During lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV), a nuclear viral long noncoding RNA known as PAN RNA becomes the most abundant polyadenylated transcript in the cell. Knockout or knockdown of KSHV PAN RNA results in loss of late lytic viral gene expression and, consequently, reduction of progeny virion release from the cell. Here, we demonstrate that knockdown of PAN RNA from the related Rhesus macaque rhadinovirus (RRV) phenocopies that of KSHV PAN RNA. These two PAN RNA homologs, although lacking significant nucleotide sequence conservation, can functionally substitute for each other to rescue phenotypes associated with the absence of PAN RNA expression. Because PAN RNA is exclusively nuclear, previous studies suggested that it directly interacts with host and viral chromatin to modulate gene expression. We studied KSHV and RRV PAN RNA homologs using capture hybridization analysis of RNA targets (CHART) and observed their association with host chromatin, but the loci differ between PAN RNA homologs. Accordingly, we find that KSHV PAN RNA is undetectable in chromatin following cell fractionation. Thus, modulation of gene expression at specific chromatin loci appears not to be the primary, nor the pertinent function of this viral long noncoding RNA. PAN RNA represents a cautionary tale for the investigation of RNA association with chromatin whereby cross-linking of DNA spatially adjacent to an abundant nuclear RNA gives the appearance of specific interactions. Similarly, PAN RNA expression does not affect viral transcription factor complex expression or activity, which is required for generation of the late lytic viral mRNAs. Rather, we provide evidence for an alternative model of PAN RNA function whereby knockdown of KSHV or RRV PAN RNA results in compromised nuclear mRNA export thereby reducing the cytoplasmic levels of viral mRNAs available for production of late lytic viral proteins. Herpesviruses produce noncoding RNAs, some of which are essential to the viral life cycle. One such noncoding RNA from Kaposi’s sarcoma-associated herpesvirus is the polyadenylated, nuclear (PAN) RNA, which is required for production and release of progeny virions from infected cells. In this study, we demonstrate that although lacking nucleotide sequence conservation, PAN RNAs from two related viruses–when knocked down–exhibit the same phenotype, loss of late lytic viral gene expression and progeny virion production. Moreover, they can functionally substitute for each other to rescue this phenotype. We demonstrate that, in contrast to published literature, the reduction in viral gene expression upon PAN RNA knockdown is not due to loss of PAN RNA association with conserved, specific chromatin loci, nor does PAN RNA expression affect the viral transcription factor complex required for generation of the late lytic viral mRNAs. We present data suggesting that PAN RNA instead serves as a binding platform to sequester cellular proteins that are mislocalized to the nucleoplasm. These herpesviral noncoding RNAs can serve as models for the mechanistic study of human noncoding RNAs. Crystallographic analysis of the PAN ENE complexed with an A9 oligonucleotide revealed that the U-rich internal loop of the ENE forms a triple-stranded interaction with the poly(A) tail of its own transcript [Kaposi’s sarcoma-associated herpesvirus (KSHV) is an opportunistic pathogen of human immunodeficiency virus (HIV) patients and the etiological agent of several human cancers, including Kaposi sarcoma and primary effusion lymphoma . The KSHanscript . This transcript –8 robustAlthough the ENE has been well studied, we still know little about the function of PAN RNA. Previous work has demonstrated that loss of PAN RNA, either through genetic knockout from the viral genome or antisense depletion of the transcript, results in misregulation of late lytic viral genes and host immune response genes , 9. The In vivo data suggested that PAN RNA may regulate chromatin states by competitively preventing LANA from associating with histone H3, an interaction required for regulating and maintaining latency [PAN RNA is exclusively nuclear, which prompted efforts to demonstrate that KSHV PAN RNA associates directly with the human and viral genomes , 11. A p latency . On the  latency . KSHV PAGenes encoding PAN RNA homologs map to syntenic regions within gammaherpesvirus genomes. As is common for many long noncoding RNAs (lncRNAs) , 15, PANRRV PAN RNA is a ~1.3-kb transcript found in the nuclei of lytic RRV-infected cells. As is the case for KSHV PAN RNA, the master herpesviral lytic activator ORF50 binds the promoter and activates expression of RRV PAN RNA . AlthougKSHV and RRV have well-established cell culture models. The BCBL-1 cell line is a clinical isolate from a body cavity-based lymphoma and is a naturally KSHV-infected human B-lymphocyte cell line. A comparable RRV-infected rhesus B-cell line does not exist; all known rhesus B-lymphocyte lines were immortalized using another human herpesvirus, Epstein-Barr virus (EBV). EBV and KSHV co-infections are known to collude and produce results different from those with KSHV alone  and simiIn this study, we demonstrate that knockdown of PAN RNA from the related herpesvirus RRV phenocopies that of KSHV PAN RNA knockdown. Furthermore, despite lacking nucleotide sequence conservation, expression of either KSHV or RRV PAN RNA can rescue production of progeny virions by either KSHV or RRV bacmids lacking the PAN RNA locus. Using CHART  , we reviTo determine whether KSHV and RRV PAN RNA homologs perform the same function during the viral life cycle, we characterized the phenotype associated with knocking down RRV PAN RNA expression in lytic BJAB RRV cells. Anti-RRV antibodies useful for investigating the effect of PAN RNA expression on late lytic gene expression are not available at this time; however, quantitative PCR (qPCR) can assess viral DNA replication and virion production to confirm the downstream phenotypic changes associated with KSHV PAN RNA loss .O-methylated and phosphorothioate-substituted antisense oligonucleotides (ASOs) that target RRV PAN RNA for RNaseH cleavage. 40 h after lytic induction with trichostatin-A (TSA), total RNA was harvested from a subset of the cells and analyzed by Northern blot to confirm knockdown of RRV PAN RNA expression  revealed that the reciprocal PAN RNA homolog failed to rescue the loss of released encapsulated virus observed after PAN RNA knockdown . Yet, leConsequently, we performed rescue experiments with PAN RNA knockout bacmids . We atteNext, we tested the ability of KSHV versus RRV PAN RNA to rescue the corresponding PAN RNA deletion bacmids. HEK293T cells were first transiently transfected with KSHV BAC36CRΔPAN bacmid , the KSHWe then performed the converse PAN RNA rescue in the context of the RRVΔPAN genome. HEK293T cells transiently transfected with RRVΔPAN bacmid were induced into the lytic phase by transiently transfecting rORF50/RTA in the presence of 100 nM TSA. The complete knockout of RRV PAN RNA from the bacmid reduced the level of encapsulated virus released into the medium in the presence of a vector control to about 10% relative to a RRV PAN RNA expression vector . SimilarPrevious studies using ChIRP suggested that KSHV PAN RNA associates broadly with host and viral chromatin , 11. We Expression profiles of PAN RNA during the lytic phase were analyzed after induction of BJAB RRV cells or KSHV-carrying BCBL-1 cells with 500 nM TSA or 600 μM valproic acid, respectively; RNA was collected at multiple time points. For the CHART studies, we assessed PAN RNA-chromatin association when the lncRNAs were at 25%, 75% and 100% of the maximal expression level. Northern blot analysis of KSHV and RRV PAN RNA revealed that these values were achieved at 18, 24 and 48 h for KSHV  and 16, Candidate CHART capture oligonucleotides were designed according to specifications of the CHART protocol  and theiThe pulldown efficiencies of RNase H-candidate CHART capture oligonucleotides were determined by incubating biotin-labeled oligonucleotides with streptavidin beads and nuclear lysate pre-cleared