Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 29-O-methylation of C 32 and N 34 of tRNA Phe , tRNA Trp , and tRNA Leu(UAA) to form Cm 32 and Nm 34 , and trm7-D mutants have severe growth and translation defects, but the reasons for these defects are not known. We show here that overproduction of tRNA Phe suppresses the growth defect of trm7-D mutants, suggesting that the crucial biological role of Trm7 is the modification of tRNA Phe . We also provide in vivo and in vitro evidence that Trm7 interacts with ORF YMR259c (now named Trm732) for 29-O-methylation of C 32 , and with Rtt10 (named Trm734) for 29-O-methylation of N 34 of substrate tRNAs and provide evidence for a complex circuitry of anticodon loop modification of tRNA Phe , in which formation of Cm 32 and Gm 34 drives modification of m 1 G 37 (1-methylguanosine) to yW (wyebutosine). Further genetic analysis shows that the slow growth of trm7-D mutants is due to the lack of both Cm 32 and Nm 34 , and the accompanying loss of yW, because trm732-D trm734-D mutants phenocopy trm7-D 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-D mutants, which lack m 2,2 G 26 in their tRNAs. We speculate that 29-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.

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.

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed TRAID-Seq, a screening strategy in S. cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressing 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 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. elegans MUT-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 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.

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 Using RNA Tagging, we identified a "super-network" comprised of four subnetworks: Puf3p, Puf4p, and Puf5p subnetworks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, is 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.

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 tRNA His guanylyltransferase accumulate uncharged tRNA His lacking the G −1 residue and subsequently accumulate additional 5-methylcytidine (m 5 C) at residues C 48 and C 50 of tRNA His , due to the activity of the m 5 Cmethyltransferase Trm4. We show here that the increase in tRNA His m 5 C levels does not require loss of Thg1, loss of G −1 of tRNA His , or cell death but is associated with growth arrest following different stress conditions. We find substantially increased tRNA His m 5 C 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 m 5 C in tRNA His after starvation for glucose and after starvation for uracil. In virtually all cases examined, the additional m 5 C on tRNA His occurs while cells are fully viable, and the increase is neither due to the GCN4 pathway, nor to increased Trm4 levels. Moreover, the increased m 5 C appears specific to tRNA His , as tRNA Val(AAC) and tRNA Gly(GCC) have much reduced additional m 5 C during these growth arrest conditions, although they also have C 48 and C 50 and are capable of having increased m 5 C levels. Thus, tRNA His m 5 C levels are unusually responsive to yeast growth conditions, although the significance of this additional m 5 C remains unclear.

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.

Nearly all tRNAHis species have an additional 59 guanine nucleotide (G À1 ). G À1 is encoded opposite C 73 in nearly all prokaryotes and in some archaea, and is added post-transcriptionally by tRNA His guanylyltransferase (Thg1) opposite A 73 in eukaryotes, and opposite C 73 in other archaea. These divergent mechanisms of G À1 conservation suggest that G À1 might have an important cellular role, distinct from its role in tRNA His 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 39-59 (reverse) polymerization in vitro at the 59 end of activated tRNAs. Here we show that thg1-D strains are viable, but only if histidyl-tRNA synthetase and tRNA His are overproduced, demonstrating that the only essential role of Thg1 is its G À1 addition activity. Since these thg1-D strains have severe growth defects if cytoplasmic tRNA His A 73 is overexpressed, and distinct, but milder growth defects, if tRNA His C 73 is overexpressed, these results show that the tRNA His G À1 residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 39-59 polymerization in vivo on tRNA His C 73 , but not on tRNA His A 73 , demonstrating that the 39-59 polymerase activity is pronounced enough to have a biological role, and suggesting that eukaryotes may have evolved to have cytoplasmic tRNA His with A 73 , rather than C 73 , to prevent the possibility of 39-59 polymerization.

We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep.