Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot. To slow its spread, large-scale diagnostics and the enforcement of strict public health measures were implemented in many countries. The current standard test for SARS-CoV-2 detection and diagnosis is based on viral RNA extraction from a pharyngeal swab followed by highly sensitive reverse transcription and quantitative PCR (RT-qPCR). Several primer sets targeting one or more of the SARS-CoV-2 genes—nucleocapsid (N), envelope protein (E), S glycoprotein (S), or RNA-dependent RNA polymerase (RdRp)—have been used  led to ts buffer . To achis buffer , a tip  and is based on the magnetic bead-based nucleic acid extraction protocol that was published by He et al.  and on tA selection of upper respiratory tract specimens  sent to the diagnostics laboratory of the Heidelberg University Hospital between April and May 2020 for SARS-CoV-2 PCR were used for the study. Surplus material from a total of 77 swab samples were collected from 17 SARS-CoV-2 positive and 60 negative patients, which were used for SARS-CoV-2 testing by RT-qPCR. The swab samples were either used at the day of collection  or were frozen, stored at −20 °C, and thawed just before the RNA extraction . Positive and negative samples were further diluted and used in replica to generate 88 positive and 76 negative samples in order to facilitate replicate testing.The following steps were performed in a biosafety level 2 (BSL-2) laboratory according to standard microbiological and diagnostic practices. To extract SARS-CoV-2 RNA from pharyngeal swabs, a lysis buffer containing 5 M guanidinium thiocyanate, 40 mM dithiothreitol, 20 µg/mL glycogen, 1% Triton X-100, and buffered with 25 mM sodium citrate to pH 8 was used. Internal control (IC) for RT-qPCR (5 µL/sample)  was added into the lysis buffer just before use. 140 µL lysis buffer and 140 µl sample were vigorously vortexed in a 1.5 mL Eppendorf tube for 10 sec and incubated for 10 min at room temperature inside a BSL-2 laminar flow cabinet to ensure both rapid virus deactivation and RNase denaturation. Lysates (280 µL) were transferred into a 96 deep-well plate  with a maximum working volume of 500 µl per well, which is compatible with the Liquidator 96 pipetting system.Just before the RNA extraction, SiMAG-N-DNA magnetic beads  were washed three times in RNase-free water. The aqueous magnetic bead stock solution of 100 µg/µl was added into absolute ethanol in order to obtain a working solution of 5 µg/µL. Using a multichannel pipette, 200 µL of magnetic bead solution was transferred into the 96 deep-well plate containing the pharyngeal sample lysates. To facilitate the adsorption of the nucleic acid onto the magnetic beads, the 96-deep-well plate was placed on an orbital shaker MS3  for 8 min at 500–1000 rpm, before the mixture was resuspended (10×) using a Liquidator 96, Model 200 µl  A,B and pThe QIAamp Viral RNA body fluid kit was carried out with manual lysis according to the manufacturer’s protocol to compare the performance of the magnetic bead RNA extraction . The sample input volume was 140 µl, the volume of IC per sample was 10 µL, and the elution volume was set to 100 µL.® Modular SARS and Wuhan CoV E-gene  and 0.5 µl of LightMix® Modular EAV RNA Extraction Control  was mixed with 4.9 µl RNase-free water, 4 µl LightCycler® Multiplex RNA Virus Master , and 0.1 µl Reverse Transcriptase Enzyme  per sample. The following primers and probe were used : fwd 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′, rev 5′-ATATTGCAGCAGTACGCACACA-3′, probe FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ; These primers are specific for a 113 bp amplicon from position 26141–26253 of the SARS-CoV-2 genome (GenBank NC_004718). 10 µl of Mastermix was distributed into a 96-well PCR plate and 10 µl of purified RNA from patient samples was added using the Liquidator 96, model 10 µl. RT-qPCR was performed using a LightCycler® 480 Instrument II  with 5 min of reverse transcription at 55 °C, initial denaturation at 95 °C for 5 min, and subsequent 45 amplification cycles with 95 °C for 5 sec, 60 °C for 15 sec, 72 °C for 15 sec, and finally cooling to 40 °C for 30 sec. Cycle threshold (CT) was determined, where the fluorescence signal of the amplification reaction was above the background fluorescence using the LightCycler software (Roche). Data analysis on raw CTs was performed in Excel and GraphPad Prism , and 95% “exact” Clopper-Pearson confidence intervals were calculated using MedCalc .For RT-qPCR detection of the SARS-CoV-2 envelope protein gene (E gene), we adopted a widely used protocol based on Corman et al. [1 to 6.1 × 106 molecules was spiked into 140 µl lysis buffer before a 140 µl patient sample was added. Magnetic bead RNA extraction was performed as described in 1 to 106 MS2 RNA molecules per reaction. RT-qPCR cycling program for MS2 RNA amplification was performed as described in Five µl diluted MS2 RNA  containing 6.1 × 10® Colorimetric LAMP 2X Master Mix M1800  and 1.25 µl 10× LAMP primer mix targeting the N gene [® Cyto or Infinite M200 plate reader . Phenol red absorbance spectra change in response to the acidification of the reaction (the absorbance at 434 nm wavelength is increased and the absorbance at 560 nm wavelength is decreased). To measure pH changes during the reaction, the difference between optical densities was calculated (ΔOD = OD434nm − OD560nm) and samples with ΔOD < 0.3 were classified as negative.RT-LAMP detection of the SARS-CoV-2 N gene was based on the protocol by Dao Thi et al. . The RT-e N gene  per reac® LAMP Kit 2X Master Mix E1700 , 1.25 µl 10× LAMP primer mix targeting the N gene [® 480 Instrument II . Real-time fluorescence was detected with intervals of 1 min for a duration of 90 min. Time of amplification (TOA) was determined based on a fluorescence threshold, where the fluorescence signal of the amplification reaction was above the background fluorescence, using the LightCycler software. Samples with TOA > 25 min were classified as negative.For the fluorescent LAMP assay, the same primer set as described in 2.6 was used. The master mix consisted of 6.25 µl WarmStartThe presented work was done with the intention to support SARS-CoV-2 diagnostics and improve emergency preparedness and response to COVID-19. Pseudo-anonymized surplus material from samples that had been collected for SARS-CoV-2 testing were used to establish and validate the protocol presented here. This work complies with the German Act concerning the Ethical Review of Research Involving Humans, permitting use of patient samples collected to perform the testing in question, for development and improvement of diagnostic assays.The magnetic bead RNA extraction protocol was established in a 96-well plate format as part of the detection workflow . The com5 fold, in order to compare the magnetic bead RNA extraction protocol with the commercial QIAcube extraction method. The extracted RNA was subjected to RT-qPCR using E gene primers. Viral RNA was detected in samples diluted up to 105 fold after either QIAcube or magnetic bead RNA extraction  was less than 10 RNA copies/reaction  after magnetic bead extraction was the same as compared to the average IC CT of 29.3 from the QIAcube RNA extractions   B,C, showBecause assays performed in a 96-well plate format can be prone to cross-contaminations, we evaluated the sensitivity and specificity of the three independent magnetic bead RNA extractions. The samples were distributed in small groups of positive and negative samples on the 96-well plate. After magnetic beads RNA extraction and RT-qPCR or RT-LAMP analysis the results were compared to the results of the RT-qPCR after QIAcube extraction to determine true and false positives (TP and FP) and true and false negatives (TN and FN). For the magnetic beads extracted samples, a CT cutoff of 40 was used to determine if a sample is defined as positive. The sensitivity was calculated (TP/(TP+FN)) for subsets of samples in specific CT ranges  D. For alWe also recently explored the RT-LAMP assay as a valid alternative RNA detection method due to shortages in RT-qPCR reagents . We compDuring DNA synthesis, the formation of a phosphodiester bond results in the release of a molecule of pyrophosphate and a proton causing a gradual acidification of the reaction mix. The detection principle of colorimetric RT-LAMP is based on monitoring pH changes during the DNA amplification, which only occurs in positive samples. Typically, phenol red, which changes color from red to yellow when the pH is lowered, is used as a pH indicator . The colThe fluorescent RT-LAMP assay is based on a fluorescent dye that intercalates into amplifying DNA strands, allowing for their detection in real time. Magnetic bead isolated samples with CT ≈ 30 led to a fluorescent signal at early time points (approx. 20–25 min) as compared to negative samples (>40 min). Therefore, we used the time point 25 min as a threshold for positive samples. Similar to the colorimetric read-out, the majority of samples with higher CT values, especially between CT 35 and CT 40, scored negative (> 40 min) D. All neThese results are well in agreement with the recently reported sensitivity cutoff at CT 30 for the RT-LAMP assay  and, thuWithin the last decades, the frequency of emerging virus outbreaks has increased globally ,20, possfrozen- CTfresh = 7.4; SD = 3.5, n = 19). In contrast to other magnetic bead based nucleic acid purification protocols [Here, we provide a magnetic bead-based RNA extraction protocol that is, to a large extent, producer independent, does not rely on unique components that are difficult to replace, and is scalable for mass testing. Because of the large surface binding capacity and rapid separation in solution by magnetic field, silica coated magnetic beads are compatible with a variety of plasticware and they can be used in a high throughput multiwell format  as well rotocols  that relRT-LAMP relies on a different DNA polymerase than RT-qPCR and, thus, provides a supplier-independent alternative to RT-qPCR. Additionally, RT-LAMP is faster and cheaper when compared to RT-qPCR and it does not require a thermocycler . As we hAlthough it is possible to perform RT-qPCR ,24 and RIn conclusion, we report a detailed step-by-step RNA extraction protocol from pharyngeal swabs, which is based on magnetic beads and does not depend on commercial extraction kits and reagents. Our protocol was validated in a 96-well plate format by the detection of SARS-CoV-2 RNA by RT-qPCR and RT-LAMP and provides reliable RNA extraction for a diagnostic pipeline that can be rapidly deployed during the SARS-CoV-2 pandemic and is also accessible to regions with insufficient laboratory capacities. This might be of importance in case of possible future shortages of commercial RNA extraction kits and enables diagnostic laboratories to be well prepared in case of a new rise of SARS-CoV-2 cases. Our protocol can be easily adapted to fully automated liquid handling robotic systems and is very likely suitable for isolation and downstream detection assays for any kind of RNA virus isolated from pharyngeal swabs.


Small non-coding RNAs (sRNAs) are ubiquitous components of bacterial adaptive regulatory networks underlying stress responses and chronic intracellular infection of eukaryotic hosts. Thus, sRNA-mediated regulation of gene expression is expected to play a major role in the establishment of mutualistic root nodule endosymbiosis between nitrogen-fixing rhizobia and legume plants. However, knowledge about this level of genetic regulation in this group of plant-interacting bacteria is still rather scarce. Here, we review insights into the rhizobial non-coding transcriptome and sRNA-mediated post-transcriptional regulation of symbiotic relevant traits such as nutrient uptake, cell cycle, quorum sensing, or nodule development. We provide details about the transcriptional control and protein-assisted activity mechanisms of the functionally characterized sRNAs involved in these processes. Finally, we discuss the forthcoming research on riboregulation in legume symbionts. Some soil-dwelling species of the large classes of α- and β-proteobacteria, collectively referred to as rhizobia, establish mutualistic symbioses with legumes. The outcome of these interactions is the organogenesis of specialized nodule structures on the roots or, less frequently, the stems of their specific host plant. Invading rhizobia colonize nodules intracellularly and differentiate to bacteroids that achieve the nitrogenase-mediated reduction of the atmospheric dinitrogen to ammonia for the benefit of the plant. Symbiotic nitrogen (N) fixation thus renders plant growth independent of exogenously applied combined N, which is commonly provided to crops in the form of pollutant and costly chemical fertilizers. Besides this doubtless agronomical and ecological significance, the rhizobia–legume symbiosis provides a complex biological experimental model to explore the molecular mechanisms underlying bacterial adaptations during chronic intracellular infection of eukaryotic hosts ,2,3.The saprophytic and symbiotic competence of free-living rhizobia in soil is largely determined by their capacity to cope efficiently with the abiotic variables shaping this environment, e.g., oligotrophy, drought, salinity or acidity, which are known to influence negatively the physiology of both symbiotic partners and the N fixation process itself . Competitrans (trans-sRNAs). Complementarity between the asRNA and its mRNA target is perfect and may extend over regions even longer than 200-nt. Conversely, regulation by trans-sRNAs typically involves short and discontinuous series of complementary nucleotides in both molecules, requiring the assistance of proteins  as RNA matchmakers [The development of high-throughput sequencing technologies has revolutionized our classic view of the prokaryotic transcriptome. Different, well-established strategies for the generation and deep-sequencing of cDNA libraries (RNA-Seq) allow for genome-wide mapping of transcription start sites (TSS), thus uncovering unexpected operon structures and novel transcribed regions systematically overlooked in the primary genome annotation  Figure . Many ofchmakers . Regardlchmakers .Escherichia coli. Soon afterwards, studies in related enterobacteria  and other clinically relevant microbes revealed the unprecedented prominent roles of sRNAs in the establishment of host-pathogen interactions [Sinorhizobium meliloti, the symbiotic partner of the forage legume alfalfa (Medicago sativa L.) and other related Medicago species [Research on prokaryotic riboregulation has been pioneered in the early post-genomic era by work on the model bacterium ractions . In the  species ,16,17. Htrans-acting class. Specifically, three studies published almost concurrently used the IGRs from the S. meliloti reference strain 1021 (Rm1021) as queries to interrogate the genomes of α–proteobacterial relatives, i.e., plant symbionts , phytopathogens (Agrobacterium tumefaciens) and animal pathogens (Brucella species) [trans-sRNAs, namely association with orphan transcription signatures  and conservation of thermodynamically stable secondary structures. Collectively, these approaches predicted more than a hundred IGRs in the three replicons of the Rm1021 genome  putatively encoding a trans-sRNA. Northern blot hybridization of total RNA, RACE (Rapid Amplification of cDNA Ends) mapping of transcripts boundaries and/or microarray probing experimentally confirmed that a few dozens of these candidate IGRs did express sRNA species from independent transcription units. These studies also anticipated the symbiotic and/or stress-dependent expression of subsets of the identified sRNAs. In the absence of further functional insights, these trans-sRNAs were initially referred to as either Smr [R. etli CFN42 (termed ReC sRNAs), Bradyrhizobium japonicum USDA110 (BjrC sRNAs) and M. huakuii 7653R (MH_s sRNAs) [Phaseolus vulgaris), soybean (Glycine max), and milkvetch , respectively.Non-protein coding genes largely escape classical genetics screens and the primary annotation of a single bacterial genome sequence, which is essentially limited to the prediction of open reading frames (ORFs), tRNAs, and rRNAs. Before the advent of high-throughput sequencing, computational comparative genomics was thus the tool of choice to identify conserved regions with putative functions in the unannotated portions of the genome. Accordingly, pioneering seminal genome-wide searches for sRNAs in rhizobia relied on the comparison of intergenic sequences  from phylogenetically close species to unveil sRNAs of the species) ,19,20. Ather Smr  or Sm [1s sRNAs) ,23. Thestrans-acting, conserved riboregulators. Straightforward experimental identification of TSS associated to coding sequences, untranslated mRNA regions, and non-coding RNA genes in the prokaryotic genomes is now feasible with the implementation of oriented differential RNA-Seq (dRNA-Seq) [S. meliloti and B. japonicum and further uncovered the complex rhizobial transcriptomes by adding hundreds of unknown trans-sRNAs, as well as thousands of mRNA-derived sRNAs and asRNAs [S. meliloti total RNA [Inherent limitations to both computational searches and microarray designs necessarily biased these early genome-wide screens for sRNAs in rhizobia to the identification of putative intergenic, RNA-Seq)  or CappaRNA-Seq)  strategid asRNAs ,27,28,29d asRNAs ,31. Howeotal RNA .S. meliloti (strains Rm1021 and Rm2011) and B. japonicum USDA110 genomes [M. truncatula by Rm2011 [E2) or RpoN (σ54) in the promoter regions of some of the identified sRNAs, thus placing these RNA regulators in major stress response and/or symbiotic regulons [Prokaryotic gene prediction pipelines such as EuGen-P have incorporated the novel gene structural features uncovered by dRNA-Seq for the accurate reannotation of the  genomes ,28,29. S genomes . Even th genomes ,28,29,32y Rm2011 . On the regulons ,28,29. Thttps://rfam.xfam.org/) [Reverse comparative RNomics contributes to unravel sRNA function by identifying either functionally characterized homologs to the query transcripts or conservation patterns potentially related to the particular lifestyle of phylogenetically close bacterial species. Homology predictions typically rely on stochastic covariance models (CMs) capturing both sequence and secondary structure (folding) conservation from a multiple alignment to the query sRNA. CMs can be automatically generated by INFERNAL, which builds RNA families collected by the Rfam database (am.org/) . Regardl- RNase P (Rfam family model RF00010), which is the ribozyme that cleaves off 5’-extra sequences on tRNA precursors .trans-translation for stalled ribosome recycling [B. japonicum [S. meliloti tmRNA homolog is expressed in a growth- and stress-dependent manner as an unstable precursor, further processed into two readily detectable 214 and 82 nt-long RNA species likely derived from the mRNA and tRNA domains of the primary transcript [- tmRNA or SsrA (RF00023), which has a dual function as transfer and mRNA, operating in ecycling . Of noteaponicum . The S. anscript .- The 4.5S RNA (RF00169), which associates with the multidomain Ffh protein, in the bacterial SRP  ribonucleoprotein complex, universally required for cotranslational protein targeting .70 RNA polymerase holoenzyme by a target mimicry mechanism, thus contributing to the transcriptional reprogramming during transition from exponential to stationary growth [Legionella pneumophila within macrophages [- The 6S RNA or SsrS (RF00013) RNA, which relies on an open promoter-like structure to counteract the activity of the σy growth . Interesrophages , which cS. meliloti trans-sRNAs without associated functional evidence has been also addressed by several independent studies [trans-sRNA input sequences into 43 family models, indicating that several of these sRNA loci occur with different levels of paralogy in the Rm1021 genome. Distribution of the majority of these families was restricted to members of the family Rhizobiaceae within α-proteobacteria, including Agrobacterium species. Only a few chromosomally encoded sRNAs are conserved beyond Rhizobiaceae, occurring in representatives of Brucellaceae or even Bartonellaceae, Xanthobacteriaceae, Beijerinckaceae, or Bradyrhizobiaceae. In contrast, sRNA loci mapping to S. meliloti pSymB or pSymA are almost unique to Sinorhizobium spp. and S. meliloti, respectively, further supporting that these two megaplasmids mostly encode accessory functions, with pSymA being the most recently acquired replicon in genus Sinorhizobium. Overall, the distribution patterns of these α–proteobacterial sRNA families hint at a major contribution of vertical inheritance and frequent ancestral duplications events to the evolution of these class of riboregulators in α-rhizobia. S. meliloti trans-sRNAs.CM-based phylogenetic distribution of other 57  studies ,44,45,46 studies ,44. TDMsS. meliloti and related α–proteobacteria. The challenge now is to decipher the biological functions of this transcriptional output, which can be still regarded as dark matter of rhizobial genomes. In prokaryotes, a large fraction of these transcripts are catalogued as trans-sRNA regulators, which is an intensively investigated class of sRNAs since the early studies of the non-coding transcriptome in classical model bacteria [trans-sRNAs is essentially characterised by their differential accumulation in response to environmental signals and target mRNA regulation by protein-assisted base-pairing mechanisms . Experimental and in silico approaches to functionally characterize trans-sRNAs in rhizobia are described with detail elsewhere [trans-sRNAs can be tracked by classical molecular-genetics methods that include the use of promoter-reporter fusions and probing of total RNA on Northern blot membranes [trans-sRNA targetomes, i.e., the set of mRNA targets, can be characterized on a genome-wide scale by profiling of either transcriptome alterations upon pulse expression of the tested sRNA or the subset of mRNAs picked up by affinity chromatography using an aptamer-tagged  version of the sRNA as a bait [RNA-Seq and comparative genomics have delivered extensive repertoires of sRNAs in bacteria . The biochanisms . Therefolsewhere ,17,47,48embranes ,50. The s a bait ,52. Rematrans-sRNAs identified in S. meliloti. In the following paragraphs we describe how these sRNAs contribute to the regulation of cellular processes relevant to both the free-living and endosymbiotic rhizobial states.A combination of these strategies has been used to gain insights into the function and activity mechanisms of a handful of Rhizobia must cope with a broad range of limiting nutrients in soil and specific nutritional needs in the rhizosphere of their host plants. Therefore, nodulation competitiveness first depends on the adequate regulation and coordination of efficient nutrient uptake and metabolism. Thus, it is not surprising the large number of ABC (ATP-binding cassette) transporters encoded by rhizobial genomes ,53,54. AtrpE(G), directly interacts with the polycistronic trpDC mRNA resulting in negative post-transcriptional regulation in S. meliloti. Considering that trp is the most costly-to-synthesize aa and the conservation of this mechanism in Agrobacterium and Bradyrhizobium but not in E. coli, this precedent provides an example of how soil bacteria have evolved post-transcriptional strategies to coordinate transport and metabolism in complex ecological systems as soils [Nutrient uptake through ABC transporters primarily relies on periplasmic solute binding proteins (SBP) that determine substrate-specificity and are induced by its own ligand, typically two or more molecules ,55. Thisas soils .E. coli and Salmonella by characterization of the GcvB sRNAs, which regulon potentially includes ~1% of all mRNAs in these bacteria, with prevalence of mRNAs from ABC transporters of aa and short peptides, but also containing mRNAs coding for aa biosynthesis-related proteins and transcription factors [A. tumefaciens, B. abortus and S. meliloti, and were termed AbcR sRNAs (ABC transporter RNA) [S. meliloti Rm1021 encodes three AbcR homologs that belong to the αr15 sRNA family, whose members have partial homology to the SuhB sRNA (Rfam model RF00519) [lsrB (trans-sRNAs are enriched in ABC transporter mRNAs (35–45% of the top scored). Proteomics and further genetic verification identified prbA, SMa0495 and livK, all coding for the SBP of aa transporters, as common targets of these sRNAs- except livK that is specifically repressed by AbcR1 [Riboregulation of nutrient uptake was first described in  factors . Functioter RNA) ,61,62,63RF00519) . AbcR1 a9) [lsrB . Their pby AbcR1 ,62.S. meliloti sRNAs are unknown. Analysis of the abcR2 promoter shows high conservation of motifs recognized by the heat-shock σ factor RpoH, whereas the Brucella abcR1 is directly and positively regulated by the LsrB homolog VltR, encoded by the neighbor gene. Interestingly, while VltR is involved in virulence, LsrB is critical for the successful symbiosis in alfalfa plants by suppression of host defense responses that contribute to nodule premature senescence [S. meliloti on alfalfa roots or free-living growth. However, AbcR homologs in Brucella or Agrobacterium are involved in virulence [S. meliloti liv ABC system has no contribution to symbiosis, its bra homolog in R. leguminosarum is needed for a Fix+ phenotype in pea [AbcR1 and AbcR2 expression patterns are divergent, suggesting differential transcriptional regulation from independent promoters. Indeed, both 5’-end RACE mapping and dRNA-Seq consistently identified a TSS for each transcript ,27. AbcRnescence ,65,66,67irulence . Notablye in pea ,69,70. TS. meliloti 77 nt-long trans-sRNA of the αr8 RNA family  under conditions of C surplus, thereby limiting intracellular accumulation of PHB granules. Together, these data suggest that mmgR expression depends on cellular N and C status [phbC has different effect depending on rhizobial species. Therefore, MmgR may have a different impact in other rhizobia that is worth to investigate [Proper regulation of N and carbon (C) metabolism is also required for successful bacteroid differentiation and N fixation. C storage in nodules occurs in the form of polyhydroxybutyrate (PHB) granules which are produced during the invasion process and degraded during bacteroid differentiation. PHB is likely used as a C source at this stage, being critical for symbiotic performance . MmgR  that regulate multiple mRNAs, including four low cell density master regulators that repress LuxR by four distinct mechanisms [Bacteria are able to communicate with their pairs and adjust their expression profiles to cell density thanks to the quorum sensing (QS) response. This collective behavior system has been extensively studied in Gram-negative bacteria and it is mainly based on the synthesis of autoinducers, such as acyl-homoserine lactones (AHLs) . These mchanisms .S. meliloti, AHLs are synthesized by the LuxI-type synthase SinI, which is regulated by other components of the Sin QS system, like SinR and ExpR, under cell density-dependent and stress conditions [sinI mRNA in an hfq knock-out mutant together with co-immunoprecipitation with Hfq anticipated QS riboregulation in S. meliloti [sinI 5’-UTR degradation, thereby influencing mRNA turnover and AHLs levels [trans-sRNA that regulates S. meliloti sinI post-transcriptionally has been also unveiled. This sRNA, initially referred to as SmelC587 (sinI mRNA sequence as query) as a putative sinI base-pair riboregulator [sinI 5’-UTR. This interaction was genetically confirmed in vivo and by in vitro by RNA degradation assays using native RNase E. Interestingly, the results show that RcsR1 competes with the endonuclease for sinI 5’-UTR cleavage site, impairing SinI translation [trpL-derived attenuator RNA rnTrpL. Ectopic RcsR1 overproduction results in trpDC mRNA level decrease as rnTrpL down-regulates sinI, suggesting a co-regulation of both QS and tryptophan biosynthesis [In nditions . Accumulmeliloti ,80. On ts levels . A transSmelC587 , was ideegulator . Abundanegulator  and, accegulator . RcsR1 inslation . This woynthesis .S. meliloti chromosome and GcrA has been reported to control expression of several genes involved in the segregation machinery during S-phase in Caulobacter [gcrA-depleted cells resemble bacteroid genome content and morphology in S. meliloti [S. meliloti trans-sRNAs have been related to the adjustment of cell cycle progression under adverse conditions by different mechanisms  as possible CtrA riboregulator. EcpR1 homologs are broadly distributed among Rhizobiales, whilst GspR occurrence is likely restricted to the genus Sinorhizobium [trans-sRNAs stand for the main phenotypes associated to their gain-of-function. EcpR1 overexpression results into an elongated cell phenotype, along with cell filamentation and genome endoduplication, mimicking the phenotypes of bacteroid and GcrA-depleted cells [S. meliloti derivatives are outcompeted by their parent strains when co-cultured, suggesting that both sRNAs confer a fitness advantage to bacteria. Expression of both EcpR1 and GspR is induced in growth-limiting stationary phase in rich media and is not detected in mature nodules [70-dependent promoter and this sRNAs is also activated by other stresses like cold and heat shocks or nutrient deprivation, stimulated by the stringent response signal ppGpp. GspR strongly accumulates in bacteria grown in minimal medium. In agreement with in silico predictions and profiling of their dependent proteomes, EcpR1- and GspR-mediated regulation of cell cycle-related genes was experimentally validated by an eGFP-based genetic reporter system [dnaA and gcrA) base pair to the same GC-rich conserved SL in the sRNA, whereas GspR represses expression of ctrA and cspA5 using different domains. Furthermore, considering the targeted mRNA region, dnaA and cspA5 are canonically regulated by sRNA binding to the vicinity of the RBS and the start codon. However, gcrA and ctrA are silenced by a still unknown mechanism that involves base pairing to nucleotides located far upstream of the translation initiation region within the long 5’-UTR of the mRNAs. CspA5 homologs usually act as RNA chaperones facilitating translation, which is probably the observed effect of CspA5 on the long 5’-UTR of the ctrA mRNA. Therefore, GspR and CspA5 are arranged in a coherent feed-forward loop that adds a novel post-transcriptional layer to the intricate CtrA regulation [Target predictions for conserved sRNA families in related α-proteobacteria showed an enrichment of cell cycle-related mRNAs for EcpR1  to reflect this fact [S. meliloti Rm1021 and Rm2011 genomes, but in the conditions tested only one of those is transcribed to levels reliably detected by northern blot probing [70-dependent promoter containing several nucleotide residues that are well-conserved in promoter regions of αr14 sRNAs. This conserved motif is the determinant of both differential expression and high transcription rates, which render NfeR1 as one of the transcripts with highest coverage scores among the identified S. meliloti sRNAs by RNA-Seq surveys [S. meliloti and alfalfa, i.e., rhizoplane colonization, root hair infection, bacteroid differentiation and symbiotic N fixation [S. meliloti survival in hypersaline medium, nodule development and overall symbiotic efficiency [S. meliloti nfeR1 deletion mutant on alfalfa roots are apparently colonized by fully differentiated bacteroids, but look round shaped rather than elongated, and smaller than wild-type indeterminate mature nodules. Their histology resembles that of nodules developed by M. truncatula mutants in marker genes for intermediate and late symbiotic stages [S. meliloti derivatives engineered to overexpress N assimilation pathways concurrently with symbiotic N fixation [To date, phenotyping of knock-out mutants has revealed putative symbiotic functions for only one chromosomally encoded his fact ,26,92. Nhis fact ,46. αr14his fact ,46. NfeR surveys ,29,34. Ic stages ,93 or infixation . In siliB. japonicum [Nodule development is largely controlled by complex organogenesis programs induced by rhizobia in their compatible legume host . In thisaponicum . These mS. meliloti anticipate a broad impact of cis-acting asRNA regulators on both the free living and host-associated lifestyles of this bacterium [S. meliloti expresses more than 3000 asRNAs that are linked to ~35% of the predicted protein-coding genes [S. meliloti, like in other microorganisms, were firstly reported associated to extrachromosomal replicons [Several studies on acterium ,27,97. aacterium . Such RNng genes ,27. Noneeplicons .trans-sRNAs are overrepresented  in S. meliloti pSymA symbiotic megaplasmid, with asRNAs particularly biased to nod and nif genes. Further characterization of seven pSymA-borne asRNAs showed that most accumulate in response to different biological conditions, anticipating functional roles [nodD2 3´-UTR, is slightly upregulated at high optical densities and upon luteolin addition (30%). It is worth noting that NodD2 is the only transcriptional regulator of S. meliloti nod genes that is not activated by this plant flavonoid. Several asRNAs opposite to genes encoding the nitrogenase components NifE and NifK show also distinct expression profiles. SmelA031 and SMa_asRNA_277, also accumulate in early and late stationary phase of growth, respectively, while SMa_asRNA_279 responds to other stresses. Interestingly, the three transcripts are repressed in nodules and under low O2 and N2 concentrations, and plants inoculated with S. meliloti overexpressing SmelA031 are moderately affected in N fixation when compared to controls. Nitrogenase activity is extremely sensitive to O2 and needs N2 as substrate to function inside the nodules. Therefore, it is feasible that such post-transcriptional regulation modulates translation of this enzyme to avoid energy loss. These results first evidenced symbiotic-related asRNA regulation in rhizobia, but hundreds of rhizobial asRNAs await functional characterization. There are some works arguing that the majority of cellular asRNAs just arise from noisy transcription and are non-functional [A systematic screening strategy to identify novel RNA regulators of N fixation revealed that both asRNAs and al roles . Among tnctional ,101. Hownctional . NeverthE. coli, phylogenetically close enterobacteria and other pathogens [Proteins involved in RNA activity and metabolism are universal players in riboregulation at different levels ,103,104.athogens . Nonetheathogens .trans-sRNAs) and stabilize diverse RNA species including mRNAs and tRNAs [E. coli and S. meliloti Hfq homologs have been shown to be functionally interchangeable [The host factor for the replication of the Qβ bacteriophage or Hfq is encoded by 55% of the bacterial genomes sequenced so far . The funnd tRNAs . These Rnd tRNAs ,113,114.nd tRNAs . This prangeable .hfq knock-out causes severe pleiotropic phenotypes in S. meliloti that include altered growth, increased sensitivity to abiotic stress, changes in the profile of QS signals, reduced competitiveness for nodulation, compromised survival of bacteroids inside nodule cells and defects in N fixation [S. meliloti [A. tumefaciens or Brucella species [Like in enterobacteria, fixation ,116,117.fixation ,117,118.meliloti ,116,119.meliloti ,120. Alt species ,121,122.S. meliloti, genome-wide profiling of RNA species co-immunoprecipitated with a functional FLAG epitope-tagged Hfq variant (CoIP-RNA) uncovered massive binding of the protein to mRNAs [S. meliloti mRNAs were enriched in Hfq CoIP-RNA, most of which functionally related to major symbiotic and stress-response pathways. Many of these mRNAs, such as those encoding NifA, FixK or stress-related alternative RNA polymerase σ factors of extracytoplasmic function  are positively regulated by Hfq by a mechanism that probably involves protection of the transcripts from degradation by cellular ribonucleases in a trans-acting sRNA-independent manner [trans-sRNAs AbcR1 and AbcR2 are particularly abundant among the Hfq-binding transcripts. In these cases, Hfq most likely acts promoting both trans-sRNA stability and base-pairing to mRNA targets to boost post-transcriptional silencing [trans-sRNAs and 2% of asRNAs currently annotated in the S. meliloti genome were scored as Hfq ligands [S. enterica ProQ protein [R. leguminosarum bv. viciae among α–rhizobia [Regulation of at least part of the Hfq-dependent mRNAs is expected to involve its cognate partner sRNAs. In to mRNAs ,123. Spet manner . Conversilencing . Nonethe ligands ,27,29,32 protein ,124. Howrhizobia . Thus, ttrans-sRNA and its target(s) as a component of a multiprotein complex called RNA degradosome that also contains a 3′-exoribonuclease (polynucleotide phosphorylase or PNPase), a RNA helicase (RhlB) and an enolase as the core proteins [As major determinants of the steady-state level of all cellular transcripts, ribonucleases (RNases) are critical elements in post-transcriptional regulatory networks . Decay oproteins . On the proteins . RNA decproteins .trans-sRNA stability and regulation in both Hfq-dependent and independent manner [E. coli, are both involved in rRNA maturation but their putative role in riboregulation has not been investigated yet [S. meliloti rnc gene has been biochemically and genetically characterized [E. coli ortholog but with different requirements for optimal activity regarding metal co-factor preference or pH tolerance that could be related to the rhizobial lifestyle. Indeed, although rnc deletion does not compromise viability and morphology of S. meliloti cells, it is detrimental for growth and overall symbiotic performance on alfalfa roots [Rhizobial genomes encode a set of more than 20 ribonucleases that include all the aforementioned proteins, but for the vast majority there is a lack of functional information . Nonethet manner ,130,131.ated yet ,133. RNacterized . Its catfa roots .SMc01113 in the chromosome of the S. meliloti reference strain Rm1021 is almost ubiquitous in bacteria and has been included in the proposed minimal prokaryotic genome [S. meliloti Hfq and YbeY mutants show strikingly similar free-living and symbiotic phenotypes as well as misregulation of several predicted sRNA-mRNA regulatory pairs [E. coli YbeY ortholog was later shown to be a metal-dependent endoribonuclease that specifically acts on ssRNA [S. meliloti YbeY also supports a universal role of this protein as RNase [S. meliloti YbeY is also competent for cleaving dsRNA [S. meliloti YbeY mutant predict a role of this enzyme in trans-sRNA and asRNA mediated silencing of genes involved in nutrient uptake and symbiotic N fixation, respectively [prbA mRNA. Further supporting a role in dsRNA degradation, S. meliloti YbeY has been also shown to co-purify with several asRNAs and their mRNA partners, including the symbiotically-relevant nifA [The gene annotated as c genome . It encoc genome . S. meliry pairs ,137. Thery pairs . Howeveron ssRNA . Biochemas RNase ,140. Howng dsRNA . Transcrectively . In thisant nifA . It woulS. meliloti. This bacterium expresses hundreds of trans-sRNAs, but only a handful of those have been characterized and functionally related to free-living and symbiotic rhizobial traits such as metabolism, cell-cycle, quorum sensing or nodule development (S. meliloti, with a remarkable bias towards symbiotically relevant genes. However, the functional significance of asRNA-mediated regulation has not been investigated further neither at genome-wide scale nor for particular symbiotic genes. Other key players in riboregulation, like RNA-binding proteins and ribonucleases remain also underexplored. Unlike in enterobacteria, the well-characterized RNA-chaperone Hfq seems to have a rather limited role in assisting trans-sRNA activity in rhizobia and the catalytic features of the set of predicted ribonucleases and its impact in RNA silencing are essentially unknown. Therefore, much work remains to be done to characterize the content and function of the sRNAs-associated proteomes.RNA-mediated regulation of gene expression, which is ubiquitous in all living organisms, contributes to fine-tune most, if not all, prokaryotic physiological processes. However, genome annotations of the majority of bacterial species do not include sRNA genes yet, so that the structure and function of the non-coding transcriptomes remains unaccountably unexplored. Knowledge gaining on how the transcriptomes shape the functions of bacteria populating diverse niches or even establish complex relationships with other organisms is now timely and technically feasible. In plant-nodulating rhizobia, most of the knowledge about riboregulation derives from work on the alfalfa symbiont elopment . Like inS. meliloti should be necessarily extended to other rhizobia and symbiotic systems. Genomes of other model N-fixing symbionts must be accurately annotated using the most recent RNA-Seq protocols to uncover the primary transcriptome. The resulting catalogs of sRNA genes must be extensively compared and characterized to assess the contribution of riboregulation to the symbiotic diversity. Deciphering the biogenesis, function, and mechanistic principles of all types of sRNAs, i.e., trans-sRNAs, asRNAs, riboswitches, 5’/3’-UTRs, other mRNA leaders, and tRFs will contribute to explore the plasticity of RNA molecules as regulators of gene expression. Of particular interest would be to investigate the RNA trafficking between rhizobia and the plant host cells as a novel layer of dialogue between the symbiotic partners. Finally, the biotechnological implications of post-transcriptional RNA-mediated regulation for the engineering of symbiosis and other environmentally relevant systems should be also explored.The knowledge gained in


Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application.In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods.The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value.SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.The online version contains supplementary material available at 10.1186/s12967-021-02775-9. Recently, increasing studies reported that circulating RNA is abundant and could be as prognosis marker. For example, serum high-temperature-required protein A2 (HtrA2) was reported to associate with the progression of breast cancer, especially could increase the diagnostic efficiency when combined with CA15-3 and CEA , 5. ExtrSince many studies have reported that EVs could be closely related to disease progression and suggested their roles as novel biomarkers for various diseases, optimal strategy for EVs purification shall be further developed as soon as possible. Although there have been several methods available to purify the EVs, including ultracentrifugation, filtration, precipitation, chromatography, immunoaffinity capture, and commercialized kits derived from the above technologies –17. Howehttp://www.exocarta.org), up to 3000 mRNAs and 2800 miRNAs have been identified in EVs. In addition, a growing number of evidence suggests that lncRNAs can be sorted by EVs [According to public database  from the same donor was equally divided into 4 aliquots, each of which was subjected to one specific extraction method. Samples that come from the same donor, with the consistent pretreatment conditions, will minimize the personal equation during the experiments. The isolation of EVs was performed forth without frozen so that the impact of freezing on sample quality would be minimized. Whole blood samples were temporary stored in EDTA collection tubes at room temperature (< 30 min). Before processing, it is necessary to ensure no visible hemolysis was observed. Then, the blood sample was centrifuged at 1600 g for 15 min at room temperature to spin down cell pellet. The supernatant was transferred to a new Eppendorf tube and centrifuged at 10,000 g for 30 min to remove debris and large vesicles at room temperature. Plasma samples shall be prepared before being used.The EVs isolated by UC: Plasma samples were first centrifuged at 2000 g for 10 min. The supernatant was filtered using a 0.22 µm constant well filtration system . The plasma were then centrifuged in a polymer centrifuge tube  at 100,000 g and 4 °C for 2 h with a rocking rotor , then washed with PBS at 100,000 g and 4 °C for 2 h. The precipitate was resuspended by 1 × PBS.The EVs isolated by exoEasy: The sample was centrifuged at 3000 g for 15 min at 4 °C using pre-filtered plasma. 1 portion of XBP buffer were added to the sample of 1 volume. The tube was gently inverted 5 times to mix the solution immediately and the mixture was placed at room temperature. The mixture was applied to an exoEasy spin column and centrifuged at 500 g for 1 min. The flow-through solution were discarded and the spin column was placed in the same collection tube. The residual volume in the spin column was removed by adding 3.5 mL of XWP buffer and centrifuging at 5000 g for 5 min. All flow-through solution were discarded. The spin column was moved to a new collection tube. The EVs adsorbed on column could be eluted by Buffer XE for BCA, NTA, and TEM assays, or RNA isolation by QIAzol regent.The EVs isolated by SEC: SEC was performed according to the manufacturer’s instructions (QEV (Izon), Exosupur ). Briefly, plasma samples were first centrifuged at 2000 g for 10 min. The supernatant was filtered using a 0.22 µm constant well filtration system after centrifugation. Then, plasma was added on the top of the column. The EVs containing fraction were collected together by 1 × PBS. The collected fractions were concentrated by ultrafiltration with 100 K Amicon Ultra-15 centrifugal filter units (EMD Millipore).The EVs isolated by ExoQuick: The plasma samples were centrifuged 3000 g for 15 min, and the supernatant was transferred to a sterile tube. The ExoQuick reagent was added, and inverted the centrifuge tube upside down several times to mix the solution well. The cells were allowed to stand at 4 °C, and then centrifuged at 1500 g for 30 min to wash the supernatant, and then centrifuged at 1500 g for 5 min and aspirated the supernatant. The precipitate was resuspended by adding 1 × PBS.A total of four EVs isolation methods were compared in this study: ultracentrifugation (UC), exoEasy kit (QIAGEN), exclusion chromatography , ExoQuick kit (Thermo Fisher).The concentration standard sample and the particle size standard sample were used for parameter calibration before the test. The data were collected for one minute by nano-flow cytometry. The particle concentration and particle diameter of the sample were determined according to the standard sample .For transmission electron microscopy, freshly isolated EVs suspensions were fixed in 4% paraformaldehyde for 1 h. The EVs suspensions from different samples (approximately 5 μL) were applied to copper mesh Formvar coated carbon stabilized grids, were allowed to adsorb to the grid for 4–5 min and then wicked off with filter paper. For negative staining of EVs, 1% aqueous uranyl acetate (5 μL) was applied to the grid for 30 s, then wicked off with Whatman filter paper. Grids were thoroughly dried before viewing .Protein samples were prepared by adding 200 μL of ice-cold NP-40 buffer with protease inhibitor to 200 μL of extracted EVs sample suspended in appropriate buffer. Mix and shake once every ten minutes then incubate on ice for 50 min. The protein concentration was measured by using BCA . Then add 2 × loading buffer (200 μL), and mix well, and heat to boil. The protein sample (50 μg) was separated with an 8–10% SDS gel, and blotted on an immuno-blot PVDF membrane. Then the BSA was applied to block for 1 h and the membrane was incubated with primary antibody  overnight at 4 °C. The membrane was then washed with Tris buffered saline containing 0.1% Tween and incubated with the secondary antibody for 1 h at room temperature. Then, the film was washed again and exposed to ECL (electrochemiluminescence).Total RNA was extracted and purified from plasma EVs using miRNeasy® Mini kit  according to the kit instruction. RNA degradation and contamination, especially DNA contamination, was monitored on 1.5% agarose gels. RNA concentration and purity were evaluated using the NanoDrop 2000 Spectrophotometer  and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System . All the RNA sequencing was performed using the single sample.A total of 5 ng RNA per sample was used as input material for sequencing libraries using the Ovation® SoLo RNA-Seq Library Preparation Kit  following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. For small RNA libraries, a total of 2.5 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Multiplex Small RNA Library Prep Set for Illumina  following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR. The clustering of the index-coded samples was performed on acBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced using an Illumina Hiseq platform and paired-end reads were generated , 26.Comparison of the variable average was performed using SPSS 19.0 statistical software. Data were plotted as mean ± SEM. GraphPad Prism was used for statistical analysis and graph generation . After testing the normality and the same variance using the Shapiro–Wilk and Levene tests, the difference between the abnormal averages was analyzed by using the Kruskal–Wallis test. A P value less than 0.05 was considered to be statistically significant.In order to find out the optimal isolation method for RNomics from EVs, we compared four existing methods, including UC, SEC, exoEasy, and ExoQuick. The whole experimental design and the operation procedures was shown in Fig. Bradford protein assays (BCA) and nano-flow cytometry (nanoFCM) were applied to analyze the total amount of protein, particle concentration, and size distribution of the EVs products obtained via UC, SEC, exoEasy and ExoQuick. As showed in Fig. To serve the purpose of RNomics, several characteristics of RNA products need to be evaluated, such as quantity, purity, types of RNA and so forth. As showed in the Fig. In order to investigate the expression profile of different types of RNAs, the properties of RNA sequencing in the EVs isolated by the four approaches were examined. The miRNA sequencing was performed to comprehensively evaluate the characteristics of RNA products. According to the consistency results in Fig. RNA sequencing analysis was also performed with the long chain RNAs (mRNA and lncRNA). As shown in Fig. Regarding lncRNA, there was little difference in the types of the total mapped lnRNA and lncRNAs with a FPKM > 5 among the four methods since most of the reads in long chain RNA sequencing belonged to mRNA and the total reads mapped to lncRNA were not enough for effective comparison (1.41–8.47%) , while the values of UC and ExoQuick were 37.86% and 34.24%, respectively .In terms of the purity and recovery rate, the results suggested that the property of SEC-derived EVs was variable, which was related to the kit selection and the fractions collected in the experiment. When using SEC for further EVs-related analysis, it is of vital importance to choose a reliable SEC column. It was reported that majority of the RNA in plasma were not packaged by EVs, but presented as a complex with RNA binding proteins . The abundance of such proteins could reflect the influence of EVs free RNAs in further RNA sequencing work. Therefore, in present study, the contents of potential protein contamination markers were evaluated. As showed in Fig. According to the protein concentration and PCR quantification assay, SEC-purified RNA showed a better vesicle enrichment characteristic. As the majority of miRNAs in plasma are AGO2 associated rather than EVs’ cargoes, SEC could wipe off free proteins efficiently and ensure that most of the miRNAs in further sequencing are EVs-specific. However, both SEC and exoEasy have the ability to exclude AGO2 protein, which might cause the miRNA detected in sequencing are not EVs-specific. Furthermore, in long chain RNA sequencing, most of the mapped reads in SEC enriched RNA were located in exon, indicating less influence from cfDNA or other non-classical long chain mRNA in plasma. Although SEC did not show outstanding advantage with respect to the total number of mRNA kinds in RNA sequencing, the detected mRNAs were preferring to be with considerable concentration with ideal FPKM values. This suggested differential expressed mRNAs in SEC based RNA sequencing will showed better reproducibility in further PCR validation work.Though RNA sequencing in plasma sample was the most widely concerned research area in liquid biopsy, differential EVs extraction method has different efficiency. RNA distribution characteristics and coverage in RNA sequencing make the analysis results showing inadequate repeatability in different groups. For the first time we provided the RNomics panorama including transcriptome sequencing of mRNA, miRNA and lncRNA of all the used EVs extraction methods, which will provide important reference value for plasma EVs based on RNA sequencing work. Moreover, we not only proved SEC based EVs isolation was most superior for RNA sequencing, but also supplied an artificial fluorescently-labeled liposome to evaluate the merit advantage of SEC column in EVs isolation. This system will supply important reference for the SEC based EVs research.For the first time we evaluated four EVs isolation methods comprehensively in RNomics including miRNA, mRNA and lncRNA. This is the most systematic work that comparing EVs isolation methods in the respect of RNomics. Other researches on the comparison of RNA extracted from EVs, were that comparison of miRNA and mRNA from urinary extracellular vesicle extracted by Hydrostatic Filtration Dialysis (HFD), ultracentrifugation (UC), and a commercial kit‐based isolation method (NG) , or compIn summary, it is recommended that SEC shall be used for the RNA sequencing works in EVs. However, it is worth noting that the conclusion shall not be extended to the EVs-related proteomics work in plasma, as RNA and protein have variant distribution pattern. Furthermore, due to the similar particle sizes of LDL, HDL, and EVs, it is inevitable to introduce LDL/HDL to the EVs fractions during SEC process.In this study, we compared four different methods (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy)) in the isolation of EVs for RNomics analysis in plasma comprehensively. The results showed that EVs obtained by SEC had the minimum miRNA binding protein AGO2 and the highest amounts of EVs specific miRNA and lncRNA, but the lowest nonspecific miRNA. Further RNA-Seq data revealed that RNAs from SEC presented more useful reads for both miRNA and mRNA. Furthermore, the detected mRNA in SEC delivered RNA were preferring to be with considerable concentration with ideal FPKM values. In conclusion, we recommend SEC for EVs RNomics in plasma.Additional file 1: Fig. S1. EVs isolated by SEC. A. Particle fluorescence value in SEC fractions 0.5–11.5 ml, which can represent particle number. B. Protein amount and particle number of EVs isolated by kits Exosupur, QEV and 4ff. Protein was measured by BCA assays and particle number by detection of A480. C. Particle/protein ratio for EVs isolated by kits Exosupur, QEV and 4ff. Exosupur Kit was chosen because of its high particle/protein ratio. D. TEM images of Exosupur fractions 1.5 and 2.0 ml from plasma, urine and supernatant of cell culture. E. Western blotting analysis of specific markers Alix, Tsg101, CD63 and inspecific marker Calnexin on cell lysates and EVs lysates from plasma, urine and supernatant of cell culture. CL: Cell lysates as control.


A large number of human immunodeficiency virus 1 (HIV-1) integrase (IN) alterations, referred to as class II substitutions, exhibit pleiotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of the three distinct mechanisms: (i) markedly reducing IN levels thus precluding the formation of IN complexes with viral RNA; (ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; and (iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in the mislocalization of viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation. At the plasma membrane of an infected cell, Gag and Gag-Pol molecules assemble around a vRNA dimer and bud from the cell as a spherical immature virion, in which the Gag proteins are radially arranged . As the the core .After infection of a target cell, RT in the confines of the reverse transcription complex (RTC) synthesizes linear double-stranded DNA from vRNA . The vDNA number of IN substitutions which specifically arrest HIV-1 replication at the integration step have been described . These sHIV-1 IN consists of three independently folded protein domains: the N-terminal domain (NTD), catalytic core domain (CCD), and C-terminal domain (CTD) , and vRNEccentric virions generated via class II IN substitutions or ALLINI treatment are defective for reverse transcription in target cells  despite In this study, we aimed to determine the molecular basis of how class II IN substitutions exert their effects on HIV-1 replication. In particular, by detailed characterization of how class II substitutions impact IN multimerization, IN-RNA interactions, and virion morphology, we aimed to dissect whether loss of IN binding to vRNA or aberrant IN multimerization underlies the pleiotropic defects observed in viruses bearing class II IN mutations. Remarkably, we found that class II substitutions either prevented IN binding to the vRNA genome or precluded the formation of IN-vRNA complexes through reducing or eliminating IN from virions. We show that IN tetramers have a strikingly higher affinity toward vRNA than IN monomers or dimers, and many class II IN substitutions inhibited IN binding to RNA indirectly through modulating functional IN tetramerization. By contrast, R262A/R263A and R269A/K273A substitutions within the CTD and the K34A change within the NTD did not perturb IN tetramer formation, and thus likely directly interfered with IN binding to RNA. Irrespective of how IN-RNA binding was inhibited, all class II IN mutant viruses formed eccentric particles with vRNPs mislocalized outside the CA lattice. Subsequently, this led to premature loss of the vRNA genome as well as IN, and spatial separation of RT and CA from the vRNPs in target cells. Taken together, our findings uncover causal mechanisms for the class II phenotype and highlight the essential role of IN-RNA interactions for the formation of correctly matured virions and vRNP stability in HIV-1-infected cells.Substitutions in IN that exhibited a class II phenotype i.e. assembly, maturation or reverse transcription defects  or affecIN mutations were introduced into the replication-competent pNL4-3 molecular clone and HEK293T cells were transfected with the resulting plasmids. Cell lysates and cell-free virions were subsequently analyzed for Gag/Gag-Pol expression, processing, particle release, and infectivity. While substitutions in IN had no measurable effect on Gag (Pr55) expression, modest effects on Gag processing in cells was visible for several missense mutant viruses including H12N, N18I, K34A, Y99A, K103E, W108R, F185K, Q214L/Q216L, L242A, V260E, as well as the ΔIN mutant . NeverthThree distinct phenotypes became apparent by assessing the amount of virion-associated IN and RT enzymes . First, With the exception of E96A, nearly all of the IN substitutions reduced virus titers at least 100-fold compared to the WT , which cUsing complementary in vitro and CLIP-based approaches, we have previously shown that viral genomic RNAs (vRNA) constitute the primary RNA species bound by IN in virions and that IN interacts with the viral genome through multiple basic residues  in its CTD . In addiIN-vRNA complexes were immunoprecipitated from UV-crosslinked virions and the levels of coimmunoprecipitating vRNA were assessed. Note that substitutions that significantly reduced the amount of IN in virions  were excNL4-3 virions were treated with ethylene glycol bis(succinimidyl succinate) (EGS) to covalently crosslink IN in situ and virus lysates were analyzed by immunoblotting. IN species that migrated at molecular weights consistent with those of monomers, dimers, trimers, and tetramers were readily distinguished in WT virions , those that may have directly inhibited IN binding to RNA , and those that primarily altered IN multimerization , was assessed by transmission electron microscopy (TEM). As expected, the majority of WT particles contained an electron-dense condensate representing vRNPs inside the CA lattice, whereas an ΔRT-IN deletion mutant virus produced similar levels of immature particles and eccentric particles . RemarkaNext, we tested whether inhibition of IN-RNA interactions through class II substitutions changes the localization of IN in virions. The premise for this is based on our previous finding that disruption of IN binding to vRNA through the IN R269A/K273A substitution leads to separation of a fraction of IN from dense vRNPs and CA containing complexes . Thus, wTo test these possibilities, we combined the CA destabilizing K203A substitution, which leads to premature disassembly of the CA lattice in vitro , with clWe have previously shown that vRNA and IN are prematurely lost from cells infected with the R269A/K273A class II IN mutant . Given tThe fates of viral core components in target cells were tracked using a previously described biochemical assay . For theAs previously reported , in cellNL4-3 in the presence of nevirapine to prevent vRNA loss due to reverse transcription, and vRNA levels associated with cells immediately following synchronization (0 hr) and 2 hr post-infection were evaluated , 2 and 6 hr post-infection, and the quantity of vRNA measured by Q-PCR. In line with the above findings, vRNA levels decreased at a faster rate with the class II IN mutants as compared to WT viruses, with half as much cell-associated vRNA remaining at 2 and 6 hr post-infection for the class II IN mutants  depIn regard to the first case (i) above, it was previously shown that IN deletion leads to the formation of eccentric particles . Thus, iOur results with different oligomeric states of WT IN show that the tetramers rather than dimers or monomers can effectively bind and bridge cognate RNA oligonucleotides . ConsistBased on MS-based footprinting experiments in vitro, we previously found that positively charged residues within the CTD of IN  directly contact RNA, as was also validated by CLIP experiments . Our finThe mechanism by which IN-vRNA interactions mediate the encapsidation of vRNPs inside the CA lattice remains unknown. One possibility is that the temporal coordination of proteolytic cleavage events during maturation is influenced by IN-vRNA interactions . In thisWhile the mislocalization of the vRNA genome in eccentric particles can be accurately assessed by TEM analysis, precisely where IN is located in eccentric particles remains an open question. Earlier studies based on the biochemical separation of core components from detergent-treated IN R269A/K273A virions indicated that IN may also mislocalize outside the CA lattice . In thisWhy is the unprotected vRNA and IN prematurely lost in target cells? It seems evident that the protection afforded by the CA lattice matters the most for vRNP stability, though we cannot rule out that IN binding to vRNA may in and of itself stabilize both the genome and IN. Alternatively, the AU-rich nucleotide content of HIV-1 may destabilize its RNA , similarAlthough the basic aspects of virion maturation are conserved, particle morphologies are vastly different across retroviruses . It is cIn conclusion, we have identified IN-vRNA binding as the underlying factor for the role of IN in virion morphogenesis and show that virion morphogenesis is necessary to prevent the premature loss of vRNA and IN early in the HIV-1 lifecycle. Despite relatively high barriers, drugs that inhibit the catalytic activity of IN do select for resistance, and additional drug classes that inhibit IN activity through novel mechanisms of action would be a valuable addition to currently available treatments. The finding that IN-vRNA interaction can be inhibited in multiple ways – by directly altering residues in the IN CTD or by altering IN multimerization in virions – can help guide the design of future anti-retroviral compounds.NL4-3 -derived Gag-Pol sequence inserted into the pCR/V1 plasmid backbone  by overlap extension PCR. Briefly, forward and reverse primers containing IN mutations in the pol reading frame were used in PCR reactions with antisense and sense outer primers containing unique restriction endonuclease sites , respectively. The resulting fragments containing the desired mutations were mixed at a 1:1 ratio and overlapped subsequently using the sense and antisense primer pairs. The resulting fragments were digested with the corresponding restriction endonucleases and cloned into pNLGP and pNL4-3 plasmids. IN mutations were introduced into the pLR2P-vprIN plasmid using the QuickChange Site-Directed Mutagenesis kit (Agilent Technologies). The presence of the desired mutations and the absence of unwanted secondary changes were verified by Sanger sequencing. HIV-1 CA K203A substitution, which destabilizes the CA lattice  and verified to be free of contamination during these studies. HEK293T cells (ATCC CRL-11268) and HeLa-derived TZM-bl cells (NIH AIDS Reagent Program) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. MT-4 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. CHO K1-derived pgsA-745 cells  that lack a functional xylosyltransferase enzyme and as a result do not produce glycosaminoglycans were maintained in Dulbecco’s modified Eagle’s/F12 (1:1) media supplemented with 10% fetal bovine serum and 1 mM L-glutamine. Single-cycle GFP reporter viruses pseudotyped with vesicular stomatitis virus G protein (VSV-G) were produced by transfection of HEK293T cells with pNLGP-derived plasmids, the CCGW vector genome carrying GFP, and VSV-G expression plasmid at a ratio of 5:5:1, respectively, using polyethyleneimine . Full-length viruses pseudotyped with VSV-G were produced by transfecting HEK293T cells with the pNL4-3-derived plasmids and VSV-G plasmid at a ratio of 4:1 (pNL4-3:VSV-G).Viral and cell lysates were resuspended in sodium dodecyl sulfate (SDS) sample buffer and separated by electrophoresis on Bolt 4–12% Bis-Tris Plus gels (Life Technologies), blotted onto nitrocellulose membranes and probed overnight at 4°C with the following antibodies in Odyssey Blocking Buffer (LI-COR): mouse monoclonal anti-HIV p24 antibody , mouse monoclonal anti-HIV integrase antibody , rabbit MT-4 cells were grown in 24-well plates and infected with VSV-G pseudotyped pNL4-3 viruses (either WT or class II IN mutant) at a multiplicity of infection (MOI) of 2 in the presence of polybrene. After 6 hr, post-infection cells were collected, pelleted by brief centrifugation, and resuspended in PBS. DNA was extracted from cells using the DNeasy Blood and Tissue Kit (Qiagen) as per kit protocol. Quantity of HIV-1 vDNA was measured by Q-PCR using primers specific for early reverse-transcripts.NL4-3 IND116N) was trans-complemented with class II mutant IN proteins as described previously , VSV-G, and derivatives of the pLR2P-vprIN plasmid bearing class II IN mutations at a ratio of 6:1:3. Two days post-transfection cell-free virions were collected from cell culture supernatants. Integration capability of the trans-complemented class II IN mutants was tested by infecting MT-4 cells and measuring the yield of progeny virions in cell culture supernatants over a 6 d period as described previously  and UV-crosslinked. Following lysis in RIPA buffer, IN-RNA complexes were immunoprecipitated using a mouse monoclonal anti-IN antibody , a membrane-permeable crosslinker, at a concentration of 1 mM for 30 min at room temperature. Crosslinking was stopped by the addition of SDS sample buffer. Samples were subsequently separated on 3–8% Tris-acetate gels and analyzed by immunoblotting using a mouse monoclonal anti-IN antibody (HEK293T cells grown on 10 cm dishes were transfected with 10 μg pNL4-3 plasmid DNA containing the WT sequence or indicated antibody .6 tagged pNL4-3-derived IN by QuikChange site-directed mutagenesis kit (Agilent) (6 tagged recombinant pNL4-3 WT and mutant INs were expressed in BL21 (DE3) E. coli cells followed by nickel and heparin column purification as described previously (All of the mutations were introduced into a plasmid backbone expressing HisAgilent) . His6 taeviously . Recombi6 tagged IN fractions  were incubated with nickel acceptor beads while increasing concentrations of biotinylated-TAR RNA was incubated with streptavidin donor beads in buffer containing 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 mg/mL BSA, and 25 mM Tris (pH 7.4). Followed by 2 hr incubation at 4°C, they were mixed and the reading was taken after 1 hr incubation at 4°C by PerkinElmer Life Sciences Enspire multimode plate reader. The Kd values were calculated using OriginLab software.Following SEC of IN as above, individual fractions of tetramer, dimer, and monomer forms were collected and their binding to TAR RNA was analyzed by an Alpha screen assay as described previously . Briefly2, 1 mM DTT, 1 mg/mL BSA, and 25 mM Tris (pH 7.4). After 2 hr incubation at 4°C, indicated concentrations of IN were added to the reaction mixture and incubated further for 1.5 hr at 4°C. AlphaScreen signals were recorded with a PerkinElmer Life Sciences Enspire multimode plate reader.The RNA bridging property of IN was analyzed by AlphaScreen-based assay as described . Briefly7 cells per dish) were transfected with 30 μg full-length proviral plasmid (pNL4-3) DNA containing the WT sequence or indicated pol mutations within IN coding sequence using PolyJet DNA transfection reagent as recommended by the manufacturer (SignaGen Laboratories). Two days after transfection, cell culture supernatants were filtered through 0.22 μm filters, and pelleted by ultracentrifugation using a Beckman SW32-Ti rotor at 26,000 rpm for 2 hr at 4°C. Fixative  was gently added to resulting pellets, and samples were incubated overnight at 4°C. The following steps were conducted at the Harvard Medical School Electron Microscopy core facility. Samples were washed with 0.1 M sodium cacodylate, pH 7.4 and postfixed with 1% osmium tetroxide/1.5% potassium ferrocyanide for 1 hr, washed twice with water, once with maleate buffer (MB), and incubated in 1% uranyl acetate in MB for 1 hr. Samples washed twice with water were dehydrated in ethanol by subsequent 10 min incubations with 50%, 70%, 90%, and then twice with 100%. The samples were then placed in propyleneoxide for 1 hr and infiltrated overnight in a 1:1 mixture of propyleneoxide and TAAB Epon (Marivac Canada Inc). The following day the samples were embedded in TAAB Epon and polymerized at 60°C for 48 hr. Ultrathin sections (about 60 nm) were cut on a Reichert Ultracut-S microtome, transferred to copper grids stained with lead citrate, and examined in a JEOL 1200EX transmission electron microscope with images recorded on an AMT 2 k CCD camera. Images were captured at 30,000× magnification, and over 100 viral particles per sample were counted by visual inspection.Cell-free HIV-1 virions were isolated from transfected HEK293T cells. Briefly, cells grown in two 15 cm cell culture plates  and ultracentrifuged using a Beckman SW55-Ti rotor at 28,500 rpm for 16 hr at 4°C. Fractions (500 μL) collected from the top of the gradients were analyzed for IN, CA, and MA by immunoblotting as detailed above.Equilibrium density sedimentation of virion core components was performed as previously described . BrieflyBiochemical analysis of retroviral cores in infected cells was performed as described previously . BrieflyNL4-3 virus stocks were prepared as described above and concentrated 40X using a lentivirus precipitation solution (ALSTEM). PgsA-745 cells were plated on 1.5 mm collagen-treated coverslips  placed in 24-well plates 1 d before infection. Synchronized infections were performed by incubating pre-chilled virus inoculum on the cells for 30 min at 4°C. Cells were infected with WT virus at an MOI of 0.5, or with an equivalent number  of IN mutant viral particles. After removal of the virus, inoculum cells were washed with PBS and either immediately fixed with 4% paraformaldehyde, or incubated at 37°C for 2 hr before fixing. To prevent loss of vRNA due to reverse-transcription, cells were infected and incubated in the presence of 25 μM nevirapine. Following fixation, cells were dehydrated with ethanol and stored at −20°C. Before probing for vRNA, cells were rehydrated, incubated in 0.1% Tween in PBS for 10 min, and mounted on slides. Probing was performed using RNAScope probes and reagents (Advanced Cell Diagnostics). Briefly, coverslips were treated with protease solution for 15 min in a humidified HybEZ oven (Advanced Cell Diagnostics) at 40°C. The coverslips were then washed with PBS and pre-designed anti-sense probes  protocol . VSV-G pe probes  specificImages were taken using a Zeiss LSM 880 Airyscan confocal microscope equipped with a × 63/1.4 oil-immersion objective using the Airyscan super-resolution mode. Ten images were taken for each sample using the × 63 objective. Numbers of nuclei and vRNA punctae in images were counted using Volocity software (Quorum Technologies). The number of vRNA punctae per 100 nuclei were recorded at 0 hr post-infection (hpi) and 2 hpi for each virus, and the number at 2 hpi compared to the number at 0 hpi.MT-4 cells were infected with VSV-G pseudotyped HIV-1 NL4-3 WT or an equivalent number of mutant viruses  synchronously at 4°C. After removal of virus inoculum and extensive washes with PBS, cells were incubated at 37°C for 6 hr in the presence of 25 μM nevirapine. Immediately after synchronization (0 hr) and at 2 and 6 hr post-infection samples were taken from the infected cultures and RNA was isolated using TRIzol Reagent. The amount of vRNA was measured by Q-RT-PCR.For each experiment, IN signal was normalized to CA signal for each virus, and the resulting value compared to that of WT (set at 100%). Reported values are the average value (as a percent of WT) and standard deviation (SD) between four independent experiments. Mutants with less than 20% IN signal of WT are highlighted in gray. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The results presented in this manuscript point to an essential role for integrase/RNA interactions during maturation of virions. The experimental data is solid and clearly presented.Decision letter after peer review:eLife. Your article has been reviewed by Päivi Ojala as the Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal their identity: Eric Freed (Reviewer #2).Thank you for submitting your article "Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study, Elliott and colleagues investigate the properties of "class II" HIV-1 integrase (IN) mutants. These class II HIV-1 integrase mutants do not compromise the catalytic activity of the enzyme but severely impair viral replication. The underlying mechanisms are not well understood, but aberrant virion morphology is a hallmark of this class of mutant.The authors suggest that these mutants interfere with binding between IN and the viral RNA by one of three mechanisms: (1) reducing IN levels in virions, (2) inhibiting IN multimerization, and/or (3) directly disrupting IN-RNA interactions. The results point to an essential role for integrase/RNA interactions during maturation of virions.This study provides significant new insights into the properties of class II IN mutants, which have for long been of interest and display properties that are mirrored by allosteric IN inhibitors. The experimental data is solid and clearly presented.Essential revisions:The authors analyze a diverse collection of previously obscure class II IN mutants and provide evidence that many of them are defective for the formation of tetramers and that it is the tetrameric form that binds viral RNA. Their data seem to clarify the location of defective IN, which they show is associated with HIV-1 cores , while the RNA genome is outside . It was refreshing to see all these mutants, including δ-IN and H12N, analyzed using several complementary approaches side-by-side. However, a few issues outlined below need to be carefully addressed.1) The conclusion that RNA binding is exclusive to the tetrameric form of IN is very important in this work, and it does make sense. Please add more evidence that the HIV needs IN tetramer to bind RNA. In the current manuscript, this conclusion is based on a single experiment : WT IN was separated into tetramer, dimer and a monomer by SEC, and the three fractions are tested for the ability to bind RNA. Are these forms really not supposed to interconvert? For example, what if dimers do bind RNA, but they fall apart during chromatography and/or downstream processing? Would it not be better to test RNA binding with class II mutants that fail to form tetramers ? In addition, there are HIV-1 IN mutants that do not form tetramers as recombinant proteins ; would these not bind RNA? It would be essential to expand Figure 4D including mutations of residues that are not expected to interact with vRNA directly. Because IN mutants stick with cores whilst vRNA is not , they may be physically separated within virions. The CLIP data  is consistent with this and could be a consequence of the physical separation. This is another reason to test RNA binding by these mutants directly.The crosslinking results in Figure 4A do not seem very convincing: there are high-MW IN species visible for all mutants. Perhaps some of them are hetero-multimer crosslinks (i.e. with other proteins in the virion?). SEC data, such as in panel B, are much clearer; I would suggest including more mutants in this analysis.Moreover, it is not clear how reproducible the cross-linking patterns are (and the WT in the third panel looks very different from the WTs in the first and second panels). The cross-linking results, to the extent that they can be interpreted, appear to be very different from the SEC profiles shown in Figure 4B. The authors conclude (subsection “IN multimerization plays a key role in RNA binding”) that the majority of class II IN mutants are predominantly monomeric in virions, yet the majority of the protein is found in high-MW aggregates. This issue needs to be addressed.2) The key issue of whether the IN mutants are excluded or not from viral cores needs to be clarified. The sedimentation of the majority of IN mutants in the dense, CA-containing fractions in Figure 6A would seem to suggest that these mutant IN proteins are located within the viral capsid. However, in the cell-based uncoating assay, the IN mutants are degraded  while a high percentage of capsids remain intact at the 2 hour time point . These data suggest that the mutant IN proteins are excluded from the protective environment of the capsid and are thus rapidly degraded. Indeed, in the model figure , these mutants are depicted as being outside the cores. One possible solution to this conundrum is that the IN mutants are prone to aggregation under the conditions used in Figure 6 and thus co-sediment with CA without actually being inside the capsid. In any case, as currently presented, the data appear to be in contradiction and leave this important question unresolved.The data presented in Figure 8 suggest that the class II IN mutations do not affect the assembly or stability of the CA lattice either in vitro or in target cells . Yet, in the Figure 8 model, the capsid is shown as