Influenza A virus (IAV) causes seasonal epidemics and sporadic pandemics, therefore is an important research subject for scientists around the world. Despite the high variability of its genome, the structure of viral RNA (vRNA) possesses features that remain constant between strains and are biologically important for virus replication. Therefore, conserved structural motifs of vRNA can represent a novel therapeutic target. Here, we focused on the presence of G-rich sequences within the influenza A/California/07/2009(H1N1) genome and their ability to form RNA G-quadruplex structures (G4s). We identified 12 potential quadruplex-forming sequences (PQS) and determined their conservation among the IAV strains using bioinformatics tools. Then we examined the propensity of PQS to fold into G4s by various biophysical methods. Our results revealed that six PQS oligomers could form RNA G-quadruplexes. However, three of them were confirmed to adopt G4 structures by all utilized methods. Moreover, we showed that these PQS motifs are present within segments encoding polymerase complex proteins indicating their possible role in the virus biology. Influenza virus, due to its pandemic potential, became an interesting subject in various biological research projects. There are three types of influenza virus: A, B and C. However, the influenza A virus (IAV) is gaining a lot of attention because of its responsibility for most of the pandemic outbreaks , the abiThe IAV negative-sense RNA genome consists of eight segments encoding 11 proteins: three polymerase complex proteins: polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2) and polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), two matrix proteins (M1 and M2) and non-structural proteins  . InteresImportantly, there is growing interest in the unique non-canonical structures called G-quadruplexes (G4s) ,14. TheyIn the current study, both bioinformatics analysis and biophysical methods allowed us to show that conserved PQS motifs occur within the influenza A/California/07/2009(H1N1) genome and indeed have the ability to fold into G-quadruplexes. Based on these results and taking under consideration the G4s localization within the vRNA segments, it can be assumed that these structures can play an important role in the regulation of different steps of the viral life cycle.http://scottgroup.med.usherbrooke.ca/G4RNA_screener) and the Quadruplex-forming G-Rich Sequences (QGRS) Mapper (accessed on 15 December 2020)  were used to identify PQS motifs across the whole IAV genome. Our bioinformatics analysis revealed the presence of twelve PQS candidates that can potentially adopt RNA G4 structures. The PQS motif characteristics are summarized in In this work, we inspected the IAV genome for the presence of the unique G-rich sequences. For this purpose, the sequence of the influenza A/California/07/2009(H1N1) genome was retrieved from the NCBI  database (RefSeq assembly accession: GCF_001343785.1). There are several bioinformatics tools allowing to search for G-rich regions in RNA sequences prone to fold into G-quadruplex structures . Herein,www.fludb.org) to retrieve complete genomic sequences of the eight IAV segments. Subsequently, the multiple sequence alignments were carried out for each segment separately, and the frequency of nucleotide residues at each position was established using MAFFT software and BioEdit 7.1 program, respectively. The percentage of the PQS motifs similarity across the IAV strains was calculated and is presented in All PQS motifs found were located within the protein-coding regions of the IAV genome. Obtained results encouraged us to ask the question of whether these PQS candidates are conserved among the IAV strains. To answer this question, we first used the IRD (Influenza Research Database) database (accessed on 15 December 2020) , ultraviolet–visible (UV-Vis) melting, circular dichroism (CD) and fluorescence assays) as well as native gel electrophoresis to examine the structure of PQS motifs identified in our bioinformatics analysis1H NMR spectra of DNA or RNA samples, we can gain information whether the molecule forms quadruplex structure under appropriate solution conditions. The core of the quadruplexes are G-tetrads in which guanosine residues are associated through Hoogsteen hydrogen bonds. Signals from the imino protons involved in this type of bond are usually in the area of 10–12 ppm, whereas those from 12–15 ppm are characteristic for Watson–Crick base pairs. The imino proton region of NMR spectra between 10 and 15 ppm provides information about the nature of hydrogen bonds between DNA or RNA base pairs [NMR spectroscopy is a powerful tool to study nucleic acids secondary structures including G-quadruplex formation ,25. By r1H NMR experiments to justify the selection of RNA G4-forming motifs. Our NMR analysis of PQS candidates provided direct evidence for the formation of RNA G4 structures by several oligomers. Based on the results obtained, the model PQS oligomers were divided into three groups depending on their predicted structure. The first group includes sequences with the highest propensity to fold into G-quadruplex structures, including 1KW, 7KW and 11KW variants  that might be in dynamic equilibrium between two different structures, including RNA G-quadruplex and hairpin. Their 1H NMR spectra demonstrated the signals within the range of 10–12 ppm, suggesting Hoogsteen hydrogen bonds involved in G4 structure formation  that is a dye selectively binding to G-quadruplexes. Experiments were conducted for PQS oligomers with the addition of NMM in a 1:1 molar ratio. In general, the recorded UV spectroscopy is a fast and simple technique to confirm G4 structure formation and evaluation of its thermal stability. Typically, the G-quadruplex unfolding is associated with a decrease in absorbance at 295 nm wavelength, at which the thermal denaturation of the G4 structure is manifested in an over 50% change in the absorbance amplitude ,27. Recom) that are presented in As a result, we were not able to obtain reliable thermodynamic parameters and decided to focus on the melting profiles and changes in the melting temperature determined for model PQS variants. The obtained results demonstrated that the melting curve profiles of four PQS oligomers  spectroscopy is a simple and relatively fast biophysical method to characterize the G-quadruplex folding topology and assess its structural geometry. It is based on a phenomenon that chiral molecules absorb left-handed and right-handed circularly polarized light differently . GeneralOne negative band around 240 nm and one positive band near 265 nm were observed for studied PQS variants . SurprisTo further confirm that PQS motifs adopt a G-quadruplex structure, thioflavin T (ThT) fluorescence intensity measurements were performed. In 2013 ThT was applied as a specific ligand towards G-quadruplex folds . BecauseAs a result, the fluorescence emission spectra of ThT in the presence of various RNA oligomers at wavelengths ranging from 448 nm to 700 nm and the fluorescence emission of ThT at 482 nm are presented in Comparing the fluorescence intensity at 482 nm for tested PQS variants we found a significant increase in ThT fluorescence upon binding to almost all of them  was performed to confirm the RNA G4s formation. DNA or RNA migrates at different rates in an electric field depending on its secondary structure. Moreover, based on the electrophoretic mobility, we can obtain detailed information about the conformation changes that determine both folding topology and molecularity in G4 structure. Therefore, we used this method to study RNA G-quadruplex folding patterns.Due to the fact that NMM is a highly selective quadruplex-specific ligand with a preference to bind to the parallel G4s , we applThe gels were first visualized under the UV light a. As expAccording to the gel stained with ThT dye presented in In summary, all the results obtained by using different spectroscopic methods and electrophoretic techniques, allowed us to discover that three PQS motifs from the influenza A/California/07/2009(H1N1) genome adopt RNA G-quadruplex structures. Nonetheless, these G4s can differ in their stability and also folding topology depending on the experimental conditions.Influenza virus continuously remains a serious threat for public health. The number of annual deaths worldwide caused by infection with this pathogen reaches above 600,000 cases. Due to the high viral genetic variability, new vaccines need to be developed every year and their effectiveness rarely exceeds 60% . TherefoBased on the literature we know that the secondary structure of vRNA is highly conserved among viral strains and biologically important for virus life cycle . The virIn virus particles and in cells, the vRNA segments are encapsidated as individual RNP complexes. The molecular model of interaction between the vRNA and NP revealed that RNA can loop out from this complex and interact with other vRNA segments or host and virus factors ,40,41. FFurthermore, taking into account described by Lee et al. NP-vRNA association profiles (including nucleotide content of NP peaks) in the A/California/07/2009 strain, we can suggest that PQS motifs may occur in G-rich regions that are NP binding positions on the vRNP. However, NP association does not exclude RNA–RNA interactions between viral RNA segments, as reported by Le Sage et al. . Due to Interestingly, previous research revealed that potential G4 sequences can be found in Zika virus and Zaire ebolavirus (EBOV) genomes and their potential biological functions were studied ,18. BurrIn this present study, we used bioinformatical and biophysical methods to search the influenza A/California/07/2009(H1N1) genome for PQS motifs and confirm the G4 structure formation. There are many PQS searching bioinformatics tools reviewed by Lombardi et al. . Here, wHere, we also analyzed the similarity of PQS motifs across the IAV genomic sequences retrieved from the IRD database. Additionally, the PQS conservation analysis based on LOGO sequences showed that G residues within G-tracts, which could be involved in G4 folding, were conserved. Recently, it has been reported that G4-prone sequences from the genomes of RNA viruses show high level of conservation among different genotypes, suggesting their crucial role in the virus life cycle ,18,43. FNidovirales [Nidovirales genomes, in contrast to IRs. However, location of PQS sites before 3′ UTR, inside 5′ UTR and before mRNA suggests their important regulatory role [More recently, some papers described bioinformatics analyses of viral genomes  and the PQS occurrence in G4-EA-H1N1 genomes [To the best of our knowledge, the current investigations provide the first evidence that conserved PQS motifs are present in the influenza A/California/07/2009(H1N1) genome and have a high propensity to fold into RNA G-quadruplex structures. Comparing our investigations with an already published study on the genotype 4 reassortant Eurasian avian-like H1N1 virus  and the Quadruplex forming G-Rich Sequences (QGRS) Mapper (accessed on 15 December 2020) .The influenza A/California/07/2009(H1N1) genome was obtained from the NCBI database (RefSeq assembly accession: GCF_001343785.1). To identify the PQS motifs, the genome was analyzed using two different tools for G-quadruplex prediction, i.e., G4RNA screener web interface (accessed on 15 December 2020) (www.fludb.org). Two types of comparative analysis of sequence data were conducted with the help of MAFFT software  and the Bioedit 7.1 program . The multiple sequence alignments using MAFFT were performed for all IAV genomic sequences deposited in the IRD database and separately for all those of IAV H1N1 subtypes. Next, the aligned sequences were loaded into the Bioedit 7.1 program for the frequency of nucleotide residues analysis. The data obtained were used to examine the PQS motifs similarity across both the IAV subtypes and particularly the IAV H1N1 subtype. Then PQS motifs were searched in aligned segment sequences, LOGO representations were constructed using WebLogo application (accessed on 15 December 2020)  and the conservation levels were determined.The nucleotide sequences for each segment of the IAV were downloaded from the Influenza Research Database (IRD) (accessed on 15 December 2020)  synthesizer using standard phosphoramidite chemistry on solid support . All oli1H NMR spectra were acquired on a Bruker AVANCE III 700 MHz spectrometer equipped with a QCI CryoProbe . The 3 mm thin wall tubes were used with the final sample volume of 200 μL H2O/D2O . RNA oligonucleotides were dissolved in a 10 mM potassium phosphate buffer (pH 6.8) containing 50 mM KCl and 0.1 mM EDTA (ethylenediaminetetraacetic acid). The experiments were conducted at 25 °C using RNA oligomers with or without the addition of N-methyl mesoporphyrin IX  in a 1:1 molar ratio. The samples were annealed by heating at 90 °C for 5 min and then slowly cooled to room temperature. Water suppression was achieved using excitation sculpting. The spectra were processed and prepared with TopSpin 3.0 Bruker Software .https://www.idtdna.com/calc/analyzer . Samples were denatured for 5 min at 90 °C and then slowly cooled to room temperature. Measurements were performed in triplicate using quartz cuvettes of 0.1 cm path length with a sample volume of 30 µL. Absorbance versus temperature curves were obtained by the UV melting method at 260 nm and 295 nm wavelengths in the temperature range 5–90 °C with a heating rate of 0.2 °C/min on a Jasco V-650 spectrophotometer  equipped with a thermoprogrammer. Thermal denaturation curves were analyzed and the melting temperatures (Tm) of model PQS oligomers were determined. The Tm values were obtained as an arithmetic average from three repeats.RNA oligonucleotides were dissolved in the same buffer as used for NMR studies. Oligonucleotide single strand concentrations were calculated based on both, absorbance measured above 80 °C and extinction coefficients, which were approximated by a nearest-neighbor model using the Integrated DNA Technologies calculator on the website 2 mmol−1) of the right-handed and left-handed circularly polarized light and normalized for plotting and comparative purposes using Origin Pro 9.8 software (version Origin Pro 2021 (9.8), Northampton, MA, USA).CD spectra were recorded on a Jasco J-815 spectropolarimeter  using 1.5 mL quartz cuvettes with a 5 mm path length and the sample volume of 1300 µL. RNA oligonucleotides were dissolved in the same buffer as used for NMR studies, to achieve a sample concentration of 14 µM. All samples were denatured for 5 min at 90 °C and then slowly cooled to room temperature overnight before data collection. Measurements were collected at 10 °C in the 220–340 nm wavelength range with a 1 nm data interval. CD curves were established as an average of three CD measurements. The buffer spectrum was subtracted from the sample spectra. CD spectra were expressed as the difference in the molar absorption  at concentration of 0.5 µM. Next, the samples were centrifuged and loaded into a 96-well plate  in three repeats. The fluorescence measurements were carried out with CLARIOstar Plus multimode plate reader  at room temperature. ThT fluorescence emission spectra were acquired over the range of 448 to 718 nm. The excitation wavelength was 425 nm. The fluorescence intensity of ThT was measured at 482 nm with excitation at 425 nm. The data analysis was performed using the Origin Pro 9.8 software (version Origin Pro 2021 (9.8), Northampton, MA, USA).−1 NMM in 1X TBE for 10 min or in solution of 0.5 µM ThT in 1X TBE for 15 min. After staining, the gels were destained for 10 min in 1X TBE. The resultant gels were visualized with the help of Fujifilm Phosphorimager  and analyzed using MultiGauge Fujifilm software .Native PAGE was conducted to monitor the mobility of RNA oligomers and confirm the RNA G-quadruplex formation. RNA oligonucleotides at concentration of 0.4 µg/mL (absorbance of 0.1 at 260 nm) were dissolved in the same buffer as used for NMR studies, to obtain final volume of 4 µL. Samples were denatured for 5 min at 90 °C and then slowly cooled to room temperature. After folding, 2 μL of 35% glycerol solution was added. Samples were centrifuged and loaded into a 20% native polyacrylamide gel prepared in 1X TBE (Tris-Borate-EDTA) buffer, pH 7.0. The electrophoresis was performed at 150 V for 3 h in a cold room using XCell SureLock Mini-Cell Electrophoresis System . For analysis the gel was stained in a solution of 0.1 mg ml


Human activities and regional-scale climate variability drive changes in the ecology of coastal and marine ecosystems. Ecological restoration has emerged as a best-management practice to combat habitat degradation and restore lost ecological functions. However, relatively short project monitoring timeframes have limited our understanding of the effects of interannual climate cycles on water quality and restoration dynamics. We collected measurements on a 23-ha oyster reef constructed in the Gulf of Mexico to determine the relationship between El Niño Southern Oscillation (ENSO)-driven climate variability and local salinity patterns, and to evaluate the effects of this climate variability and salinity on oyster population dynamics and faunal community composition over a medium-term (five-year) timeframe. The role of ENSO-driven climate variability on local salinity patterns (primarily from changes in precipitation and evaporation) and faunal dynamics was investigated using the Oceanic Niño Index (ONI). Salinity was negatively correlated with ONI with an approximately 4-month lag. Higher ONI values (El Niño periods) were followed by reductions in salinity, increases in oyster recruitment and density, and reductions in resident motile fauna density and species richness. Lower ONI values (La Niña periods) had higher and less variable salinities, and higher areal coverage of restoration substrates by large oysters. ENSO-driven salinity reductions in the second year after reef construction coincided with a shift in resident motile faunal community composition that was maintained despite a second strong salinity reduction in year 5. Our results indicate that it is important to expand the typical monitoring timeframes to at least five years so that resource managers and restoration practitioners can better understand how both short-term environmental variability and longer-term climate cycles can affect the outcomes of restoration actions. Substantial degradation of estuarine and coastal ecosystems is a global problem that is heavily influenced by multiple human activities . Many deIn the U.S. Gulf of Mexico (GoM), El Niño Southern Oscillation (ENSO) influences local and regional weather patterns , 7. ENSOSuccessful restoration planning and implementation require an understanding of ecological responses to climate variability . BecauseCrassostrea virginica populations have experienced severe declines over the past two centuries -1) from -1), and salinity were made at the surface and the bottom of the water column at multiple sampling sites on each sampling date using a YSI data sonde (no replicates at each site). Discrete salinity data was more representative of the salinity at Half Moon Reef than data from the closest continuous (daily) monitoring station (Site NCM4) [Discrete measurements of temperature (°C), dissolved oxygen (mg lte NCM4)  because te NCM4) . The potte NCM4) . The rel2) with 2.5 cm length × 2 cm width grid size was overlaid on two areas of each substrate piece to estimate percent areal cover. The area measured was selected haphazardly from the any area of the substrate pieces that were directly exposed to the water column . All live oysters were enumerated and measured for shell height. The pieces were returned after measurements were taken. Up to 20 oysters (> 25 mm) per sampling event were held on ice and brought back to the laboratory to determine presence and severity of the protozoan parasite Perkinsus marinus using the culture method of [Perkinsus marinus causes severe oyster mortalities in the Gulf of Mexico and disease dynamics can be strongly influenced by ENSO cycles; La Niña conditions create higher water temperatures and salinities, which are favorable for P. marinus susceptibility and disease transmission [P. marinus was calculated by dividing the number of infected oysters by the number of oysters sampled. Weighted prevalence, a measure of the relative severity of P. marinus infection in a population, was calculated by multiplying the mean infection intensity of all oysters at a site on a certain date the by prevalence at the same site and date.Divers retrieved at least two representative pieces of substrate (30 to 50 cm diameter) from each constructed reef site in each sampling event  to characterize and quantify the oyster population. A piece of flexible mesh  variables with oyster density and areal coverage of reef construction substrates by oysters. Temporal summaries of mean oyster densities and disease variables were made using annual means, starting from when the reef was constructed . Oyster size-frequency distributions were made by binning all oysters within each sampling year. Univariate analyses and data management were performed using SAS 9.4 .2) were deployed in April 2014 at each sampling site and were filled with concrete cobble (reef construction substrate) at the constructed reef sites and soft sediment at the control sites. The purpose of the trays filled with cobbles was to enable a quantifiable area of the reef to be sampled for resident motile fauna. Placing two sampling trays at each site allowed for each tray to be sampled only once every six months, to minimize the disturbance of removing most motile fauna. to assess differences in habitat provision. Resident motile fauna (> 1mm) were collected quarterly from a single sampling tray at each site starting in July 2014 by divers using a suction sampler (Honda 160cc semi-trash pump with 5.1 cm ports) [-1 HCl.Two sampling trays of dimensions 46 x 61 cm (0.28 mm ports) . Trays re(x+1) transformed and biomass data were fourth root transformed. Similarity percentage analyses (SIMPER) were used to describe taxa that were characteristic of, and different among, treatments and dates. The BIO-ENV procedure was used to correlate community composition with combinations of environmental and climate variables using Spearman-rank correlations [Spearman correlations were calculated to determine the relationship between environmental and climate (ONI) variables with faunal density, biomass, and species richness. Non-metric multidimensional scaling analysis (nMDS)  with a Belations . Temporaelations .This study was carried out in strict accordance with recommendations in the Public Health Service Policy on Humane Care and Use of Laboratory Animals from the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Texas A&M University-Corpus Christi . Field collection of organisms was conducted in accordance with Texas Parks and Wildlife Department Scientific Permit Number SPR-0911-344.-1 and were inversely related to temperature, ranging from 5.7 mg l-1 in July 2014 to 10.9 mg l-1 in January 2018. pH averaged 8.1 ± 0.1 and ranged from 7.8 at bottom waters in January 2019 to 8.7 in July 2014. pH was similar at the top and bottom on all dates aside from in January 2019 when there was vertical salinity stratification.Salinities averaged (mean ± standard error) 21.3 ± 5.6 and fluctuated widely over the course of the study, ranging from 1.8 at the surface in October 2018 to 30.9 in October 2014 . Two notEl Niño conditions were present in the first and second years after reef construction (mid-2014 to mid-2016) and transitioned to La Niña conditions in the third and fourth years after reef construction (mid-2016 to mid-2018); El Niño conditions returned during the fifth year (mid-2018 to 2019). Salinity was negatively correlated with ONI with 2-month , 3- month , 4-month , and 5-month  lags . These c-2, all size classes) in the first year after reef construction, and the lowest mean density  in the fifth year (-2) in the first year, and consistently low in the third through fifth years after reef construction  and lower in years 3–5 (81–96 m-2). Market size (≥ 76 mm) oysters were present at the lowest density (2 ± 2 ind. m-2) in the first year after reef construction and the highest density in the fourth year (167 ± 18 ind. m-2).Oyster density on the constructed reef decreased through time, with the highest mean density  and fifth (798 ± 102 ind. m-2) years after reef construction, and lowest in the third year (240 ± 27 ind. m-2) after reef construction (-2) in the first year and lowest (102 ± 20 ind. m-2) in the third year after reef construction. An extraordinarily large recruitment of Astyris sp. dove snails into the control area caused the isolated peak in resident motile fauna density in May 2015 (2021 ind. m-2). The most abundant species at the constructed sites was the decapod Petrolisthes sp. , and at the control sites was the gastropod Astyris sp.  in the second year and lowest (9.0 ± 2.7 g m-2) in the first year after reef construction (-2) in the third year and lowest (1.2 ± 0.4 g m-2) in the fourth year after reef construction. Presence of large adult M. adina stone crabs drove the peaks in biomass observed in winter samples at constructed reef sites  and control sites  was dominated by the decapod M. adina  in the first year after reef construction , 5-month , and 6-month  lags, and weakly but positively correlated with salinity  . SpeciesResident motile fauna community composition separated into three main groups with at least 55% similarity  composed 82% and 40% of all oysters on Half Moon Reef. Initial recruits likely benefited from clean substrates with relatively little fouling by organisms or sediments that can limit larval settlement of oyster larvae , 60 and M. adina; however, because of their seasonality on the constructed reef, additional research across longer time scales would allow for an improved understanding of these relationships.Interspecific and intraspecific interactions likely also contributed to oyster population dynamics on the constructed reef. High post-settlement mortality of oysters immediately following reef construction appeared to be density dependent as is characteristic of oyster populations when recruitment is high . High deP. marinus parasites was essentially zero until the fourth year after reef construction, which coincides with low ONI values and the occurrence of neutral and La Niña ENSO conditions from mid-year 3 to mid-year 5. La Niña conditions generally correspond to warmer, drier weather conditions in the Gulf of Mexico [P. marinus: higher water temperatures and salinities [P. marinus infection coinciding with the onset of La Niña conditions, the delayed spread to the reef and low intensity at Half Moon Reef compared to other reefs with lower salinities [P. marinus infection at Half Moon Reef.Infection of oysters on the constructed reef by f Mexico , which clinities . Our qualinities . Howeverlinities  means thPerkinsus marinus infections are primarily spread by proximity to feces, pseudofeces, and decomposing tissues of infected oysters [P. marinus to Half Moon Reef, and progression in the constructed reef oyster population. Additional research is warranted to assess the role of geographic isolation in slowing P. marinus disease transmission to constructed reefs as a strategy to reduce the potential for severe infection-related reductions in the oyster population [P. marinus is prevalent on reefs throughout Matagorda Bay and the Gulf of Mexico [P. marinus prevalence and severity [ oysters , and are oysters , althoug oysters . In addipulation . It is lf Mexico , 62, 67,f Mexico . Howeverseverity , 40, 69.Astyris sp. in April 2015 to control sites. As the size frequency distribution shifted toward larger oysters, the constructed reef continued to support distinct faunal assemblages compared to the controls, however, motile fauna densities did not increase over time. The increase in oyster size equates to an increase in habitat complexity. Although more structurally complex habitats may be expected to sustain higher densities of organisms [The constructed reef provided immediate habitat benefits to resident motile fauna, which was associated with ENSO-driven effects on local salinity. In the first year after reef construction, resident motile faunal community composition and species richness were similar to those in the control area, indicating linkages between the constructed reef and surrounding unstructured habitat . Coincidrganisms , 72, denrganisms , 74. It Although typical funding cycles prioritize short-term ecological monitoring, longer term studies are critical for understanding responses to large-scale estuarine variability , 31, manS1 Table(PDF)Click here for additional data file.S2 TableSee (PDF)Click here for additional data file.S3 TableSee (PDF)Click here for additional data file.S4 TableSee (PDF)Click here for additional data file.S1 FigMean temperature , dissolved oxygen  and pH (C) at the surface (< 1 m depth) and bottom (> 1 m depth) at Half Moon Reef, measured quarterly from January 2014-January 2019.(TIF)Click here for additional data file.S2 FigSee (TIF)Click here for additional data file.S3 FigSee (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigBlue shading indicates one standard deviation on either side of the mean. The maximum weighted prevalence possible is 5.(TIF)Click here for additional data file.S6 FigSee (TIF)Click here for additional data file.S7 FigMean (SE) Xanthidae density (A) and biomass (B), and salinity and ONI (C) measured quarterly from January 2014-January 2019. ONI data are from .(TIF)Click here for additional data file.S8 FigMenippe adina density (A) and biomass (B), and salinity and ONI (C) measured quarterly from January 2014-January 2019. ONI data are from [Mean (SE) are from .(TIF)Click here for additional data file.


Saccharomyces cerevisiae, different aspects and similarities in higher eukaryotes are also addressed. We will first focus on how the different phases of the cell cycle affect the RNA polymerases activity and then how RNA polymerases status impacts on cell cycle. A good example of how RNA polymerases functions impact on cell cycle is the ribosome biogenesis process, which needs the coordinated and balanced production of mRNAs and rRNAs synthesized by the three eukaryotic RNA polymerases. Distortions of this balance generates ribosome biogenesis alterations that can impact cell cycle progression. We also pay attention to those cases where specific cell cycle defects generate in response to repressed synthesis of ribosomal proteins or RNA polymerases assembly defects.Eukaryotic life is possible due to the multitude of complex and precise phenomena that take place in the cell. Essential processes like gene transcription, mRNA translation, cell growth, and proliferation, or membrane traffic, among many others, are strictly regulated to ensure functional success. Such systems or vital processes do not work and adjusts independently of each other. It is required to ensure coordination among them which requires communication, or crosstalk, between their different elements through the establishment of complex regulatory networks. Distortion of this coordination affects, not only the specific processes involved, but also the whole cell fate. However, the connection between some systems and cell fate, is not yet very well understood and opens lots of interesting questions. In this review, we focus on the coordination between the function of the three nuclear RNA polymerases and cell cycle progression. Although we mainly focus on the model organism  The eukaryotic cell cycle is controlled by a regulatory network, whose general features are conserved from yeast to humans . It procRNA synthesis in the eukaryotic nucleus is carried out by three multisubunit complexes. RNA polymerase II (RNA pol II) transcribes the vast majority of genes, including all protein coding and many other non-coding RNAs (ncRNAs) such as snRNAs, miRNAs, and snoRNAs. RNA polymerase I (RNA pol I) transcribes ribosomal RNAs (rRNA) as a single polycistronic gene: rRNA 35-47S, which is processed into 3 mature rRNAs: 28S (25S in yeast), 18S and 5.8S. This gene appears repeatedly in all eukaryotes with hundreds of copies arranged in tandem. RNA polymerase III (RNA pol III) transcribes an intermediate number of small, non-coding genes (150–400 different), including 5S rRNA and tRNAs . RNA pol I transcription accounts for almost 60% of global transcription and RNA pol III for around 25%. Of the latter, the 5S rRNA constitutes between 10–15%; and the rest, mostly corresponds to tRNAs. Finally, RNA pol II transcription corresponds to approximately 15% of the total. An important part of this corresponds to RNAs that encode ribosomal proteins .S. cerevisiae yeast genome is transcribed periodically during each cell division cycle. Abundant information is available on the waves of genes expression associated to the different phases , the complex regulatory connection between them, and on the technological approaches to study this phenomenon . This mitotic repression has been observed tructure . We willS. cerevisiae  to tDNA genes; Cdc28 promotes the recruitment of TFIIIC and stimulates the interaction TFIIIC/TFIIIB which directly increases the dynamics of RNA pol III in vivo. Bdp1, a component of the TFIIIB complex, has been proposed as the direct target for Cdc28  by an inhibitory cdc2 mediated phosphorylation . Then, t35S rDNA . More reanaphase .in vivo by strongly inhibiting inosine monophosfate (IMP) dehydrogenase, a rate-limiting enzyme in the novo synthesis in guanine nucleotides  and mycophenolic acid (MPA) interfere with transcription elongation leotides . Our stund mRNAs . Using cnd mRNAs .Ribosome biogenesis is a highly resource-consuming process and, therefore, involves the tight regulation and balanced synthesis of all its components. This complicated pathway requires the coordinated assembly of rRNAs, synthesized by RNA pol I and III, and ribosomal proteins (r-proteins), whose mRNAs are transcribed by RNA pol II. This coordination is critical for an effective utilization of cell resources and requires a balanced function