with unbound beads. After stringent high-salt washing, RNAs purified on the streptavidin beads were analyzed by Northern blot . To reduWe evaluated PAN RNA association with chromatin for both KSHV and RRV using two separate antisense capture oligonucleotide sets and several time points representing different levels of PAN RNA expression. BCBL-1 cells containing KSHV and BJAB cells containing RRV were induced into the lytic phase with 600 μM valproic acid or 500 nM TSA, respectively, for up to 48 h prior to performing CHART enrichment of PAN RNA-associated genomic DNA. CHART peaks were called for each time point, relative to input, using MACS2 software see . Any genThe lack of overlap between binding sites on host chromatin for the two PAN RNA homologs was not limited by the high-throughput CHART procedure because after isolating genomic DNA associated with KSHV PAN RNA, putative KSHV CHART peaks, but not RRV CHART peaks, could be verified as enriched by qPCR . PublishTo evaluate whether the same protein factor might bind both PAN RNA homologs, but recruit the lncRNAs to separate, non-overlapping sites on the genome, we compared the PAN RNA CHART peaks to 275 ENCODE eCLIP (enhanced crosslinking and immunoprecipitation) datasets and 162 ENCODE ChIP (chromatin immunoprecipitation) datasets. Of the 276 factors represented in these two analyses, none overlapped with greater than 15% of either the KSHV or RRV CHART peaks . Additio4Cl) were used in the same fractionation protocol to minimize the chance that the absence of PAN RNA signal in the chromatin was due to the fractionation buffer. These salts lie on different points along the Hofmeister series–a classification that orders ions by their ability to stabilize and solubilize proteins [Finally, we fractionationated lytic BCBL-1 cells and analyzed the distribution of PAN RNA between the nucleoplasm and chromatin by Northern blot . Surprisproteins . The quaproteins , 29, indLoss of KSHV PAN RNA results in reduced K8.1 and ORF65 late lytic protein expression , Figs 2  2 and S1To test whether KSHV PAN RNA affects vPIC transcriptional activity, we (1) assessed whether knockdown of PAN RNA alters the steady-state mRNA level of any vPIC component; (2) analyzed vPIC-regulated transcript levels upon PAN RNA knockdown; and (3) monitored the influence of PAN RNA expression on a dual luciferase reporter system that expresses firefly luciferase from a vPIC-dependent promoter. At 48 h post lytic induction of BCBL-1 cells, when late lytic transcription is underway, qPCR analyses revealed that the steady-state levels of neither vPIC component mRNAs nor vPIC-regulated transcripts were significantly altered upon knockdown of PAN RNA, as compared to control knockdown samples . The appTo directly test whether PAN RNA expression affects the activity of the vPIC, we took advantage of a luciferase reporter system whose expression is activated only in the presence of all six viral transcription factors . Upon trTo determine how loss of PAN RNA results in misregulation of late lytic viral genes, we induced BCBL-1 TREx cells into the lytic phase for 24 h with 1 μg/mL doxycycline following knockdown of PAN RNA or, as a control, GFP mRNA. Doxycycline regulates expression of the inducible gene encoding the lytic activator RTA/ORF50. As observed previously, knockdown of KSHV PAN RNA using two separate antisense oligonucleotides resulted in a specific reduction in late lytic viral protein expression (K8.1 and ORF65)  and in pPAN RNA is predominantly, if not exclusively nuclear. Therefore direct effects on the cytoplasmic processes of protein production or decay are unlikely. Nonetheless, we assessed whether protein decay rates are altered in the absence of PAN RNA expression. After 48 h of lytic induction, BCBL-1 cells were treated with the translation elongation inhibitor cycloheximide. At 0, 8, 24 and 30 h of translation inhibition, equivalent numbers of cells were harvested and analyzed by Western blot for the early viral protein ORF6, late viral proteins K8.1 and ORF65, as well as the host protein GAPDH . Little To evaluate whether PAN RNA affects export of viral mRNA from the nucleus, we fractionated lytically-induced BCBL-1 cells following knockdown of either PAN RNA or, as a control, GFP mRNA. The quality of fractionation was assessed by Western blot and by qRT-PCR  for a cyWe have shown that two herpesviral PAN RNA homologs are functionally interchangeable, despite lacking appreciable nucleotide sequence conservation. Knockdown or knockout of KSHV PAN RNA was previously shown to result in loss of late lytic protein expression, and consequently, a reduction in release of new virions into the surrounding media , 10. In The herpesviral noncoding PAN RNA is essential to the viral life cycle, but the molecular mechanism by which this lncRNA acts remains unclear. Seven hypothetical models for PAN RNA function have been proposed –44. ThreWe tested whether the two PAN RNA homologs might be localized on separate, non-overlapping regions in the genome either by a DNA binding protein or an RNA binding protein associated with nascent RNA. We compared our PAN RNA CHART peak datasets to all ENCODE CHIP and eCLIP datasets available at the time of our analysis. Of the 264 proteins included in this analysis, none exhibited greater than 15% overlap with either of our datasets. The protein that showed the greatest overlap (14.3%) with the KSHV PAN RNA dataset, but was nearly absent from that of RRV, was FOXK1. The binding site for a different forkhead protein, FOXD3, was enriched in PAN RNA ChIRP peaks . ForkheaPrevious work studying PAN RNA association with chromatin used a technique similar to CHART, known as ChIRP . The ChIin vitro binding studies, to be due to inhibition of virion production. The unpackaged state of newly synthesized viral genomes in the absence of PAN RNA may lead to increased accessibility for LANA binding.Campbell and colleagues found that the knockdown of PAN RNA leads to the recruitment of LANA protein at viral promoters, an activity normally associated with latency, suggesting a role for PAN RNA in latent-lytic transitions and chromatin association . HoweverAlthough PAN RNA is considered exclusively nuclear, ribosome footprint profiling detected ribosome-protected regions of KSHV PAN RNA within three open reading frames (ORFs) at the 5′ end of the transcript, which overlaps with K7 . Due to We suggest that the absence of PAN RNA during the viral lytic phase perturbs efficient nuclear export of mRNAs. A modest reduction in nuclear export of several viral and host mRNAs was observed when either KSHV or RRV PAN RNA was eliminated indicating that this is a conserved phenotype of PAN RNAs. Despite the change in the nuclear-cytoplasmic distribution of early viral transcripts, and host GAPDH and actin mRNA, we did not observe a change in corresponding protein levels. A reduction in mRNA export likely has a more pronounced effect on diminishing the level of late lytic viral proteins due to the absence of late lytic transcription and translation prior to PAN RNA expression. In contrast, expression of host transcripts and early viral transcripts may generate a protein population that remains unchanged for the duration of the PAN RNA knockdown experiments. Consistent with this hypothesis, the half-life of GAPDH has been estimated to be ~38 h–significantly longer than the time frame of our assays . WhetherWe hypothesize that PAN RNA’s function is to associate with RNA binding proteins, such as PABPC , that arPAN RNA represents a cautionary tale for the investigation of RNA association with chromatin, whereby sequence reads of DNA cross-linked to an abundant RNA give the appearance of specific interactions. Rather than the latest technological advances, well-controlled, unbiased approaches should be applied to determine the true biological role of the ncRNA under investigation. Protein-RNA co-purification studies may also be subject to false positive interactions. This has been especially true of PAN RNA, which–by virtue of its abundance and diffuse nuclear distribution–can transiently and non-specifically associate with many factors.Molecular tools available for the study of lncRNAs such as PAN RNA are still under development. Most lncRNAs are of low abundance and lack obvious nucleotide sequence conservation that aids in identification of essential sequence elements. Here, we have exploited the use of a ncRNA from a related virus to provide a genetically-tractable model system. Viruses thus offer a unique advantage over host ncRNAs for interrogating questions in RNA biology. PAN RNAs may be multifunctional and fulfill different roles at each stage of the lytic phase. Moving forward, carefully designed and innovative approaches are needed to expand insights into the multifaceted functions of gammaherpesviral PAN RNAs.Body cavity based lymphoma-1 (BCBL-1) , BCBL-1 The RRV wild-type bacmid was a kind gift from the Desrosiers lab (University of Miami) . This ba7 cells were resuspended in 100 μL/107 cells of Sucrose Buffer I  NP-40). After centrifuging the lysate at 500 x g for 5 min at 4°C, the supernatant was removed. Nuclei were washed in 1 mL Sucrose Buffer I lacking NP-40. After removing the supernatant, the nuclei pellet was resuspended in 20 μL/107 cells of Low Salt RNA Buffer . 1/5 volume of High Salt RNA Buffer  was added five times. The nuclear extract was then incubated at 4°C on a rotary platform for 20 min and diluted 1:2.5 with RNA Nuclear Diluent . 10 μL 100 μM DNA oligonucleotides were added to 90 μL of nuclear extract and incubated for 30 min at 37°C. Reactions were stopped by adding 0.5 mL TRIzol and purified according to the manufacturer’s protocol. Samples were run on a 1.2% formaldehyde agarose for a northern blot analysis.Endogenous RNaseH cleavage was assayed as described . BrieflyCHART was carried out as described . CHART chttps://pypi.python.org/pypi/MACS2) [www.encodeproject.org and compared to the KSHV and RRV peak genomic loci using the BEDTools suite intersect sub-command [50-bp deep sequencing reads were mapped using Bowtie2 default settings to the custom indices containing both the appropriate viral genome  and the human genome. Reads that mapped to multiple genomic loci were removed using samtools (samtools view input.bam | grep AS:i:0 | grep -v XS:i:0). MACS2 (i/MACS2)  was usedi/MACS2)  CHIP and-command .2, 10 mM beta-glycerophosphate, 0.5% Nonidet P-40). Digitonin  was added to a final concentration of 25 μg/mL while wheat germ agglutinin  was added to a final concentration of 1 μg/mL (BCBL-1) or 1 mg/mL (BJAB-RRV) and the cells were incubated on ice for 5 min to allow cell permeation. For lysis, the cells were carefully layered over 300 μL of RLB buffer containing sucrose  sucrose, 10 mM beta-glycerophosphate, 0.5% NP-40) and then centrifuged at 13,000 x g for 10 min at 4°C. After centrifugation, supernatants were transferred into a fresh microcentrifuge tube and designated as cytoplasmic fractions. The nuclear pellets were washed three times in 100 μL of RLB buffer and spun down at 400 x g for 4 min at 4°C; the first of which contained digitonin at a final concentration of 25 μg/mL. The pelleted nuclei were resuspended in 30 μL of NUN1 buffer  and incubated on ice for 5 min. The nuclei were then lysed by adding 300 μL of NUN2 buffer . After incubation on ice for 15 min with occasional vortexing and centrifugation at 15,000 x g for 15 min at 4°C, the supernatants were transferred to fresh microcentrifuge tubes as the nucleoplasmic fractions. The chromatin-associated pellets were washed three times with 50 μL of NUN2 buffer, spun at 15,000 x g for 4 min at 4°C, and 100 μL of chromatin buffer  added. The chromatin-associated pellet was then sonicated at 4°C for 10 cycles of 30 sec on, 30 sec off, using a Diagenode Bioruptor Pico sonication device. Input samples were sonicated using the same conditions. NaCl was replaced with LiCl or NH4Cl in all buffers for indicated fractionations. One half of each fraction was prepared for RNA and protein analysis. RNA was extracted with TRIzol per the manufacturer’s instructions. The purity of the resulting fractions was assessed by qPCR and Western blot.Fractionation was performed on ice with pre-chilled buffers using either 36 h-induced KSHV-infected BCBL-1 cells or 43 h-induced BJAB-RRV cells. The washed and pelleted cells were resuspended in 100 μL of RLB buffer . Total DNA was kept constant in each sample by adding pBluescript SK+. Viral transcription factor plasmids were transfected in equimolar amounts. Dual luciferase assays were conducted 24 h post transfection with the Dual-Luciferase Reporter Assay System . The reagents were prepared according to the manufacturer’s instructions and measurements were conducted with a GloMax-Multi Detection System (Promega). Luciferase measurements were recorded as the ratio of firefly to Renilla luciferase activity and normalized to sample 7 (pGL4.16 K8.1 vPIC). Data presented were from four independent experiments, each using mean values of technical triplicate samples.9 x 1016 K8.1; ) and a RRNA was purified with TRIzol and treated with RQ1 DNase (Promega) according to the manufacturer’s protocols. 1 μg of RNA was used to generate cDNA with random hexamer primers and Superscript III (Invitrogen) using the recommended protocol. cDNA was diluted 3-fold and 0.75 μL was analyzed in a 15-μL qPCR reaction using FastStart Essential DNA Green Master (Roche) SYBR reagent on a Roche Lightcycler 96. RNA levels were normalized to RNaseP RNA levels, which do not change during lytic induction. To determine the extent of PAN RNA overexpression in HEK293T cells relative to BCBL-1 cells, PAN RNA levels were normalized to the average CT value of five viral transcripts , which accounts for variances in viral genome copy number and induction efficiency between samples. For samples obtained from the same cell line, the fold-change in PAN RNA levels calculated using RNaseP RNA was comparable to the calculation using the five viral transcripts.°C, phenol extracted, ethanol precipitated and diluted to 30 ng/μL prior to analysis by qPCR. The average signal from two primer pairs specific to the viral genome was normalized to the average signal from two primer pairs specific to the human genome.Seven days after lytic phase induction, 3 million cells were pelleted, washed with PBS and resuspended in genomic DNA isolation buffer  with 0.1 mg/mL proteinase K. The solution was incubated overnight at 40°C. Proteinase K lysis buffer  was added to a final volume of 2 mL and then incubated at 40°C for 1 h. 1 ng/mL of a control plasmid (psiCHECK-2) was added to each sample as a normalization control for loss of DNA during subsequent phenol chloroform extraction and ethanol extraction. DNA was resuspended in 15 μL of ddH2O (resulting in 100-fold concentration) and analyzed by qPCR. The average signal from two primer pairs specific to the viral genome was normalized to the signal from the control plasmid.Seven days after lytic phase induction, 1.5 mL of supernatant was collected, passed through a 0.45 micron filter and incubated with 20 units/mL DNase One (New England Biolabs) for 1 h at 37S1 Appendix(XLSX)Click here for additional data file.S2 Appendix(XLSX)Click here for additional data file.S3 Appendix(XLSX)Click here for additional data file.S4 Appendix(XLSX)Click here for additional data file.S1 Fig(A) BCBL-1 TREx cells were electroporated with oligonucleotides antisense either to GFP mRNA (control KD) or to KSHV PAN RNA, as well as increasing amounts of plasmid encoding RRV PAN RNA under the control of the KSHV PAN RNA promoter. The total DNA concentrations were kept constant by adding empty pBluescript vector. Following electroporation, cells were induced into the lytic phase with 1.5 μg/mL doxycycline. 