A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2.

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.

Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase.

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.

Architecture and dynamics of overlapped RNA regulatory networks.

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 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.

tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae.

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)C-methyltransferase 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.

Yeast Trm7 interacts with distinct proteins for critical modifications of the tRNAPhe anticodon loop.

Post-transcriptional modification of the tRNA anticodon loop is critical for translation. Yeast Trm7 is required for 2'-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-Δ 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-Δ 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 2'-O-methylation of C(32), and with Rtt10 (named Trm734) for 2'-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-Δ mutants is due to the lack of both Cm(32) and Nm(34), 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 m(2,2)G(26) 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.

The requirement for the highly conserved G-1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase.

Nearly all tRNA(His) species have an additional 5' guanine nucleotide (G(-1)). G(-1) 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(-1) conservation suggest that G(-1) 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 3'-5' (reverse) polymerization in vitro at the 5' end of activated tRNAs. Here we show that thg1-Delta 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(-1) addition activity. Since these thg1-Delta 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(-1) residue is important, but not absolutely essential, despite its widespread conservation. We also show that Thg1 catalyzes 3'-5' polymerization in vivo on tRNA(His) C(73), but not on tRNA(His) A(73), 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 tRNA(His) with A(73), rather than C(73), to prevent the possibility of 3'-5' polymerization.

Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae.

tRNAs in yeast and vertebrate cells move bidirectionally and reversibly between the nucleus and the cytoplasm. We investigated roles of members of the beta-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 beta-importin family. The beta-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.

Failure to monitor ticlopidine: the case for clopidogrel.

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 <0.5 x 10(9)/mm(3)). Patient compliance with full blood count monitoring was 85% at two weeks and 80% at four weeks. Two patients (1.4%) suffered subacute stent thrombosis. At six months, there were no deaths, one (0.7%) myocardial infarction and nine patients (6%) requiring target vessel revascularization. CONCLUSION: A two-week course of ticlopidine is well tolerated and does not appear to be associated with an increase in adverse cardiac events. However, even with a dedicated monitoring team, adequate haematological follow-up was achieved in only 80% of patients. The need for a licensed antiplatelet agent which has a lower side-effect profile and does not require haematological monitoring is obvious.