of the RNA pol I, II, and III transcription activities. Thus, the synthesis of rRNAs and r-proteins are two coordinated pathways that lead to efficient ribosome biogenesis [ and refeS. cerevisiae. In this study, most of the repressed genes involved a G1 arrest (nine encoding 60S subunit components and twenty-two encoding r-proteins of the 40S subunit) and only nine repressed genes encoding components of the 60S subunit resulted in a G2/M delay  in both systems the outcome is a G1 arrest; ii) in both organisms, the G1 arrest responses are mediated by a ribosomal stress; iii) in both scenarios the accumulation of free r-proteins (as L5) is essential for coupling to cell cycle. This strong parallelism between the mechanisms responding to nucleolar stress in yeast and metazoan suggests that it reflects a general control strategy extended throughout eukaryotes. However, a major difference between the two systems exists: yeast does not contain p53 or MDM2. The interpretation of these differences has been extensively discussed and other systems exhibiting nucleolar stress without p53 have been described .rpc128-1007 presents a severe defect in RNA pol III assembly as well as an expected reduction in tRNA levels , also led to a delay in the G1 phase of the cell cycle  to the G1 arrest. The different elements that participate in the G1/S transition regulatory network, are good candidates. This knowledge would have a relevant translational potential as more than 50% of human cancers lack functional p53. Identification of new p53-independent response pathways could potentially reveal new therapy strategies for p53-defective cancer.viceversa, a precise knowledge of this complex regulatory interplay will be achieved with a huge translational potential that it has already begun promisingly.In summary, only understanding both regulatory aspects of this crosstalk, how cell cycle modulates transcription and


RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes. This concept extends to the investigation of the transcriptome by RNA sequencing (RNA-Seq), with this field already moving away from simply examining differential gene expression (DGE) of messenger RNA (mRNA), and towards investigation of other species of cellular RNA. Increasingly, non-coding RNA (ncRNA) have been shown to have numerous and varied biological roles. They have been implicated in disease aetiology and pathogenesis3, have high level of evolutionary conservation4 and stability8 and as such are attractive targets of research. Capturing these ncRNA species can, however, be more challenging than capturing mRNA. Many commercially available ncRNA sequencing kits exist, including but not limited to Illumina TruSeq Small RNA Sample kit, PerkinElmer NEXTFLEX Small RNA Library Prep Kit, and NEBNext Small RNA-Seq Kit. All of these methods rely on specifically isolating small RNA transcripts  by size selection and excision of the region of interest from a solid matrix, followed by precipitating the RNA9. While this can allow deep sequencing of RNA within that size range, it is limited in two respects. First, a significant amount of information on the transcriptome is lost through RNA falling outside of the excised size range. Second, the precipitation of RNA from the gel will never be entirely efficient, resulting in unavoidable loss of material. It would be ideal, therefore, to develop a protocol that allows the researcher to simultaneously identify small non-coding RNA transcripts, as well as larger coding- and non-coding RNA, without material loss due to size selection.Over the last 50 years, it has gradually been accepted that previously-dubbed “junk DNA” plays vital biochemical roles in higher organisms. This DNA does not directly code for proteins yet makes up ~ 80% of the human genome. The gathering consensus is that by taking an holistic approach to the genome, that is not just examining protein-coding genes, it is possible gain a better understanding of the whole12. Current RNA-Seq protocols generally follow one of two rRNA removal methods—enrichment of polyadenylated (poly-A) RNA or depletion of ribosomal RNA (rRNA). Poly-A selection relies on the use of oligo dT primers to capture polyadenylated transcripts. This population is largely made up of mRNA, but does not capture all mRNA. Indeed, there is considerable evidence that a significant proportion of brain-derived mRNA is non-polyadenylated, further complicating the use of poly-A selection for investigating brain transcriptomes17. As a result, RNA-Seq data generated by positive capture of polyadenylated RNA do not represent information from non-polyadenylated transcripts, degraded RNA transcripts, and the vast majority of non-coding RNA species. By contrast, depleting total RNA samples of rRNA allows quantification of a more varied population of RNA species. rRNA depletion can be achieved by a variety of means, including dedicated rRNA removal kits. For example, Ribo-Zero Plus (Illumina), captures rRNA by hybridization to complimentary oligonucleotides (ONTs) coupled to magnetic beads that, when precipitated, remove the rRNA from the rest of the RNA. Another method relies on hybridizing rRNA to complementary DNA oligonucleotides. This is followed by RNaseH digestion of the RNA:DNA hybrids . Takara/Clontech SMARTer Stranded Total RNA-Seq kit also includes a proprietary method for rRNA removal that uses ZapR to degrade cDNA originating from rRNA. These methods show different rRNA depletion efficiency, depending on input RNA quality21 and furthermore, some variability in rRNA depletion efficiency has been reported between the implementation of the same protocol at different physical locations18.The removal of ribosomal RNA (rRNA) from RNA samples is a crucial step in RNA-Seq methods. Ribosomal RNA is a considerable roadblock to the detection of other functionally relevant RNA species, as it makes up to 80–90% of total RNA in a cell (by mass)22. With sequencing protocols usually generating in the vicinity of 20–30 million reads per sample, this can equate to 4–6 million reads mapping to rRNA. Better rRNA removal efficiency would result in those reads becoming available for mRNA and non-coding RNA sequence reads, of greater experimental interest to researchers. Furthermore, many of these techniques include multi-step protocols, often requiring precipitation steps (in the case of bead-based systems) and/or digestion or degradation steps. This often results in the loss of RNA material through purification, precipitation, or digestion. Thus the ideal rRNA removal technology would minimise workflow steps, sample handling, and reduce loss of material from precipitation or purification steps.Generally, the literature reports rRNA making up anywhere from 0.5 to 20% of final rRNA-depleted sequencing librariesIn a new development, Qiagen has released the QIAseq FastSelect rRNA removal kit, which utilizes complementary ONTs that bind to rRNA and prevent their reverse transcription to cDNA. The two main draws of this technology are its seamless integration into existing library preparation protocols (a single pipetting step and 14 min protocol), and the fact that it does not require any additional purification, precipitation, or enrichment steps, thereby minimizing sample loss. This considerable reduction in sample handling is key to accurate and efficient detection of especially low-abundance transcripts.24. This is not to say, however, that difficulties do not exist when performing RNA-Seq using samples of lower quality27. Firstly, as noted above, degraded RNA proscribes the use of Poly-A selection for rRNA removal, as the process of degradation renders poly-A selection inefficient, and introduces a strong 3’ gene end bias to sequenced reads29. Second, studies report that RNA samples of low quality  consistently show decreased proportions of mappable reads and a perceived reduction in sample complexity, with fewer highly-expressed genes and an abundance of low-expression genes26.Another perceived hurdle in effective RNA-Seq is quality of the input RNA. While standardized methods exist for assessing RNA quality and the level of RNA degradation , there is no well-defined consensus on what constitutes a sample that is too degraded for RNA-Seq. Any cut-off for sample exclusion used in the literature is, therefore, almost entirely arbitraryAs part of an ongoing investigation into the transcriptome in Alzheimer’s disease, we have developed an end-to-end RNA-Seq workflow that addresses some of the shortcomings of currently available protocols, in particular for rRNA depletion, minimisation of sample loss, and handling of varying input RNA quality. We demonstrate that this protocol is capable of identifying and quantifying both coding and non-coding RNA  simultaneously from both high-quality and degraded RNA samples.To determine whether this protocol  incubation at 16 °C, rather than the recommended 30 °C for 30 min. This markedly increased adapter ligation efficiency 15-fold , with < 50% of cDNA fragments falling under 160 base pairs (bp)  loci, and 5–12% unmapped reads  how many different transcripts can be identified from the RNA-Seq data and (b) what kind of transcripts can be identified.18. Mouse samples identified > 15,000 unique transcripts expressed at greater than one RPKM and an additional 4000 expressed at greater than 0.1 RPKM  for mouse and human RNA samples to normalise the number of unique transcripts detected for sequencing depth and gene length. RPKM was chosen here over the more commonly used transcripts per million reads (TPM) to allow direct comparison to other published reports of genes detectedPKM Fig. a. For thPKM Fig. a. The nunes Fig. a. These Mus musculus GRCm38.100 and Homo sapiens GRCh38.96—as well as piRNA and tRNA from piRNABank and UCSC Genome Browser respectively) is shown in Table 32. Additionally, the method described here, when compared to data obtained from more conventional library construction methods for both mouse34 and human samples35, results in not only a greater number of total unique genes identified, but also a greater proportion of ncRNA biotypes compared to protein-coding mRNA , SERPING1 , KRT5 , and NEUROD6 , all genes that have been reported to be altered in AD38  of Ct values against normalized read counts , in order to control for library size and gene length Fig. . Counts 43. As such, it is possible (even likely) that transcripts associated with small non-coding RNA are overrepresented in our final sequencing libraries. However, this effect is likely consistent across samples and libraries, and would certainly not affect the identification of unique transcripts.RNA sequencing is an ever-evolving technique that offers unique insights into the transcriptome. Current protocols often require the researcher to choose between investigating mRNA (by poly-A selection) or small RNA (by size selection). Either one of these alone, while offering depth of sequencing, misses out on a great deal of information from excluded transcripts. Here, we report a significant advancement in RNA-Seq methodology, a novel method to investigate the whole transcriptome, from the same sample at the same time, using ribosomal-depleted RNA. This approach takes advantage of several existing commercially available kits, with some important alterations to manufacturer’s protocols. This altered workflow resulted in high quality sequencing libraries from input RNA samples of a variety of quality, from both mouse and human tissue. Low quality input RNA had no negative effect on the final library quality. Qiagen FastSelect rRNA removal agent integrated seamlessly into the existing Ion Total RNA-Seq kit v2 library prep protocol and resulted in highly effective depletion of rRNA from the final libraries, even from degraded samples, which is often a drawback of other rRNA removal techniques. A high number of genes were identified in the RNA-Seq data, including transcripts often overlooked by more targeted RNA-Seq protocols  has been previously reportedbwa, Bowtie2, TopHat2, cufflinks, and HISAT2 for read mapping/alignment, software such as kallisto or salmon for direct quantification, differential gene expression tools such as DESeq2, Cuffdiff, and limma, and integrated pipelines such as exceRpt. Several studies have aimed to evaluate the relative sensitivity and accuracy of these analysis tools and pipelines, but for the most part, no one tool or pipeline consistently performed better than the others53. An in-depth discussion of these tools is outside the purview of this report.We recognise the variety of bioinformatics tools available for the analysis of RNA-Seq data. These include 56, knowing the changes that occur in the disease state can aid in understanding the disease, developing diagnostic tools, and hopefully developing new treatments. Since non-coding RNA are such a ubiquitous aspect of cellular function, the same approach, and therefore this method, can be applied to a variety of diseases and research areas.The ability to capture sequencing reads from a wide variety of RNA species, coding and non-coding, is valuable to investigate many aspects of the transcriptome. In our research into Alzheimer’s disease, the ability to take a snapshot of the RNA environment allows us a unique insight into AD pathology, both in the APP/PS1 mouse model and in human post-mortem brain tissue. In particular, since non-coding RNA is being increasingly implicated in the aetiology and pathogenesis of ADAltogether, we believe this workflow may be useful to researchers wishing to investigate the whole transcriptome simultaneously, with effective rRNA depletion, and without complicated and high-loss size selection protocols commonly used for small RNA-Seq, or poly-A selection for mRNA-Seq.57. In this study we utilised a double transgenic model of Alzheimer’s disease  originally sourced from The Jackson Laboratory (https://www.jax.org/strain/004462) and maintained as a colony at the University of Otago breeding facility. Mice were housed under specific pathogen-free conditions in a day-night controlled light cycle, with food and water access ad libitum. Animals underwent no additional procedures prior to their stated use. All mice were genotyped for the presence of human exon-9-deleted variant PSEN1, which co-segregates with the APPswe gene, as previously described58. Male transgenic (tg) and wild-type (wt) littermates at 15 months old (n = 4 per group) were anaesthetised with sodium pentobarbitol and the brains removed into ice cold artificial cerebrospinal fluid solution . The left hippocampus was dissected and snap-frozen on dry ice. All samples were stored at -80 °C until used. RNA extracted from mouse hippocampi is henceforth referred to by the identifier “Sample #”.All animal use was compliant with the New Zealand Animal Welfare Act 1991 and performed under guidelines and approval of the University of Otago Animal Ethics Committee . The reporting in this manuscript follows the recommendations in the Animal Research: Reporting on In Vivo Experiments (ARRIVE) guidelinesUse of human tissue was approved by and compliant with the guidelines of the New Zealand Health and Disability Ethics Committee (14/STH/20/AM07), the Human Research Ethics Committee of The University of Melbourne (1545740—for patient tissue banking and consent), and the Victorian Institute of Forensic Medicine EC 18–2019). Informed consent was obtained from all donors. Post-mortem middle temporal gyrus (MTG) samples were received from the Victorian Brain Bank (VBB). Age-matched healthy control brains  were defined as free from Alzheimer’s Disease (AD) lesions with numbers of plaques and tangles below the cut-off values for a neuropathological diagnosis of AD (NIA Reagan criteria). No other neurological diseases were present. Alzheimer’s disease brains  met the standard criteria for Alzheimer’s disease neuropathological diagnosis. There were no significant differences between the ages of the two groups . Patient sex was self-reported. All samples were stored at −80 °C until used. RNA extracted from human MTG samples is henceforth referred to by the identifier “Patient #”. Patient demographics and case information are available in Table . InformeTotal RNA was extracted from previously-frozen tissue using the mirVana™ PARIS™ RNA isolation kit , according to the manufacturer’s instructions. The concentration and purity were determined by both spectrophotometry  and capillary electrophoresis .Except where explicitly stated, all samples, regardless of species or group of origin, were treated identically. Sequencing libraries were prepared for Ion Proton using the Ion Total RNA-Seq kit v2  largely following manufacturer’s instructions. Total RNA (500 ng) was used as input to the Ion Total RNA-Seq kit v2 (356 ng input was used instead of 500 ng for Patient 1 due to low RNA yield), to which was added 1 μL of 1:100 ERCC Spike-In Mix 1 , commonly employed to control for cross-sample variation in library preparation. RNA fragmentation by RNase III was performed at 37 °C. The fragmentation time was optimised to 8 min for the mouse RNA, and 1 min for the human RNA. This will vary depending on quality and integrity of the input RNA material. The resulting fragmented RNA was purified using the Magnetic Bead Cleanup Module , and purified RNA eluted in 13 μL nuclease-free water.Ligation of Ion adapters  was performed using 3 μL of the eluted purified fragmented RNA, added to 2 μL Ion Adapter Mix v2 and 3 μL Hybridization solution, and incubated in a thermal cycler at 65 °C for 10 min followed by 30 °C for 5 min. To this hybridization reaction was added 10 μL 2× Ligation Buffer and 2 μL Ligation Enzyme Mix, and incubated at 16 °C for 16 h in a thermal cycler. Following ligation, reverse transcription (RT) and rRNA removal was performed simultaneously as follows. RT master mix was prepared on ice . QIAseq FastSelect rRNA removal agent  consists of ONTs complementary to ribosomal RNA sequences. These ONTs, when bound to rRNA sequences, prevent reverse transcription. The master mix was added to the ligation reaction, and incubated at 70 °C for 10 min, followed by a step-wise cooldown . This step is necessary for the oligonucleotides in the FastSelect rRNA removal agent to bind rRNA fragments and prevent reverse transcription. Finally, 4 μL 10× Superscript Enzyme Mix was added to each reaction and the reactions incubated at 42 °C for 30 min.The resulting cDNA was purified using the Magnetic Bead Cleanup Kit, and eluted in 12 μL nuclease-free water. In order to amplify the cDNA, 6 μL of this elution was added to a master mix of 45 μL Platinum PCR Supermix, 1 μL Ion Xpress 3’ Barcode Primer, and 1 μL Ion Xpress RNA Barcode BC## . This mixture was amplified in a thermal cycler for 14 cycles . The amplified cDNA was purified again using the Magnetic Bead Cleanup Kit, and analysed by capillary electrophoresis .The prepared sequencing libraries were diluted to equimolar concentrations of 100 pmol/L for pooling. Emulsion PCR was performed with the Ion OneTouch™ 2 system (Invitrogen) using the Ion PI™ Hi-Q™ OT2 200 kit  according to the manufacturer’s instructions. The four pairs of mouse samples (tg and wt) were processed simultaneously end-to-end, as were the seven human MTG samples. Libraries were sequenced on Ion PI™ v3 chips , prepared using the Ion PI™ HiQ™ Seq 200 kit . The mouse samples of two pools of mixed barcoded libraries were sequenced on two Ion PI v3 chips (2 wt and 2 tg per chip), avoiding the use of all sequential barcodes on the same chip. Similarly, human MTG libraries were sequenced in two pools of mixed barcoded libraries—one contained a pool of four samples (2 AD and 2 control), the other a pool of three samples (1 AD and 2 control).Hprt (Assay ID: 03024075_m1), Cst7 (00438351_m1), Tyrobp (00449152_m1), c-Fos (00487425_m1), Trem2 (04209424_m1). The reactions were amplified on the Applied Biosystems ViiA 7 system as follows: Hold 50 °C 2 min, hold 95 °C 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min.For gene expression qRT-PCR, using 350 ng starting total RNA input from mouse samples, cDNA was generated using SuperScript IV First Strand Synthesis System  per manufacturer’s instructions, utilizing priming by random hexamers. Of this cDNA, a 1:25 dilution was used for the qRT-PCR reaction, which was performed using TaqMan Gene Expression Master Mix , with the following TaqMan gene primers: Mouse For miRNA, 10 ng of total RNA from mouse samples was used. cDNA was generated using the TaqMan MicroRNA Reverse Transcription Kit  according to manufacturer’s instructions. The qRT-PCR reactions were prepared using TaqMan Universal PCR Master Mix , with the following TaqMan miRNA primers: miR-34a-5p (Assay ID: 000426), miR-34c-5p (000428), miR-129–1-3p (002298), miR-210-3p (000512). The reactions were amplified on a Applied Biosystems ViiA 7 system as follows: Hold 50 °C 2 min, hold 95 °C 10 min, and 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Genes and miRNA were chosen as a mix of housekeeping genes, genes that are known to be changed from the literature, and some genes of interest whose expression in APP/PS1 mice is unknown.MicroRNA and mRNA qRT-PCR data were processed separately to account for differing input RNA amounts, and in each case, raw Ct values were used for analysis.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)59. Adapter sequences were trimmed from reads using AdapterRemoval v2.1.7 60. Reads were then trimmed for quality using Trimmomatic v0.38 (http://www.usadellab.org/cms/?page=trimmomatic)61 using a 5-base sliding window, cutting when the average quality per base drops below 20, and dropping reads less than 17 bases long.Data from each barcoded library were separated into different data files automatically on the Ion Torrent Suite version 5.4 . The Ion Torrent Suite was also used for analysis of ERCC Spike-In controls. Sequence read quality was evaluated using FastQC v0.11.5 (Mus musculus GRCm38.95 reference genome (available on the Ensembl website: http://www.ensembl.org/info/data/ftp/index.html) and human RNA reads to the Homo sapiens GRCh38.96 reference genome using STAR v2.5.4b 62. Reference .gtf files for RNA biotypes  were extracted from the Mus musculus GRCm38.95 and Homo sapiens GRCh38.96 annotation files using the grep command.Mouse RNA reads were aligned to the https://github.com/rajewsky-lab/mirdeep2)63.MicroRNA (miRNA) were quantified from aligned counts using miRDeep2 v0.1.2 (http://pirnabank.ibab.ac.in)64.Piwi-interacting RNA (piRNA) sequences were obtained from piRNABank (http://genome.ucsc.edu).Sequences for tRNA were obtained from the UCSC Genome Browser  were also performed using R. Additional analysis and data visualisation performed using SeqMonk v1.45.1 (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/).The following packages were used to aid data handling and visualisation: Supplementary Information.


The PODEn were blended with diesel fuel at three different ratios . The test results showed that the PODEn could optimize the combustion process of diesel engines that met the China VI emission standards, effectively improve the thermal efficiencies of diesel engines, and reduce the emissions of hydrocarbon (HC), carbon monoxide (CO), and soot. With an increase in the PODEn blending ratio, the peak values of the in-cylinder pressure, average in-cylinder temperature, and instantaneous heat release rate gradually decreased, and each peak progressively moved forward. As the start of combustion gradually moved forward, the combustion duration was shortened by 0.7–2.8°CA, the heat release process became more concentrated, and the effective thermal efficiency was increased by up to 2.57%. The effective fuel consumption gradually increased, yet the equivalent effective fuel consumption gradually decreased, with the largest drop being as high as 4.55%. The nitrogen oxides (NOx) emission increased slightly, and the emissions of HC, CO, and soot gradually decreased. The emissions of CO and soot declined significantly under high-speed and high-load conditions, with the highest reductions reaching 66.2% and 76.3%, respectively.To study the influence of diesel fuel blended with polyoxymethylene dimethyl ethers (PODE Shenzhen took the lead in implementing the China VI a emission standard at the end of 2018, while Shanghai, Guangzhou, Tianjin, and other cities began to enforce the China VI b emission standard on July 1, 2019. Formulated based on both the European VI c and EPA Tier 3 emission standards in terms of the requirements for the OBD and the evaporative emissions, the China VI emission standard is known as the strictest emission standard in the world.To address the environmental pollution and energy crises prevailing worldwide, countries around the world continue to implement increasingly stringent regulations related to the discharge of pollutants. The EU began to implement European VI, VI b, and VI c emission standards in January 2013, September 2014, and September 2017, respectively. Compared with the European V emission standard, the European VI emission standard contained added emission limits for the particle number (PN), and the on board diagnostics (OBD) limit was even more stringent. Proposed by the Environmental Protection Agency (EPA) in March 2013, the Tier 3 fuel and vehicle standards will be implemented in phases from 2017 to 2025. The EPA’s Tier 3 limits the total emissions of “NMOG (nonmethane organic gas) + NO3. Facing increasingly strict emissions regulations and a gradually growing energy crisis, investigators are pursuing the efficient and clean combustion of diesel. The optimization of fuel characteristics by blending oxygenated fuels is an effective way to achieve this objective6. Once the oxygenated fuel is blended with diesel, the physical and chemical properties of the blended fuel, e.g., the oxygen content, cetane number, and latent heat of evaporation, are changed accordingly, thereby altering the spray characteristics, combustion features, power performance, and emission characteristics of diesel engines11. Alcohols , ethers , and esters  are currently the primary oxygenated fuels blended in diesel. Oxygenated fuels such as alcohols, ethers, and esters are strongly competitive for improving the combustion process and pollutant emissions of diesel engines. However, significantly different from diesel in terms of overall physical and chemical properties, these fuels are restricted in various ways in practical applications18. Therefore, it is of great practical significance to find oxygenated fuels with similar physical and chemical properties to those of diesel.Diesel engines are widely used in transportation, agriculture, and industryn) are chemically expressed as CH3O(CH2O)nCH3 . With physical and chemical properties fairly similar to those of diesel, PODEn are liquid at 20 °C and they can be miscible with diesel in any proportion. With a high oxygen content (45.2–51%), a large cetane number (above 63), and without a C–C bond in their molecular structure, PODEn are able to effectively reduce the soot emissions during the combustion process of diesel engines22. In addition, the production costs of PODEn are close to those of diesel, and with the advancement of production technology and the expanded production scale of PODEn, the production costs of PODEn are expected to be further reduced, which makes it possible for PODEn to be applied to diesel engines on a large scale. Hence, PODEn are a type of clean alternative fuel that exhibit excellent performances and great potential30.As a new type of oxygenated additive for diesel, polyoxymethylene dimethyl ethers  emissions by 18%, while if pure PODEn were used, the PM emissions could be decreased by 77%, meeting the Euro IV emission standard. By blending 7.5% PODEn into the diesel used on a Euro III light-duty diesel engine, Pellegrini et al.32 found that compared with pure diesel, diesel blended with PODEn reduced the PM emissions and the exhaust smoke by 13% and 32%, respectively, with increased emissions of polycyclic aromatic hydrocarbons (PAHs) and PM with particle sizes smaller than 30 nm. Liu et al.33 burned diesel blended with 15% and 25% PODEn in a Euro IV diesel engine, indicating that blending PODEn could significantly improve the trade-off relationship between the emissions of NOx and the soot of the diesel engine. Under the WHSC test cycle, with the NOx emissions controlled at 2.7 g/(kW·h), the soot emissions met the Euro VI emission standard. Liu et al.34 studied the effect of the PODEn blending ratio on the performance of a diesel engine with China IV emission standards, and the results showed that as the PODEn blending ratio increased, the ignition delay period shortened, the in-cylinder temperature gradually increased, the soot emission was reduced by up to 47.6%, and the effective thermal efficiency could be increased by up to 3.4%. Wang et al.36 explored the impact of blending PODEn on the combustion and emission characteristics of light-duty and heavy-duty diesel engines, and the results indicated that blending PODEn prolonged the ignition delay period and increased the peak heat release of the main injection. When 20% PODEn were blended, the soot and CO emissions of a light-duty diesel engine could be reduced by 90%, the PM emissions of a heavy-duty diesel engine during the ESC (European Stationary Cycle) test cycle were reduced by 36.2%, and the effective thermal efficiency was increased by 0.85%. Moreover, to further improve the physical and chemical properties of fuels, Huang et al.37 and Chen et al.38 studied a ternary fuel mixture of diesel, PODEn, and high-octane fuel, and the results indicated that blending a certain proportion of PODEn in the diesel or high-octane fuel could reduce the emissions of NOx and soot.Recently, a number of studies have been carried out to study the effects of blending PODEn can effectively improve the combustion and emission characteristics of diesel engines, especially when it comes to reducing the emissions of soot and PM. It also has been shown that the same fuel has different combustion and emission performances in different types of diesel engines39. Likewise, there are certain differences in the effects of blending the same proportion of PODEn into the diesel fuel on the combustion characteristics of different types or models of engines as well as the effects on the emissions of NOx, soot, and CO41. Many studies have confirmed the feasibility of adding PODEn to diesel engines, yet there are few experimental studies regarding PODEn blending with the diesel used in diesel engines that meet the China VI emission standards. Given that China is about to fully implement the China VI emission standards, it is necessary to carry out such studies on PODEn/diesel blended fuels with China VI diesel engines. To this end, with a supercharged, intercooled, high-pressure common-rail diesel engine with the China VI emission standards as the research subject, the influences of the PODEn blending ratio on the combustion and emission characteristics as well as the fuel economy of a heavy-duty diesel engine were investigated in this study. The results will provide empirical support for optimizing the fuel characteristics and combustion process of PODEn as an alternative fuel for China VI emission standards and the revision of the release control and post-treatment strategies.Therefore, blending PODEA Weichai Power WP12.460 China VI heavy-duty diesel engine, an in-line 6-cylinder, supercharged, intercooled, electronically controlled high-pressure common-rail diesel engine was used in the tests. The technical specifications of the engine are shown in Table x, HC, and CO, and the AVL 415SE smoke meter was used to measure the exhaust soot. The Siemens FC3000 mass flow meter was used to measure the fuel consumption.The testing apparatus was mainly composed of an electric eddy current dynamometer, a combustion analyzer, a mass flow meter, a coolant and oil thermostatic system, and an exhaust gas analyzer. Figure n used in the study were industrial PODEn with a purity of 99.9% that were produced by Shandong Yuhuang Chemical (Group) Co., Ltd., and they were primarily composed of PODE2, PODE3, and PODE4 with mass fractions of 2.553%, 88.9%, and 8.48%, respectively. The test diesel was 0# commercial diesel. In this test, three kinds of PODEn/diesel blended fuels were prepared, with PODEn volume percentages of 10%, 20%, and 30%, which were denoted as P10D90, P20D80, and P30D70, respectively, and the pure diesel was denoted as P0D100. The physical and chemical properties of the fuel are shown in Table 42.The PODE−1, were tested, with brake mean effective pressure (BMEP) values of 0.38 MPa, 0.76 MPa, 1.14 MPa, and 1.52 MPa. During the test, except for the fuel blend, the fuel injection strategy and the other control parameters remained constant, and the exhaust gas recirculation (EGR) valve remained closed. Under different operating conditions, the fuel injection strategy was the two-stage injection of the original machine: “pre-injection + main injection,” and the injection time and pressure were based on those of the original machine. Both the cooling water temperature and the oil temperature were controlled at about 80 °C. The ambient temperature for the test was 22 °C ± 3 °C, and the atmospheric humidity was 10% ± 3%.Four different loads under two speeds, i.e., n = 1300 and 2000 r·minPrior to the tests, all the instruments and the equipment were calibrated to ensure the reliability of the test and the accuracy of the test results. During the test, each operating point was repeatedly measured to eliminate uncertainty. After the engine was operating stably for 5 min, the fuel consumption was measured three times, and the average value was taken as the test result. The combustion analyzer collected the combustion characteristics data for 200 cycles and the average value of the data was taken for analysis. The exhaust gas analyzer and the smoke meter continued to be measured for 1 min, and with the invalid values eliminated, the average values were taken as the test results.n blending ratios when n = 2000 r·min−1, BMEP = 0.38 MPa, and BMEP = 1.14 MPa. Since the fuel injection consisted of two stages, the exothermic process was divided into two phases: pre-injection and main-injection heat release. Since the pre-injection lasted for a relatively short time, the amounts of fuel injection and heat released were small, and the blending of the PODEn exerted no significant effect on the peak value of the pre-injection heat release rate. However, since the duration of the main-injection phase was longer, the peak value of the main-injection heat release rate decreased with the increase in the PODEn blending ratio, with the peak phase gradually moving forward. Because the PODEn had a lower heat value than diesel, under the injection conditions controlled by the electronic control unit (ECU) of the original engine, the blended fuel emitted less heat than the diesel with a declining peak value of the heat release rate. Moreover, the PODEn had a large cetane number, which shortened the ignition delay period of the blended fuel and advanced the ignition timing.Figure n blending ratio increased, the maximum in-cylinder pressure decreased, with the corresponding phase of the peak in-cylinder pressure gradually moving forward. Due to the large cetane number, great self-ignitability, and good volatility of the PODEn, the ignition delay period of the blended fuel became shorter, with the corresponding phase of the peak in-cylinder pressure moving forward. The peak in-cylinder pressure decreased due to the following two factors. First, the lower heat value of the PODEn reduced the heat value of the blended fuel and the heat release during the main-injection phase. Second, the shortening of the ignition delay period of the blended fuel caused the start of combustion (SOC) to occur earlier, shortened the mixing time of the fuel and the air, and reduced the premixed combustion ratio.Figure n blending ratios when n = 2,000 r·min−1, BMEP = 0.38 MPa, and BMEP = 1.14 MPa. As the PODEn blending ratio increased, the peak in-cylinder temperature dropped and the curve shifted forward. Since the latent heat of vaporization of the blended fuel increased after the blending of the PODEn, after the fuel was injected into the cylinder, part of the heat was absorbed, resulting in a relatively significant temperature drop, which led to a decrease in the peak in-cylinder temperature. However, due to the large cetane number of the PODEn, the combustion heat release of the blended fuel was advanced, thereby shifting the peak in-cylinder temperature forward.Figure n blending ratios when n = 2,000 r·min−1, BMEP = 0.38 MPa, and BMEP = 1.14 MPa. In this study, the crank angle corresponding to a cumulative heat release rate of 10% (CA10) was defined as the SOC, the crank angle corresponding to a cumulative heat release rate of 90% (CA90) was defined as the end of combustion (EOC), and the crank angle between the SOC and the EOC was defined as the combustion duration. With BMEP = 1.14 MPa, compared with P0D100, when P10D90, P20D80, and P30D70 were used, the CA10 was advanced by 0.5°CA, 0.8°CA, and 1.0°CA, respectively, and the CA90 was advanced by 1.2°CA, 2.6°CA, and 3.8°CA, respectively, and the combustion duration was shortened by 0.7°CA, 1.8°CA, and 2.8°CA, respectively. This indicated that as the PODEn blending ratio increased, the combustion duration was gradually shortened, and the heat release was more concentrated, which was conducive to improving the thermal efficiency of the engine.Figure n blending ratios when n = 2,000 r·min−1, BMEP = 0.38 MPa, and BMEP = 1.14 MPa. The cumulative heat release decreased after the PODEn were blended, and with the increase in the blending ratio, the cumulative heat dropped more significantly. At a BMEP of 1.14 MPa, when P0D100, P10D90, P20D80, and P30D70 were used, the maximum cumulative amounts of heat released were 4,959.9 J, 4,863.6 J, 4,789.1 J, and 4,733.6 J, respectively. Compared with P0D100, the cumulative amounts of heat released were reduced by 1.94%, 3.44%, and 4.56% when P10D90, P20D80, and P30D70 were burned, respectively. At the same speed and load, the effective power values of the engine were equal for all the blends. The lower the cumulative heat release was, the higher the thermal efficiency of the engine was. This indicated that blending PODEn helped to improve the effective thermal efficiency of the engine. This conclusion is further discussed in “Figure be and the effective thermal efficiency ηet are two important parameters that characterize the fuel economy of the engine, and they are critical indicators for measuring the engine performance. Because the low heat value of PODEn differs greatly from that of diesel, directly comparing the fuel consumption of the fuels could not accurately reflect the total heat consumed by the fuels. Therefore, the concept of equivalent effective fuel consumption beq was introduced; i.e., the total heat value consumed by the PODEn/diesel blended fuel was converted to the diesel consumption corresponding to an equivalent heat value.The effective fuel consumption be with different PODEn blending ratios are shown in Fig. n blending ratio. Due to the low heat value of the PODEn, the heat values of the blended fuels P10D90, P20D80, and P30D70 were 93.6%, 87.3%, and 81.3% of that of diesel, respectively, and the amount of heat released by combustion within the unit mass of the blended fuel was reduced. Without changing the fuel injection strategy, the output torques corresponding to the same BMEP operating condition were the same. Hence, it was important to increase the injection amount of the blended fuel, resulting in an increase in the effective fuel consumption.The variations of the effective fuel consumption beq and the effective thermal efficiency ηet of the engine with different PODEn blending ratios. Given the same load, with the increase in the PODEn blending ratio, the equivalent effective fuel consumption gradually decreased, and the effective thermal efficiency increased significantly. With n = 2000 r·min−1 and BMEP = 1.52 MPa, compared with P0D100, the equivalent effective fuel consumptions of P10D90, P20D80, and P30D70 decreased by 2.20%, 3.26%, and 4.55%, respectively, with the effective thermal efficiencies increasing by 0.59%, 1.48%, and 2.57%, respectively. Due to the large cetane number of the PODEn, once the PODEn were blended, the ignition delay period was shortened, and the combustion advanced with the heat release and the peak in-cylinder pressure in the main-injection phase being closer to the top dead center , the in-cylinder temperature was low. As the PODEn blending ratio increased, the in-cylinder temperature further declined , the in-cylinder temperature increased rapidly. Although blending PODEn decreased the in-cylinder temperature, the in-cylinder temperature remained high  values of P10D90, P20D80, and P30D70 decreased by 35.1%, 62.8%, and 76.3% compared to that of P0D100. Unlike diesel, the PODEn exhibited great volatility and a low viscosity and boiling point. Therefore, blending PODEn contributed to the evaporation and atomization of the fuel, improved the homogeneity of the mixed gas, and effectively avoided fuel cracking caused by an uneven gas mixture under high-temperature and hypoxia conditions44. Furthermore, blending PODEn increased the oxygen content in the blended fuel, which supplied the oxygen during the combustion process, thereby improving the combustion conditions in some areas during the diffusion combustion process of the diesel engine and promoting the post-oxidation process of the soot. In particular, the PODEn molecules did not contain C–C bonds, which could significantly reduce the amounts of olefins and PAHs generated during the combustion reaction, thereby lowering the amount of soot. The above factors greatly improved the soot emission of the diesel engine after the blending of PODEn. Furthermore, under high speeds and loads, the soot emission decreased more substantially with the increase in the PODEn blending ratio. With increases in the speed and the load, the combustion temperature in the cylinder increased, and the high-temperature hypoxic areas increased. Hence, due to the high oxygen content and excellent volatility, the PODEn had a more evident effect on improving the combustion conditions when the gas mixture had an excessively high concentration.Figure n, with a high oxygen content and a large cetane number, are a promising additive for diesel. In this study, a series of tests were performed on a supercharged intercooled high-pressure common-rail diesel engine powered by diesel fuel blended with PODEn at different ratios, i.e., 10%, 20%, and 30%, to explore the influences of PODEn blending on the combustion and emission characteristics as well as the fuel economies of a heavy-duty diesel engine that met the China VI emission standards. The following conclusions were obtained:n significantly affected the combustion characteristics of the diesel engine. As the PODEn blending ratio increased, the peak in-cylinder pressure and the average in-cylinder temperature gradually decreased, and the phase corresponding to the peak gradually shifted forward. The peak heat release rate in the pre-injection stage varied slightly, and the peak heat release rate in the main-injection stage gradually decreased, with the phase corresponding to the peak gradually moving forward.Blending PODEn blending ratio increased, the start of the combustion gradually shifted forward, the duration of combustion was shortened by 0.7–2.8°CA, the heat release became more concentrated, the combustion heat release of the fuel was closer to the top dead center, and the maximum effective thermal efficiency could be improved by 2.57%. After blending with PODEn, the effective fuel consumption increased, but the equivalent effective fuel consumption decreased significantly, with the maximum reduction reaching 4.55%.As the PODEn into diesel could effectively improve the emission characteristics of the diesel engines. Particularly under high-speed and high-load conditions, the PODEn, due to its high oxygen content and great volatility, improved the oxygen concentration in the cylinder and the uniformity of the combustible mixture, which effectively inhibited the generation of soot and reduced the emission of soot by as much as 76.3%. With the increase in the PODEn blending ratio, the emission of NOx increased slightly, but the emissions of HC and CO gradually decreased, with the highest reduction of the CO emissions reaching 66.2%.Blending PODEPODE


Bunyavirales members. Monodispersed L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex.Arenaviruses initiate infection by delivering a transcriptionally competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA  template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication, new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA and affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase—which copies the viral genome segments and synthesizes all four viral mRNAs—bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery and thus advancing antiviral strategies targeting this essential complex. Mammarenavirus, family Arenaviridae, order Bunyavirales) are responsible for sporadic outbreaks of rodent-borne hemorrhagic fevers in humans. Notable examples include Lassa fever virus (LASV), which is responsible for an estimated 5,000 to 10,000 deaths, annually  RNA-dependent RNA polymerase (RdRP) and matrix protein (Z) within the larger RNA segment, and an RNA-binding nucleoprotein (NP) and glycoprotein precursor (GPC) within the smaller RNA segment . The gen17–7GpppN cap structures of host mRNAs for L-mediated endonucleolytic cleavage and priming of transcription initiation  of RNA via positively charged residues in a groove formed by the N-terminal domain –53. RNA –in vitro  fold with specificity for immunostimulatory double-stranded RNAs (dsRNAs) of viral origin , 60–63. 60–64–in vitro .As the sole protective coating for viral genomic and antigenomic RNA templates, the functions of negative-strand RNA virus (NSV) nucleoproteins are tightly linked to the activities of the viral polymerase. An affinity balance is required between polymerase-nucleoprotein and nucleoprotein-RNA binding and the ability of the polymerase to displace nucleoprotein subunits from the RNA during elongation. Arenavirus L can interact with NP indirectly via vRNA scaffolds and by direct binding –73. Howe–The subcellular complexes formed by L and NP during arenavirus infections remain unknown. Currently, our structural understanding of the arenavirus RTC is limited to electron microscopy (EM) observations of virion-associated RNPs. Here, we provide insight into the diverse intracellular structures formed by the polymerase and nucleoprotein of Machupo virus by reconstitution of functional MACV RNPs in hamster cells. MACV L and NP with engineered affinity tags assemble to form replication- and transcription-competent RNPs within cells. These complexes were purified and characterized by negative-stain EM. The MACV complexes adopt diverse structures ranging from monomeric L-NP particles, homotrimeric NP ring-like assemblies, and higher-order flexible filamentous nucleocapsids. These findings provide the first structural insights into the subcellular organization of actively replicating arenavirus L and NP subunits. Moreover, these findings highlight the utility of such reconstituted RNP systems for studying the replication machinery of highly pathogenic mammarenaviruses.trans, eGFP production is an indicator of L-mediated RNA synthesis activity  MACV L were modified by incorporating Flag (DYKDDDDK) and 6×His (HHHHHH) affinity tags at the C terminus of L for antibody and metal affinity protein purification, respectively -driven minireplicon system to incorporate affinity tag epitopes into the L polypeptide  Fig. 1BFig. 1B. activity . Insect ity tags , 74, 75.ectively . Prelimiectively . These rtrans with the minireplicon RNA, as has been done for other arenavirus replicon systems (S-transferase (GST)-tagged MACV Z protein (GKPIPNPLLGLDST) resulted in successful RNP replication and transcription, opening potential avenues for further affinity purification or in-cell imaging of RTCs  and transacting Pol II-expressed L and NP (pL and pNP). The mass of pL transfected into cells was tested over a range of 0 to 8 μg. MACV L was immunoprecipitated from lysates of transfected cells using a rabbit polyclonal antibody raised against recombinant L purified from insect cells. The relative abundance of immunoprecipitated and cell lysate-associated L was determined by SDS-PAGE, Coomassie blue staining, and immunoblotting. Immunoprecipitated MACV L reached saturation at approximately 6 μg of transfected pL plasmid . ConsistWT) gradually accumulated eGFP over 72 h, and this was absent in cells expressing the RdRP catalytic site mutant of L  (DYKDDDDK(×3) peptide for Ni affinity or anti-Flag purification approaches, respectively.The copurifying L and NP samples isolated using anti-MACV L polyclonal antibodies  had fewe; pLSDD) . Cell lyDYKDDDDK(×3) soluble peptide yielded highly pure samples containing both L and NP  analyses of peptide-eluted samples from wild-type L-expressing cells , lane 2 During purification optimization, we tested variations of buffer conditions for elution of the L-NP complex. Final elutions performed at neutral pH (7.0) yielded predominantly monodispersed particles, as observed by negative-stain EM  and 6A. 17–The MACV RNPs are categorized into three principal architectures: (i) medium to large circularized complexes approximately 100 to 250 nm in diameter, (ii) medium-sized linear complexes approximately 100 to 200 nm long, or (iii) smaller ring-like complexes 30 to 40 nm in diameter decorated with peripheral globular particles . The med17–84–82–In addition to the decorated ring-like structures mentioned above, we also documented the formation of smaller circular structures present in electron micrographs of the L-NP complexes purified by Ni affinity chromatography  and 8A. Here, we report a new approach for isolating and characterizing the principal core replication machinery of Machupo virus. We utilized a cell-based minireplicon system for MACV to assess the effects of affinity tags introduced into L and NP on replication and gene expression. Affinity purification of MACV L has yielded unexpected complexes of NP-bound L and RNP-like filamentous structures . These cConcrete data and conclusions on the interactions between arenavirus polymerase and nucleoproteins are lacking. It is currently not well understood whether the L-NP interaction is direct, RNA mediated, or both at times. Previous efforts characterizing interactions between L and NP from LCMV and LASV demonstrated that virus-specific RNA was required for copurification of L and NP, suggesting an indirect interaction mediated by RNA binding of both proteins . In contin vitro to yield monodispersed single particles  . The larThe smaller ring-like RNPs  could co98–All RNPs appear to be sparsely decorated with globular structures along the periphery of the flexible filaments , 81–85. 81–We also show that expression of the MACV Z protein can inhibit L-mediated replication and transcription of the minireplicon . The mul0-P complexes for nonsegmented NSVs  supplemented with glucose, l-glutamine, sodium pyruvate, and 10% fetal bovine serum (FBS). Spodoptera frugiperda insect cells (Sf21) were maintained in spinner flasks at 27°C in TC100 medium (Gibco) supplemented with 10% FBS and used for expression of apo-MACV L as described previously   to generate the pLSDD plasmids. Additionally, wild-type MACV NP  was cloned into empty pGEM3 and pCAGGS vectors (pNP) for the T7 RNAP and Pol II experiments, respectively, and supplemented during transfections with the L-expressing (pL) and minireplicon RNA-expressing (pMG) vectors described below (GKPIPNPLLGLDST) for the experiments performed in The original pGEM3-based vector for T7 RNAP-driven expression of wild-type MACV L   was modi40450.1) . New coderiments  and 4. Ted below . A pcDNAThe MACV minireplicon assays were performed essentially as described previously, with minor modifications , 74. BSRFull-length MACV L was purified from insect cells as described previously , 75 and 2HPO4, 1.47 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl [pH 7.4]), and resuspended in 0.75 ml of cold 1× PBS and kept on ice. One-third of the total resuspended cell volume (0.25 ml) was mixed 1:1 (vol/vol) with cold 2× lysis buffer  and incubated on a tube rotator at 4°C for 30 min. After lysis, the ∼0.5-ml crude lysate was centrifuged at maximum speed in a refrigerated tabletop centrifuge for 10 min. The supernatant was collected and used for subsequent MACV L immunoprecipitation and immunoblotting. All buffers were prepared with a supplemented broad-spectrum protease inhibitor cocktail (Sigma).Following RNP expression, confluent BSR-T7 cells were removed from each 60-mm dish using sterile cell scrapers, washed twice with 1× phosphate-buffered saline  from the steps described above was incubated with 5 μl of 10% SDS , 5 μl of 100× (10 mg/ml) bovine serum albumin (BSA), and 1 μl of anti-MACV L antiserum . The lysate-antiserum mixture was incubated on a tube rotator at 4°C for 2 h to allow antibody-L binding. The lysate-antibody mixture was then incubated overnight with 25 μl of prewashed protein A-agarose resin (Sigma). After overnight incubation, the protein A resin was pelleted and washed twice with cold 1× PBS supplemented with 0.1% SDS. The final ∼25 μl of washed resin was mixed 1:1 with 1× PBS and used for subsequent immunoblotting analyses.For immunoblotting analyses, 12.5 μl of each input lysate or resuspended immunoprecipitated sample was mixed with 12.5 μl of 2× SDS loading buffer  and boiled for 5 min. Sample tubes were centrifuged at maximum speed for 1 min to collect condensation, and 10 μl of each sample was loaded onto a 7.5% polyacrylamide–SDS gel. Gels were run in duplicate for Coomassie staining and immunoblotting using standard equipment (Bio-Rad). For immunoblotting, total separated proteins were transferred from polyacrylamide gels onto hydrated nitrocellulose membranes at 4°C at 300 mA for 2 h. Membranes were incubated by gentle rocking at room temperature for 1 h in a blocking solution of 5% (wt/vol) dried milk in 1× Tris-buffered saline  with 0.05% (vol/vol) Tween 20 (TBST). Blocking solution was removed, and membranes were incubated overnight in a primary antibody solution of anti-MACV L  with 1% (wt/vol) supplemented dried milk (milk-TBST). After overnight incubation, the primary antibody was removed, and blots were washed three times with 1× TBST. A secondary antibody solution of goat anti-rabbit horseradish peroxidase (HRP) conjugate (Invitrogen) diluted 1:10,000 in milk-TBST was added to the blots, and the mixture was incubated at room temperature for 1 h. The secondary solution was removed, and the blots were washed twice with 1× TBST and once with 1× TBS to remove trace amounts of detergent. Blots were developed with standard HRP chemiluminescence substrate reagents (Pierce) and imaged using an Amersham Imager 600 (GE Life Sciences).DYKDDDDK antibody-conjugated affinity resin (Pierce) or 25 μl of prewashed Ni-nitrilotriacetic acid (NTA) agarose (Qiagen). Lysate-resin mixtures were incubated on a tube rotator at 4°C overnight. Resins were pelleted by centrifugation in a refrigerated tabletop centrifuge at ∼200 × g for 1 min, and the unbound lysate fraction was removed. Resins were washed three times with 1× PBS supplemented with 0.1% SDS. The wash buffer for Ni-NTA purifications included 20 mM imidazole to reduce weak or nonspecific binding events. After the third wash, 150 μl of elution buffer  was added to each corresponding pelleted resin. The final elution pH was typically near neutral at 7.0 to 7.4; however, a broader range was tested for the experiments presented in Cell lysates were prepared from vaccinia virus-infected and -transfected BSR-T7 cells following the same initial steps outlined above for the MACV L immunoprecipitation. Following centrifugal clarification, 0.75 ml of lysate was combined with 25 μl of prewashed anti-Negative staining of the L-NP complexes was performed as described previously . Purifie