Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10␣, Hs10␤, Hs8, and Hs6, respectively), homologous to the ABC10␣, ABC10␤, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10␣, Sc10␤, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Eukaryotic mRNAs are synthesized by large transcription complexes formed by RNA polymerase II and a number of protein cofactors controlling the selectivity and efficiency of transcriptional initiation, elongation, and termination (14, 36). Purified preparations of RNA polymerase II were obtained for several eukaryotes (references 26, 37, 38, and 54 and references therein) and were found to consist of at least 10 distinct polypeptides ranging from 220 to less than 10 kDa. Their subunit structure is thus much more complex than is that of the three-component bacterial core enzyme ␣ 2 ␤␤Ј. Archaeal RNA polymerases also contain a large number of polypeptides, and most of them are related to eukaryotic subunits (21,(23)(24)(25)38).The genes encoding the 12 subunits of the yeast enzyme have all been cloned and sequenced (see Table 1). The three largest subunits, Sc1, Sc2, and Sc3, are homologous to the ␤Ј, ␤, and ␣ components of the bacterial core enzyme (28, 38, 54). Sc11 (50) is homologous to AC19, a subunit which is shared by yeast RNA polymerases I and III (15). Sc7 is similar to what is most probably the C25 subunit of RNA polymerase III (39). Sc7 was initially believed to be nonessential for mRNA synthesis in vivo (54), but further studies indicated that deletion of the corresponding gene is lethal (29). Five small subunits, Sc5, Sc6, Sc8, Sc10␣, and Sc10␤ (22,44,49,52), are present in all three nuclear RNA polymerases (11,12,37,46). These 10 specific or common subunits are essential components of the transcription apparatus, as strains carrying the corresponding null alleles are nonviable. In contrast, the deletion of the genes encoding the RNA polymerase II-specific Sc4 and Sc9 subunits leads to slowly growing but viable mutants (48, 51).The human RNA polymerase II, although less extensively characterized, contains at least 10 distinct subunits (19,26). The yeast and animal enzymes are closely related antigenically (18), indicating a strong evolutionary conservation. This was directly established by cloning and sequencing seven human cDNAs encoding RNA polymerase II subunits Hs1, Hs2, Hs3, Hs5, Hs6, Hs9,[32][33][34]47), which all showed significant homology to the corresponding yeast subunits (see Table 1). However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the human enzyme (26) failed to reveal small polypeptides of less than 10 kDa that would correspond to the Sc10␣ and Sc10␤ subunits shared by all three yeast RNA polymerases (12).It was recently demonstrated that the common subunit Sc6 of Saccharomyces cerevisiae can be functionally replaced in vivo by homologs from Schizosaccharomyces pombe and ha...

The Colorado potato beetle Leptinotarsa decemlineata is an insect pest that threatens potato crops globally. The primary method to control its damage on potato plants is the use of insecticides, including imidacloprid, chlorantraniliprole and spinosad. However, insecticide resistance has been frequently observed in Colorado potato beetles. The molecular targets and the basis of resistance to imidacloprid and chlorantraniliprole have both been previously quantified. This work was undertaken with the overarching goal of better characterizing the molecular changes associated with spinosad exposure in this insect pest. Next-generation sequencing was conducted to identify transcripts that were differentially expressed between Colorado potato beetles exposed to spinosad versus control insects. Results showed several transcripts that exhibit different expression levels between the two conditions, including ones coding for venom carboxylesterase-6, chitinase 10, juvenile hormone esterase and multidrug resistance-associated protein 4. In addition, several microRNAs, such as miR-12-3p and miR-750-3p, were also modulated in the investigated conditions. Overall, this work reveals a molecular footprint underlying spinosad response in Colorado potato beetles and provides novel leads that could be targeted as part of RNAi-based approaches to control this insect pest.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily “monocyte-like” at Day 1 to primarily “macrophage-like” at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

No abstract

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

No abstract

Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma.