation of Trm7, Trm732, and Trm734 proteins, and the corresponding modifications, and because the putative human TRM7 ortholog FTSJ1 is implicated in nonsyndromic X-linked mental retardation.

Nearly all tRNAHis species have an additional 5′ guanine nucleotide (G−1). G−1 is encoded opposite C73 in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNAHis guanylyltransferase (Thg1) opposite A73 in eukaryotes, and opposite C73 in other archaea. These divergent mechanisms of G−1 conservation suggest that G−1 might have an important cellular role, distinct from its role in tRNAHis charging. Thg1 is also highly conserved and is essential in the yeast Saccharomyces cerevisiae. However, the essential roles of Thg1 are unclear since Thg1 also interacts with Orc2 of the origin recognition complex, is implicated in the cell cycle, and catalyzes an unusual template-dependent 3′–5′ (reverse) polymerization in vitro at the 5′ end of activated tRNAs. Here we show that thg1-Δ strains are viable, but only if histidyl-tRNA synthetase and tRNAHis are overproduced, demonstrating that the only essential role of Thg1 is its G−1 addition activity. Since these thg1-Δ strains have severe growth defects if cytoplasmic tRNAHis A73 is overexpressed, and distinct, but milder growth defects, if tRNAHis C73 is overexpressed, these results show that the tRNAHis G−1 residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 3′–5′ polymerization in vivo on tRNAHis C73, but not on tRNAHis A73, demonstrating that the 3′–5′ polymerase activity is pronounced enough to have a biological role, and suggesting that eukaryotes may have evolved to have cytoplasmic tRNAHis with A73, rather than C73, to prevent the possibility of 3′–5′ polymerization.

tRNAs traffic between the nucleus and the cytoplasm in response to nutrient availability. Using a new assay to track tRNA within cells, we show that tRNA nuclear import is constitutive, whereas tRNA reexport to the cytoplasm is regulated. Msn5 functions only in tRNA re-export, whereas Los1 functions in both the primary and reexport steps., tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the β-importin family in tRNA subcellular dynamics. Retrograde import of tRNA into the nucleus is dependent, directly or indirectly, upon Mtr10. tRNA nuclear export utilizes at least two members of the β-importin family. The β-importins involved in nuclear export have shared and exclusive functions. Los1 functions in both the tRNA primary export and the tRNA reexport processes. Msn5 is unable to export tRNAs in the primary round of export if the tRNAs are encoded by intron-containing genes, and for these tRNAs Msn5 functions primarily in their reexport to the cytoplasm. The data support a model in which tRNA retrograde import to the nucleus is a constitutive process; in contrast, reexport of the imported tRNAs back to the cytoplasm is regulated by the availability of nutrients to cells and by tRNA aminoacylation in the nucleus. Finally, we implicate Tef1, the yeast orthologue of translation elongation factor eEF1A, in the tRNA reexport process and show that its subcellular distribution between the nucleus and cytoplasm is dependent upon Mtr10 and Msn5.

AIM: Ticlopidine remains unlicensed for use in coronary artery stenting. Haematological monitoring at two-weekly intervals of all patients taking ticlopidine is recommended because of the risk of neutropaenia. The authors assessed their ability to monitor a two-week course of ticlopidine given to patients undergoing coronary stenting procedures. METHODS AND RESULTS: One hundred and forty-one unselected consecutive patients undergoing coronary stenting procedures were prescribed ticlopidine 250 mg twice daily for two weeks, in addition to aspirin. Prior to discharge home they and their GPs were given written information outlining the side-effects of ticlopidine and the monitoring procedures required. One hundred and thirty-three patients (94%) completed the full two-week course of ticlopidine; three (2.4%) developed significant neutropaenia (neutrophil count

tRNAs are highly modified, each with a unique set of modifications. Several reports suggest that tRNAs are hypomodified or, in some cases, hypermodified under different growth conditions and in certain cancers. We previously demonstrated that yeast strains depleted of tRNAHis guanylyltransferase accumulate uncharged tRNAHis lacking the G−1 residue and subsequently accumulate additional 5-methylcytidine (m5C) at residues C48 and C50 of tRNAHis, due to the activity of the m5C-methyltransferase Trm4. We show here that the increase in tRNAHis m5C levels does not require loss of Thg1, loss of G−1 of tRNAHis, or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNAHis m5C levels after temperature-sensitive strains are grown at nonpermissive temperature, and after wild-type strains are grown to stationary phase, starved for required amino acids, or treated with rapamycin. We observe more modest accumulations of m5C in tRNAHis after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m5C on tRNAHis occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m5C appears specific to tRNAHis, as tRNAVal(AAC) and tRNAGly(GCC) have much reduced additional m5C during these growth arrest conditions, although they also have C48 and C50 and are capable of having increased m5C levels. Thus, tRNAHis m5C levels are unusually responsive to yeast growth conditions, although the significance of this additional m5C remains unclear.