discontinuous or "broken" in the context of the IN mutants, a feature that would presumably significantly compromise capsid stability. The model thus appears to be inconsistent with the data.3) The difference between tetramers, dimers and monomers in Figure 4C is surprising. The species would be expected to re-equilibrate after SEC. Please comment. Essential revisions:The authors analyze a diverse collection of previously obscure class II IN mutants and provide evidence that many of them are defective for the formation of tetramers and that it is the tetrameric form that binds viral RNA. Their data seem to clarify the location of defective IN, which they show is associated with HIV-1 cores , while the RNA genome is outside . It was refreshing to see all these mutants, including δ-IN and H12N, analyzed using several complementary approaches side-by-side. However, a few issues outlined below need to be carefully addressed.1) The conclusion that RNA binding is exclusive to the tetrameric form of IN is very important in this work, and it does make sense. Please add more evidence that the HIV needs IN tetramer to bind RNA. In the current manuscript, this conclusion is based on a single experiment : WT IN was separated into tetramer, dimer and a monomer by SEC, and the three fractions are tested for the ability to bind RNA. Are these forms really not supposed to interconvert? For example, what if dimers do bind RNA, but they fall apart during chromatography and/or downstream processing? Would it not be better to test RNA binding with class II mutants that fail to form tetramers ? In addition, there are HIV-1 IN mutants that do not form tetramers as recombinant proteins ; would these not bind RNA? It would be essential to expand Figure 4D including mutations of residues that are not expected to interact with vRNA directly. Because IN mutants stick with cores whilst vRNA is not , they may be physically separated within virions. The CLIP data  is consistent with this and could be a consequence of the physical separation. This is another reason to test RNA binding by these mutants directly.We thank the reviewer for the insightful comments. As suggested, we have determined the RNA binding properties of additional mutants that do not form tetramers in vitro  alongside with K34A, R262A/R263A and the canonical class II IN mutant V165A using an RNA binding and RNA-bridging assay  as shown in Figure 5 and Figure 5—figure supplement 1 of the current manuscript. The latter approach relies on IN-mediated bridging of fluorescently labeled target RNA oligonucleotides (i.e. TAR) and as such is a good proxy for assessing the multimerization-dependent RNA binding ability of IN. First, we show that all other class II IN mutants have reduced affinities towards RNAs. Second, we show that WT IN tetramers but not dimers are able to bridge between separate target RNAs. Third, we show that all of the class II IN mutants tested exhibit substantially lower levels of RNA bridging activity. Taken together, these results strongly argue that IN multimerization is critical for the RNA binding/bridging ability of IN. Responses to the other suggestions/criticisms, which were also raised by other reviewers, are detailed below.2) The crosslinking results in Figure 4A do not seem very convincing: there are high-MW IN species visible for all mutants. Perhaps some of them are hetero-multimer crosslinks (i.e. with other proteins in the virion?). SEC data, such as in panel B, are much clearer; I would suggest including more mutants in this analysis.Moreover, it is not clear how reproducible the cross-linking patterns are (and the WT in the third panel looks very different from the WTs in the first and second panels). The cross-linking results, to the extent that they can be interpreted, appear to be very different from the SEC profiles shown in Figure 4B. The authors conclude (subsection “IN multimerization plays a key role in RNA binding”) that the majority of class II IN mutants are predominantly monomeric in virions, yet the majority of the protein is found in high-MW aggregates. This issue needs to be addressed.The reviewer raises important points and we addressed these concerns as follows. First, we have now included SEC profiles of other IN mutants , which show that many formed dimers but had defects in tetramerization in vitro . Second, we have generated a table (Supplementary file 2), which summarizes published work on the multimerization properties of the class II IN mutants studied herein. Third, we have now included quantitation of IN multimers from three independent biological replicates for in virion crosslinking experiments in Figure 4A, which convincingly illustrate the multimerization defects of the IN mutants. Fourth, we have changed the text to clarify the experimental results.We agree with the reviewer that in these experiments, a large fraction of the signal is found in high MW aggregates. While the multimerization experiments in virions are more physiologically relevant, they are also inherently more complex. For example, we cannot distinguish whether these high molecular weight species represent aberrant IN multimerization products, aggregates or crosslinking between IN and other viral proteins. Another possibility is that these high molecular weight species may not resolve well in this system on the 6% Tris-acetate gels, which unfortunately is a limitation of this approach. Notwithstanding, we believe that the quantitation of data from multiple experiments as provided now reveals consistent patterns in the multimerization properties of the class II mutants in virions.Finally, we do not think that in vitro SEC profiles can be directly compared to the results obtained from in virion crosslinking experiments for several reasons. First, as chemical crosslinking is not 100% efficient and because the crosslinked proteins are separated under denaturing/reducing conditions, the in virion crosslinking assay may not report on the actual form and amount of IN multimers in virions. Second, as indicated above, crosslinking experiments done in virions are inherently more complex due to the presence of other viral proteins/RNAs that may be within close proximity of IN. Third, while the in vitro SEC experiments provide key information on the intrinsic properties of IN, they may not correlate 100% with the actual multimeric form of IN within the more complex setting of the virion. Thus, while each approach has limitations, we believe that they are complementary and highlight the importance of IN multimerization in RNA binding as a whole.3) The key issue of whether the IN mutants are excluded or not from viral cores needs to be clarified. The sedimentation of the majority of IN mutants in the dense, CA-containing fractions in Figure 6A would seem to suggest that these mutant IN proteins are located within the viral capsid. However, in the cell-based uncoating assay, the IN mutants are degraded  while a high percentage of capsids remain intact at the 2 hour time point . These data suggest that the mutant IN proteins are excluded from the protective environment of the capsid and are thus rapidly degraded. Indeed, in the model figure , these mutants are depicted as being outside the cores. One possible solution to this conundrum is that the IN mutants are prone to aggregation under the conditions used in Figure 6 and thus co-sediment with CA without actually being inside the capsid. In any case, as currently presented, the data appear to be in contradiction and leave this important question unresolved.The reviewer raises an important and interesting point that we also have thought of. We completely agree with the reviewer on the conclusions derived from the data and have clarified this point further in the text. Demonstrating precisely where within the virions class II IN mutants are located extends beyond the scope of this paper and future studies are warranted to study this process in detail. For example, we tried to directly assess where IN is located in virions by immunogold EM; however, these experiments suffered from low resolution and contrast due to the differences in this particular protocol compared to the standard EM procedures conducted in this study. This was compounded by the epitope inaccessibility for the antibodies used  under the fixation conditions required for immunogold-EM experiments.As an alternative, we took a biochemical approach to assess whether we can separate class II IN mutants from the CA lattice. To this end, we took advantage of previously defined capsid destabilizing mutation K203A. We reasoned that if the class II IN mutants are not bound to RNA and kept inside the CA lattice, CA destabilization would alter their migration pattern in biochemical fractionation experiments as done in Figure 7 of this manuscript. To our surprise, we found that CA destabilization did not alter the migration patterns of class II IN mutants. To completely rule out the possibility that class II IN mutants are still bound to RNA (which would explain their migration in dense fractions), we conducted similar experiments in viruses lacking NC, and thus are devoid of RNA, or in the presence of RNase throughout the fractionation protocol. Migration pattern of IN remained unaffected under these conditions either, making it difficult to assess whether class II IN mutants are located inside the CA lattice or are aggregated. These data are now included in the Figure 7—figure supplement 1. Based on these results, and as suggested by the reviewer, we favor the model that class II IN mutants are mislocalized outside of the CA lattice but likely form aggregates as they are prematurely degraded in target cells. This point is now clearly made in the text leading to this figure and in Discussion section.4) The data presented in Figure 8 suggest that the class II IN mutations do not affect the assembly or stability of the CA lattice either in vitro or in target cells . Yet, in the Figure 8 model, the capsid is shown as discontinuous or "broken" in the context of the IN mutants, a feature that would presumably significantly compromise capsid stability. The model thus appears to be inconsistent with the data.We agree with the reviewer that this is an important point. We modified the model such that the CA lattice is complete as the data provided here do indeed suggest that CA assembly and stability are unaffected. The possible reasons for the discrepancy between our work and Fontana et al., 2015 are discussed in the text. The current text reads:“Although this finding is in disagreement with the previously observed morphological aberrations of the CA lattice present in eccentric particles [26], it is possible that the biochemical experiments used herein lack the level of sensitivity required to quantitatively assess these aberrations or that the cryo-ET procedure impacts the structure of the CA lattice in particular in eccentric virions due to the absence of a packaged vRNP complex. A further possibility is that while the CA lattice in class II IN mutant virions may appear morphologically aberrant, they may still uncoat similar to WT virions in target cells.”5) The difference between tetramers, dimers and monomers in Figure 4C is surprising. The species would be expected to re-equilibrate after SEC. Please comment.We agree with the reviewer that this is an important point. While it is possible that the collected monomers and multimeric IN species can interconvert, we minimize this by subjecting the collected fractions from SEC immediately to the α screen assay. Thus, even if a fraction of dimers is converting to tetramers, it is unlikely to have a significant impact on the overall outcome of these experiments. The fact that we are observing a profound difference in the RNA binding properties of tetramers vs. dimers and monomers (for WT) indicates that the samples do not re-equilibrate at appreciable levels during the course of these experiments. Otherwise, we would observe similar levels of binding to viral RNAs.