48 h after induction, a subset of the cells was harvested for Northern blot analysis of PAN RNA levels and for Western blot analysis of late lytic proteins K8.1 and ORF65 and early protein ORF6. (B) RT-qPCR quantification of PAN RNA levels relative to the average of five viral transcripts . RRV PAN represents data from KSHV PAN RNA KD with 15 μg RRV PAN RNA expression vector. (C) RT-qPCR analysis of the early viral transcript ORF18 and two late viral transcripts ORF26 and ORF67A. (D) Seven days after lytic induction, DNase-resistant encapsulated viral DNA levels in the media were assessed by qPCR and normalized to an external loading control added at the onset of viral DNA isolation. (E) Seven days after lytic induction, intracellular DNA was harvested and the level of intracellular viral DNA relative to host DNA was determined by qPCR. The average signal from two primer pairs specific to the viral genome was normalized to the average signal from two primer pairs specific to the human genome. (F) BJAB RRV cells were electroporated with oligonucleotides antisense either to GFP mRNA (control KD) or to RRV PAN RNA, as well as increasing amounts of plasmid encoding KSHV PAN RNA under the control of the RRV PAN RNA promoter. The total DNA concentration was kept constant by adding empty pBluescript vector. Following electroporation, cells were induced into the lytic phase with 100 nM TSA. 40 h after induction, a subset of the cells was harvested for Northern blot analysis of PAN RNA levels and for Western blot analysis of late lytic protein expression. In the same manner as described above, PAN RNAs levels (G), viral transcript levels (H), extracellular released viral DNA (I) and intracellular viral DNA (J) were analyzed. Data are the average of at least two biological replicates; error bars represent standard deviations of the mean.(TIF)Click here for additional data file.S2 FigΔPAN RRV bacmids.(A) Sequence of the DNA cassette inserted at the RRV PAN RNA locus. The entire PAN RNA sequence was deleted, including 140 bps upstream and 22 bp downstream that are necessary to express RRV PAN RNA . A 1641-(TIF)Click here for additional data file.S3 Fig(A) qPCR of DNA isolated by KSHV PAN RNA CHART oligonucleotide set 1 see  using pu(TIF)Click here for additional data file.S4 Figwww.cbs.dtu.dk/services/SignalP/). C-score predicts cleavage site, S-score predicts signal peptide location, Y-score is a combined cleavage site score that accounts for the location of the signal peptide.(A) Sequence alignment of a portion of the KSHV and RRV PAN loci. Candidate KSHV open reading frames identified by ribosome footprint profiling  are indi(TIF)Click here for additional data file.S5 FigBCBL-1 TREx cells were electroporated with oligonucleotides antisense either to GFP mRNA (control KD) or to KSHV PAN RNA and then induced into the lytic phase with 1 μg/mL doxycycline. 48 h after induction, a subset of the cells was harvested for RT-qPCR quantification of transcript levels in PAN RNA knockdown cells, relative to those in control knockdown cells. The levels of KSHV ORF11 mRNA, and host unspliced GAPDH and unspliced actin transcripts increased upon PAN RNA knockdown.(TIF)Click here for additional data file.S6 FigBJAB RRV cells were induced with 100 nM trichostatin-A (TSA) following electroporation with antisense oligonucleotides complementary to either GFP mRNA (control KD) or RRV PAN RNA (Oligo 493). After 40 h of lytic induction, cytoplasmic and nuclear fractions were isolated. (A) Western blot analysis assesses the purity of the cytoplasmic (GAPDH) and nuclear (FUS) fractions. (B) RT-qPCR analysis of fractionated BJAB-RRV cells. The fraction of each transcript present in the cytoplasm, relative to the nucleus is plotted. Despite extensive optimization of conditions, nuclei isolated from BJAB-RRV cells were prone to RNA leakage, which obscures the results of this analysis. Unspliced actin did not yield a signal above background samples lacking reverse transcription.(TIF)Click here for additional data file.


Heterodera glycines. We found that both females and males undergo body wall muscle atrophy and loss of attachment to the underlying cuticle during immobile developmental stages. Male H. glycines undergo somatic muscle renewal prior to molting into a mobile adult. In addition, we found developmental changes to the organization and number of motor neurons in the ventral nerve cord correlated with changes in mobility. To further examine neuronal changes associated with immobility, we used a combination of immunohistochemistry and molecular biology to characterize the GABAergic nervous system of H. glycines during mobile and immobile stages. We cloned and confirmed the function of the putative H. glycines GABA synthesis-encoding gene hg-unc-25 using heterologous rescue in C. elegans. We found a reduction in gene expression of hg-unc-25 as well as a reduction in the number of GABA-immunoreactive neurons during immobile developmental stages. Finally, we found evidence of similar muscle atrophy in the phylogenetically diverged plant-parasitic nematode Meloidogyne incognita. Together, our data demonstrate remodeling of neuromuscular structure and function during sedentary plant-parasitic nematode development.The sedentary plant-parasitic nematodes are considered among the most economically damaging pathogens of plants. Following infection and the establishment of a feeding site, sedentary nematodes become immobile. Loss of mobility is reversed in adult males while females never regain mobility. The structural basis for this change in mobility is unknown. We used a combination of light and transmission electron microscopy to demonstrate cell-specific muscle atrophy and sex-specific renewal of neuromuscular tissue in the sedentary nematode  The sedentary plant-parasitic nematodes are an economically devastating group of pathogens. They are capable of modifying their host’s metabolism by establishing a permanent feeding site within the plant roots. Following infection, sedentary nematodes remain at their feeding site where they are only capable of slight movements associated with feeding. Upon reaching adulthood, females continue to remain immobile at their feeding site, while adult males regain mobility to search for females. Using both microscopic observation and examination of gene function, we found that immobility is associated with changes to the muscle and neurons controlling movement. Interestingly, we found that males are capable of reversing these changes upon reaching adulthood. Our findings may be used to develop targeted control strategies for these devastating parasitic nematodes while avoiding potential off-target effects to beneficial nematodes that do not show this unusual mode of development and behavior. Heterodera glycines, accounts for over a billion dollars in yield loss yearly . We. WeCaeno elegans ; howeverganelles . Muscle ganelles ,15.Within the phylogenetic clade comprising most plant-parasitic nematodes, the sedentary life-style has arisen at least twice ,17. WhilH. glycines. Using light and electron microscopy, we describe the anatomical changes to the neuromusculature and surrounding epidermis of H. glycines during the transition to immobility and the resumption of mobility in males. We characterize the GABAergic system during H. glycines post-hatch development. Finally, we examine whether similar changes to body wall muscles occur in the phylogenetically diverged root-knot nematode Meloidogyne incognita. Our results demonstrate extensive remodeling of neuromuscular tissue in sedentary plant-parasitic nematodes.Here, we conduct a detailed developmental examination of the structural basis for changes of mobility in the sedentary species H. glycines using both the F-actin binding fluorescent probe phalloidin as well as transmission electron microscopy (TEM). Mobile J2 H. glycines somatic muscle comprise rhomboid-shaped cells longitudinally arranged in four quadrants along the length of the body ; howevepidermis . In C. e cuticle ,24. Losse intact . This ob elegans ,26.H. glycines females continue to feed and grow in width, while J4 males begin remodeling back into a vermiform shape  [H. glycines development. We previously found that mobile J2 H. glycines contain 65 VNC neurons [H. glycines degenerate during sedentary stages of development. However, males undergo neuronal remodeling that includes the addition of neurons. This addition of neurons results in more VNC neurons in adult males than in mobile J2s. Similar to C. elegans, the males of H. glycines may include sex-specific motor neurons in the VNC [In rd (VNC) ,27. We,  neurons ,28. Here the VNC ,30.H. glycines, we characterized the GABAergic nervous system. In C. elegans, GABA is produced by approximately one-fourth of the C. elegans VNC neurons. Release of GABA in the VNC leads to relaxation of body wall muscles and acts in opposition to the excitatory neurotransmitter acetylcholine. GABA is synthesized by glutamate acid decarboxylase (GAD), which in C. elegans is encoded by unc-25 [unc-25, from H. glycines (hg-unc-25). The predicted amino acid sequence of hg-UNC-25 is over 60% identical to C. elegans UNC-25  mutant with the H. glycines homolog. unc-25(e156) mutants are completely defective for production of GABA [unc-25(e156); hg-unc-25(+) animals contained 2–4 GABA VNC, whereas unc-25(e156) mutants are completely negative for GABA-immunoreactivity  also become immobile soon after infecting the plant host [Meloidogyne spp. are phylogenetically diverged from Heterodera glycines  suggested that the absence of body wall muscle in M. hapla adult females was “a result of the sedentary mode of life”; however, this conclusion was based on light microscopy of mobile J2s and adult females. While we cannot entirely rule out that the loss of muscle mass in H. glycines is caused by a lack of movement, our data suggests this is highly unlikely. We demonstrate that muscle atrophy occurs in conjunction with loss of mobility. Furthermore, cyst nematodes, such as H. glycines, are able to survive as fully developed, but inactive J2s, for years in a pre-hatch diapause state [We demonstrate that se state . Upon acC. elegans is essential for proper movement [C. elegans unc-25 mutant with hg-unc-25 demonstrates its functional conservation and corresponds with recent data demonstrating the conservation of regulatory regions of unc-25 among diverse nematode species [unc-25 between C. elegans and H. glycines, we found differences in the number and morphology of GABA-immunoreactive cells between these species. Interestingly, we also found that the handedness of most H. glycines GABA-immunoreactive neurons in the VNC differs from A. suum and C. elegans. Our results suggest that nematodes may be an appropriate animal phylum for exploring the evolution of neuronal handedness.The GABAergic system of movement . Our res species . DespiteH. glycines are unknown. Previous RT-PCR data of several genes encoding muscle-related proteins showed reduced expression in adult females compared with mobile J2s [H. glycines. Our data suggest that neuromuscular changes in H. glycines are cell specific. The absence of atrophy in head and esophageal muscles in H. glycines allows for continued feeding and head movements during the sedentary stages. In C. elegans, disruption of the BAG2 chaperone homolog UNC-23 leads to the opposite phenotype wherein the nematode undergoes a progressive degeneration of head muscles [H. glycines muscle-related genes result in the different developmental fates of head and body wall muscles. Alternatively, the muscle cell-specific atrophy seen in H. glycines could be due to selective neuronal degeneration. The head and neck muscles of C. elegans are differently innervated than the rest of the body wall muscles [C. elegans body wall muscles are innervated by the VNC, the head muscles receive innervation directly from the circumpharyngeal nerve ring. It will be interesting to determine via TEM whether a similar wiring pattern occurs in H. glycines and if muscle atrophy is due to denervation as seen in some mammals [The mechanisms regulating neuromuscular remodeling in bile J2s . A reexa muscles . It is p muscles . Whereas mammals .H. glycines and M. incognita strongly suggests the independent evolution of this behavior. However, it is important to note that within the Tylenchida, which includes both plant and insect parasitic nematodes, sedentary behavior is found in several phylogenetically separate genera [The ability to establish a specialized feeding site in their host is a significant adaptation among plant-parasitic nematodes and is often correlated with sedentary behavior. The atrophy of neuromuscular tissue is likely due to a relaxation of selection pressure for the maintenance of mobility. In addition, the degeneration of neuromuscular tissue could allow energy derived from the catabolism of muscle protein to be shunted toward reproductive development. The presence of muscle atrophy in both e genera . It is, H. glycines was originally isolated from a soybean field in Illinois, USA and cultured in the greenhouse on soybean cultivar ‘Macon’. To collect synchronized developmental stages of H. glycines, seeds of the soybean variety ‘Macon’, were germinated in a moist paper towel for three days. Soybean seedlings were then placed in pluronic gel F-127 with hatched J2 H. glycines for 24 hours [H. glycines second-stage juveniles (J2s). After 24 hours, roots were washed with water to remove nematodes that had not infected. H. glycines-infested roots were then planted into a pasteurized sandy loam soil and kept under a 12-hour light cycle until extraction. Infested roots were macerated with a hand blender to obtain H. glycines at specific time points. The roots were processed 6–7 days after inoculation to collect J3s and 9–10 days after inoculation to collect J4 females and males [Meloidogyne incgonita was a gift from Dr. Jason Bond and maintained on ‘Rutgers’ tomato in the greenhouse. Seeds of tomato variety ‘Rutgers’ were planted into a sandy loam soil for two weeks and then inoculated with freshly hatched M. incognita J2s. After four weeks, post-infective stages of M. incognita were extracted from the tomato plants and the developmental stages were identified based on morphology [P. penetrans was a gift from Dr. Terry Niblack and was cultured on corn root explants on Murashige and Skoog (MS) media [P. penetrans were isolated by synchronizing populations from eggs. P. penetrans eggs were extracted as previously described [C. elegans strains N2 and CB156 unc-25(e156) III were cultured on NGM agar with E. coli OP50 as previously described [24 hours . Alternand males . Developnd males . Meloidorphology . P. peneS) media . Specifiescribed  Freshly escribed . Developescribed .Nematodes were fixed in 4% paraformaldehyde (Electron Microscopy Science) overnight at 4°C and washed three times with water. Nematodes were placed on a 4% agar pad with phalloidin  and DAPI  or Hoechst  to stain nuclei. An Andor system micropoint laser attached to a Zeiss Axioimager with 63x objective was used to create openings in the cuticle at regular intervals along the length of the nematode to increase the penetration of dyes. Following opening of the cuticle, nematodes were incubated in the stain overnight. Successful penetration of phalloidin in sedentary nematodes was confirmed by observing fluorescence in esophageal muscle. At least ten animals were examined for each developmental time point. All light microscopy images were captured with Zen software on a Zeiss M2 AxioImager with DIC and fluorescence optics. Images were examined in FIJI and multiple images were combined using the ImageJ stitching plugin.Nematodes were fixed in Carnoy’s fixative  overnight in a 1.5 ml centrifuge tube and allowed to settle in the bottom of the tube ,44. The H. glycines were collected from soybean roots and stored overnight at 4°C. High pressure freezing and freeze substitution were modified from previous methods used for C. elegans [E. coli strain OP50. Nematodes were loaded into carriers with 20% bovine serum albumin and frozen in an HPM 010 high pressure freezer. Freeze substitution was performed in 2% OsO4 (Electron Microscopy Sciences) and 0.1% uranyl acetate (Polysciences) in 2% H2O and 98% acetone in an FS-8500 freeze substitution system. Samples were kept at -90°C for 110 hours before being warmed to -20°C over five hours. Samples were then kept at -20°C for 16 hours before being warmed to 0°C over five hours. Samples were washed four times in pre-chilled 100% acetone at 0°C. The last wash was one hour. Samples were then transferred to room temperature and washed two times in 100% acetone. Samples were infiltrated with 1:1 Polybed812 (Polysciences) resin:acetone for 24 hours, 2:1 resin:acetone for 36 hours, 100% resin for 24 hours, and then changed to fresh resin for three days. All infiltration steps were conducted on an orbital shaker at room temperature. Samples were then submerged into embedding molds with resin and hardener and baked at 60°C for two days. 70 nm sections were collected using a PowerTome PC ultramicrotome with a diamond knife and collected onto formvar-coated copper slot grids. Sections were imaged with a Phillips CM200 TEM. Quantification of sarcomere size, epidermal thickness, and body area were calculated using FIJI measurement tools. Pseudocolor overlays were created with Adobe Photoshop using the Wormatlas tissue color code scheme (http://www.wormatlas.org/colorcode.htm). Multiple sections were examined from at least two animals at each time point.Synchronized developmental stages of  elegans ,47. MetaC. elegans and H. glycines were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde (Electron Microscopy Science) for 15 minutes at 4°C. Fixed nematodes were washed three times with PBST  [C. elegans, nematodes were washed three times after fixation with PBST and shaken in 5% β-mercaptoethanol/PBST (Sigma-Aldrich) at 37°C overnight [C. elegans were then washed three times with 1 mM CaCl2/1% Triton X-100/0.1 M Tris-HCl (pH 7.5). 200 U/ml collagenase type IV was added to C. elegans and shaken vigorously for 15–30 minutes at 37°C, and washed three times with PBST. To increase the permeability of fixed H. glycines, specimens were cut into small segments [H. glycines sections were incubated in 100 mM Tris/1mM CaCl2 and proteinase K (2 mg/ml) for 20–30 minutes at room temperature to facilitate antigen retrieval. Nematode segments were incubated in prechilled methanol on ice for one minute followed by one minute on prechilled acetone on ice. Segments were washed three times with PBST. After permeabilization, both nematode species were incubated in 1% BSA (Sigma-Aldrich) dissolved in PBST for at least one hour followed by incubation in a 1:100 anti-GABA  primary antibody at 4°C overnight. Nematodes were washed three times with PBST and incubated in 0.1%BSA/PBST with a 1:100 secondary anti-rabbit IgG-FITC  antibody at a 1:100 ratio overnight before examination. Antibody staining was observed on over 40 J2 H. glycines and 10 sedentary H. glycines. pH 7.4) . To incrvernight . C. elegsegments . H. glycH. glycines J2s were homogenized in liquid nitrogen to extract RNA using the Qiagen RNeasy Blood Kit (Qiagen) following the manufacturer’s instruction. Genomic DNA contamination was removed from the RNA using the Turbo DNA-free kit (Thermo Fisher Scientific). cDNA was synthesized using the Thermoscript RT-PCR system (Invitrogen). hg-unc-25 was amplified from H. glycines cDNA using primers 5’-ATGAAATTAAAGGAGCATAAAGAATC-3’ and 5’-TCACAAATCTTCTCCCAGTGTG-3’. The plasmid pPD157.60 (gift from Andrew Fire via Addgene) was used as the backbone for the construct. Forward: 5’-CATTTTTTCTACCGGTACCAATACG-3’ and reverse: 5’- ACATTTTTTTTTCTCTTTCCGTCTC-3’ primers were used to amplify the backbone including a 1.9 Kb unc-25 promoter region from pPD157.60 (Addgene). The backbone and hg-unc-25 cDNA were ligated using Gibson Assembly (ThermoFisher Scientific) following the manufacturer's instruction. Three 51-bp artificial introns were added into the hg-unc-25 cDNA to improve expression (Andrew Fire Vector Kit) using Gibson Assembly. The rescue construct unc-25p::hg-unc-25 (40 ng/ul) was co-injected with the coelomocyte marker plasmid coel::RFP (80 ng/ul) to rescue the C. elegans unc-25(e156) mutant using standard microinjection techniques [Fresh chniques . Five inH. glycines were homogenized in liquid nitrogen. RNA was extracted and genomic DNA contamination was removed from the RNA. RT-qPCR was performed using the Power SYBR Green RNA-to-Ct 1 step kit on an ABI PRISM 7000 Sequence Detection System. Expression of hg-unc-25 was examined using the primers qhgunc25 forward primer 5’-TCCAAAGGGATGGAAGGTTATC-3’ and reverse primer 5’-GCCTTCAGTGCGTTTGATTT-3’. far-1, a surface associated retinol and fatty-acid protein encoding gene was compared for the relative expression of hg-unc-25 [hg-unc-25, the ΔCt between hg-unc-25 and hg-far-1 at each stage was calculated, and a one-way ANOVA was performed to calculate the mean separation [Synchronized developmental stages of g-unc-25 ,50. Eachparation . NormaliS1 Fig(TIF)Click here for additional data file.S2 FigH. glycines head region demonstrating that head (arrow) and esophageal (arrowhead) muscles do not degenerate. (B) Phalloidin (green) and DAPI (blue) staining of J4 body wall muscle actin and nuclei. Scale bars, 10 μm.Fluorescent micrograph of (A) phalloidin-stained J3 (TIF)Click here for additional data file.S3 FigC. elegans, unc-25 encodes the sole enzyme glutamate acid decarboxylase for GABA synthesis. hg-UNC-25 is 69% identical to UNC-25.In (TIF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.


Herein, we comprehensively characterize and compare the activities of these two helicases on various nucleic acid substrates in vitro, with a focus on RG4 structure. We find that both MOV10L1 and MOV10 are able to resolve RG4, with MOV10L1 being more efficient in that. In contrast to MOV10, MOV10L1 prefers to bind to a junction between single-stranded RNA and RG4, which is mediated by both its N and C termini. Furthermore, we show that RG4 unwinding by MOV10L1 facilitates the cleavage of this specific RNA structure by an endonuclease.MOV10L1 and its paralog MOV10 are evolutionally conserved RNA helicases involved in distinct RNA regulatory pathways. The testis-specific MOV10L1 is essential for spermatogenesis and PIWI-interacting RNAs biogenesis, whereas MOV10 is ubiquitous and multifunctional. Although both proteins have been implied to correlate with RNA G-quadruplex (RG4)   •Both MOV10L1 and MOV10 can resolve RG4 structure in an ATP-dependent manner•MOV10L1 unwinds RG4 more efficiently than MOV10•MOV10L1 preferentially binds to an ssRNA-RG4 junction•RG4 unwinding by MOV10L1 facilitates its endonucleolytic cleavage Biological Sciences; Biochemistry; Molecular Biology Their din vitro . One of in vitro . The abubstacles .in vitro. We found that even though both of them can unwind RG4, there are a few striking differences between them. MOV10L1 could take advantage of its unique features in RG4 binding and unwinding to mediate piRNA biogenesis. Last, a proof-of-concept assay with MOV10L1 and an endonuclease supports a model that they might cooperatively unwind and cleave RG4 structures in the initial step of piRNA biogenesis.In this study, we employed robust biochemical assays to test the capability of MOV10L1 and MOV10 in binding and unwinding of RG4 structure 2+ concentration for the unwinding activity of purified full-length MOV10L1 protein in vitro  RNA (2+ and the unfolding was initiated by adding ATP. An unlabeled 25-nt single-stranded RNA (ssRNA) trap with 16 inconsecutive nucleotides complementary to the RG4 sequence was introduced along with ATP to prevent the unfolded RNA substrate from refolding  RNA . Briefly(ds) RNA . The cir(ds) RNA B (Tang a(ds) RNA , indicatefolding A. As theefolding C. Unwindefolding C.Figure To find out whether the observed RG4 unwinding was caused by MOV10L1's helicase activity instead of other mechanisms, we sought to determine whether this activity depends on certain parameters that are compatible with a helicase mechanism. First, we tested the requirement of ATP hydrolysis as an energy source for RG4 unwinding. We performed a similar experiment in which the substrate was incubated with the MOV10L1 helicase in the presence of either ATP or its non-hydrolyzable analog, adenylyl-imidodiphosphate (AMP-PNP) or ATPγS. We found that RG4 was unwound and converted to dsRNA in the presence of ATP in this condition . We obseBesides ATP, unwinding of the RNA duplex structure by MOV10L1 requires the substrate bearing a 5′ ssRNA tail . We thenNext, we sought to characterize RG4-unwinding kinetics by MOV10L1 and compared its unwinding efficiency with those of other RNA substrates or its paralog MOV10. We first conducted the RG4-unwinding assay in the presence of MOV10L1 as a function of time. As expected, the fraction of unwound RG4 substrate was increased over time A. For coin vitro. To test that, we performed the RG4 unwinding assay with purified MOV10 protein (As MOV10 shares sequence similarity, to some extent, with MOV10L1, we next interrogated whether MOV10 might also be able to resolve RG4  protein A. We fou protein B, Lane 8 protein D and 2E. protein C and S3D protein B–2D. Giv protein , the facTo gain further insight into the mechanisms of MOV10L1 in RG4 unwinding, we then investigated how 5′ ssRNA tail regulates MOV10L1 unwinding. As is the case for some helicases, they prefer to associate with ss nucleic acids first and unwind higher-order structures after translocating to the fork junction . AlternaTo further verify MOV10L1's preferential binding to a junction, we performed similar experiments with various RNA duplex substrates. As expected, MOV10L1/RNA complexes were observed with either the 5′-tailed or the forked RNA duplex substrate . HoweverTaken together, we conclude that MOV10L1 has a structure-specific binding manner, and the presence of an unpaired 5′ ssRNA region promotes its binding to the structured RNA substrate.Many helicases have been found to possess dual unwinding activities, acting on both DNA and RNA substrates. Thus far, there are seven helicases exhibiting RG4-unwinding activity and two of them have been identified to unwind DG4 structure as well . We thenIt is widely known that besides the helicase core domain, the helicase function often needs to be activated through extra domains . The N- The ancillary domains of helicase are often found to regulate helicase unwinding activities by mediating its association with substrates; we thus examined the binding affinities of these mutants to the 5′-tailed RG4 substrate using an EMSA approach similar to the one described above except with MOV10L1 truncated mutants. We found that none of these three mutants could effectively form complex with the substrate D. Taken in vivo (Previous studies suggest that the MOV10L1 helicase might participate in piRNA biogenesis in a manner that it resolves RNA secondary structures in piRNA precursor to facilitate its endonucleolytic cleavage in vivo . To dissin vivo , we chosin vivo . This suin vivo A, Lane 2in vivo B and S7.in vivo in eukaryotic cells, indicating the existence of robust and effective machineries, such as helicases (High-throughput reverse transcription sequencing methods have identified over 12,000 RG4s in the transcriptome . Moreoveelicases . Thus faelicases . Therefoin vivo study obtained from Mov10l1 mutant mice shows that loss of MOV10L1 helicase activities abolishes piRNA biogenesis accompanied by a remarkable accumulation of primary piRNA precursors and a failed production of MILI-bound PPIFs and enrichment of RG4 elements in piRNA precursors (in vivo tight correlation between MOV10L1 footprints and MILI-bound PPIFs and RG4 sites, and importantly, the necessity of RG4 resolution to form all PPIFs (A previous ecursors . An initecursors . This moecursors C: MOV10Lll PPIFs . A recenll PPIFs , promptiAlthough the genetic information regarding the piRNA pathway is ample, how primary piRNA precursor transcripts are distinguished from many other RNA transcripts to enter the piRNA biogenesis pathway is obscure . PreviouEffective nucleic acid binding and unwinding of helicase often ask for the functional interplay between the helicase core and ancillary domains as well as auxiliary partners . This noAlthough we provided evidence that in our experimental conditions the unwinding activities per microgram for each protein can be considered being comparable, a further more detailed characterization would be required to demonstrate that the recombinant proteins are folded correctly, uniformly, and that they show the same activity per microgram of protein mass.All methods can be found in the accompanying


Prochlorococcus MED4, establishing CRAFD-Seq as a new method for identifying direct targets of these sRNAs that is applicable to all bacteria, including those that are not amenable to genetic modification. The results suggest that these sRNAs are integrated within a regulatory network of unprecedented complexity in the adjustment of carbon and nitrogen-related primary metabolism.The sRNA Yfr1 and members of the Yfr2 sRNA family are almost universally present within cyanobacteria. The conserved motifs of these sRNAs are nearly complementary to each other, suggesting their ability to participate in crosstalk. The conserved motif of Yfr1 is shared by members of the Yfr10 sRNA family, members of which are otherwise less conserved in sequence, structure, and synteny compared to Yfr1. The different structural properties enable the discrimination of unique targets of Yfr1 and Yfr10. Unlike most studied regulatory sRNAs, Yfr1 gene expression only slightly changes under the tested stress conditions and is present at high levels at all times. In contrast, cellular levels of Yfr10 increase during the course of acclimation to darkness, and levels of Yfr2 increase when cells are shifted to high light or nitrogen limitation conditions. In this study, we investigated the targetomes of Yfr2, Yfr1, and Yfr10 in  Compared to other regulators of gene expression, such as transcription factors, sRNAs exhibit some unique features with respect to regulatory dynamics, i.e., regulation by an sRNA leads to a faster decrease in the level of the protein product of the target gene than does regulation by a transcription factor2. Studies of sRNAs in bacteria and archaea show that they can regulate gene expression at the post-transcriptional level1, either by occluding or exposing ribosomal binding sites or ribonuclease cleavage sites1. Other distinct modes of sRNA action have been described that often require RNA chaperones such as Hfq or ProQ for their full functionality4.Small non-coding RNAs (sRNAs) are present within all domains of life and modulate the expression of target genes5, the marine picocyanobacterium Prochlorococcus, sRNAs have been investigated since 20056 and until today more than twenty were discovered in the model strain Prochlorococcus marinus MED4 (Prochlorococcus MED4)8. In contrast to the reduction in the number of protein regulators associated with the genome streamlining observed in this clade, the number of sRNAs is not reduced in Prochlorococcus MED47. The relatively high number of sRNAs present in this bacterium may represent an adaptation to the extremely oligotrophic environment it inhabits, where nutrients are scarce and the synthesis of an RNA regulator requires fewer resources than that of a protein regulator7. Of the completely sequenced Prochlorococcus genomes, those of the low-light II/III ecotype are the only ones that do not contain yfr1 genes encoding cyanobacterial functional RNA 1 (Yfr1), which are otherwise ubiquitous in cyanobacteria9. Most yfr1 homologs are located between the genes encoding inosine-5′-monophosphate dehydrogenase (guaB) and thioredoxin (trxA)10. To date, only two porins, which originated from a gene duplication event, have been shown to be targets of Yfr1 in Prochlorococcus MED411. In addition to yfr1, the yfr2 sRNA family is similarly conserved and is present in all analysed cyanobacteria12. Unlike yfr1, yfr2 genes are present in multiple copies in cyanobacterial genomes and occur at unconserved loci12. Many Prochlorococcus strains possess two copies of yfr2, although four gene copies are present in Prochlorococcus MED4 (yfr2-5) and Synechococcus CC9611 harbours nine12. With the exception of tmRNA, the 6 S and 4.5 S sRNAs and RNase P RNA, which are universally conserved throughout all bacterial phyla, all other sRNAs identified in Prochlorococcus are restricted to the marine picocyanobacteria (Prochlorococcus and marine Synechococcus) and do not occur outside this group8.In the most abundant photosynthetic organism on EarthProchlorococcus relies heavily on sRNAs to modulate gene expression, the transcription of sRNAs is also coordinated by sigma factors and transcription factors. Despite the reduced set of regulatory proteins harboured by Prochlorococcus MED4, this bacterium encodes representatives of a variety of regulatory proteins. To activate regulatory pathways, Prochlorococcus MED4 encodes five sigma factors from groups 1 and 2, two Fur-type transcriptional regulators, three Crp-type transcriptional regulators (including NtcA), the LysR-type transcriptional regulator RbcR, the global response regulator LexA, one ArsR-type transcriptional regulator, two AbrB-type transcriptional regulators, the GntR-type transcriptional regulator PMM1637, and six two component response regulators 9.While it appears that 13. MAPS technology is based on the affinity purification of MS2-tagged sRNA and its bound targets followed by RNA-Seq, and this method can also be used to study sRNAs that function without an RNA chaperone14. Another approach, called GRIL-Seq, is based on the recombinant expression of T4 ligase and the sRNA of interest, taking advantage of proximity ligation and the subsequent enrichment of hybrid sRNA-target fragments followed by sequencing15. Target-enrichment sequencing of sRNAs (TEsR) uses a biotinylated in vitro transcript of an sRNA of interest to enrich targets that were reverse transcribed into cDNA16.Studies of sRNA targets have developed from the prediction and verification of one or a few targets towards sequencing of the entire targetome of an sRNA, for which various approaches have been developed. RNA chaperones can be immunoprecipitated to allow for subsequent sequencing of the bound RNA (RIP-Seq)cell-free RNA affinity pull-down followed by sequencing), a technique that is also applicable to organisms for which no genetic tools are available. Our CRAFD-Seq results suggest that Yfr2 interacts with both Yfr1 and the highly conserved sRNA Yfr10, which is less conserved in position and structure but contains the same conserved motif as Yfr1. Although Yfr1 and Yfr10 share a set of targets, these sRNAs also appear to have unique targets. The distinct targetomes of Yfr2, Yfr1 and Yfr10 coupled with their interdependency constitute a complex regulatory network comprising hundreds of genes in the Prochlorococcus MED4 genome.In this study, we established a new method for sRNA target enrichment that uses a hybrid sRNA-target pulldown approach we call CRAFD-Seq , which is responsible for the 3′ processing of tRNAs, in the Yfr2 dataset . The sRNA plasmids carrying yfr1, yfr2, yfr1 + yfr2, a non-interacting control RNA (control) or yfr1/yfr2+ control were cotransformed with the plasmid carrying the PMM1121 5′ UTR fused to sfgfp. In the presence of Yfr1 or Yfr1+ control, a pronounced repression in GFP fluorescence  was observed  frequently plays a role in sRNA-target interactions in bacteriar10 Fig. . To exclr10 Fig. . Cleavage E Fig. .Because of the unique interaction between the sRNAs Yfr2 and Yfr1 or Yfr10, we also used the CRAFD-Seq protocol to elucidate the targetomes of Yfr1 and Yfr10 by using these sRNAs as bait. The reciprocal fishing approach, using Yfr1 or Yfr10 as bait, largely confirmed that Yfr2 and its three homologs interact with Yfr1 or Yfr10 Fig.  and S3. intaRNA11-predicted interaction site for full-length genes of Yfr1, Yfr2 and Yfr10 with the lists of enriched/non-enriched CRAFD-Seq identified peaks , 1.74 × 10−5 (Yfr10) and 2.73 × 10−7 (Yfr2) , in a 5′ UTR (21% for Yfr1 and Yfr10), or in a 3′ UTR  Fig. . The funs Tables  and perfr2) Fig. .11 PMM1119 (the homolog of PMM1121), inorganic pyrophosphatase  and the bifunctional ornithine acetyltransferase/N-acetylglutamate synthetase   or UGGU (M2)  are targeted by Yfr1 but not Yfr10  Table . The latppa Fig.  were 6- ppa Fig.  and S3. ely Fig. . HoweverA11 Fig. . The forM2) Fig. . Neitherint Fig. . The affnts Fig. . Despitents Fig. . In contglgC, PMM0769) mRNA appears to be under the control of a highly complex regulatory network of non-coding RNAs, including Yfr1/10, Yfr2 and the cis-encoded antisense RNA (asRNA) asGlgC , pgk  and pgmI , which appear to be controlled by Yfr1/Yfr10 and corresponding asRNAs, the latter of which seems to be under the control of Yfr2  is possibly not as established as previously thought for cyanobacterial sRNAs and their targets32. The SD sequence does not occur more often than by chance in Prochlorococcus MED4 and MIT93138, and in Synechocystis sp. PCC 6803 it is only detected in 26% of all genes33. The latter number of SD sequences in Synechocystis sp. PCC 6803 correlates well with the 27% of genes that were observed to have a higher than average intergenic coverage in the ribosomal profiling experiment for which SD sequences were detected32. The genes containing SD sequences were enriched in the categories of photosynthesis and respiration and for translation, possibly because genes in these categories are highly translated32. However, this does not appear to be the case for Prochlorococcus, and further studies are required to examine the detailed influence of internally binding sRNAs in Prochlorococcus on their targets.The CRAFD-Seq results obtained in our study revealed a relatively high occurrence of enriched peaks within CDS regions. This result is in contrast to those obtained in RIL-Seq experiments, where an almost equal distribution of chimeric sRNA fragments with CDS and 5′ UTRs was observedSynechocystis sp. PCC 6803 despite the presence of elevated nblA levels27. Furthermore, nitrogen starvation is also indicative as an excess of carbon supply34. The metabolic fluxes of fixed carbon are therefore rerouted from amino acid synthesis, which cannot be sustained, towards gluconeogenesis36. The genes encoding enzymes that metabolize fixed carbon in the form of 3-phosphoglycerate, either in the direction of gluconeogenesis (such as pgk and gap2) or towards the citrate cycle (such as pgmI) are regulated by Yfr1 and/or Yfr10. However, because Yfr2 can interact with Yfr1/10, the stimulus of late nitrogen or high light stress can be integrated into these metabolic pathways via Yfr1/10 and through the Yfr2-controlled asRNAs of pgk, gap2, and pgmI. The pathway from 3-phosphoglycerate towards the citrate cycle appears to be indirectly controlled by Yfr2, because pgmI is also a target of Yfr2, possibly enhancing the shutdown of this route for fixed carbon. A highly complex regulatory circuit involving Yfr2, Yfr1/10 and asglgC can be similarly anticipated for glgC, which catalyses the first committed step in glycogen synthesis. Another target of Yfr1 is the important metabolic branch point enzyme phosphoglucomutase (encoded by pgm), which links glycolysis, the oxidative pentose phosphate pathway and glycogen metabolism, making it directly involved in the processes of polyglucan storage (glycogen synthesis) and utilization 37. Unfortunately, in this study we only determined the targetomes of Yfr1, Yfr2, and Yfr10, and a functional analysis of the mode of action of the assayed sRNAs requires further investigations.During nitrogen starvation, cells undergo chlorosis and reduce the levels of their photosynthetic apparatus, since phycobiliproteins and chlorophyll are rich in nitrogen that can be recycled. This process is abolished in mutants of the cyanobacterial GntR transcriptional regulator in ppa expression is downregulated during high light stress38 and nitrogen depletion39, both conditions where Yfr2 is upregulated. This result may suggest that Yfr1 and Yfr10 have stabilizing effects on ppa mRNA. A similar observation was made for the Yfr10-specific targets of the carboxysome-associated genes csoS1 and csoS2 and for atpA and atpC, which are downregulated during nitrogen stress39. However, because multiple regulatory inputs affect one gene, and the mode of action for sRNAs in Prochlorococcus is enigmatic, we could not detect this effect in general for the sRNA targets presented here.Comparing the expression profiles of an sRNA and its targets may provide useful information on the mode of interaction. Previous studies showed that 10. This idea is further confirmed with the discovery of a second sRNA (Yfr10) that shares the same conserved motif as Yfr17. Unlike Yfr1, synteny and sequence conservation, except from the Yfr1 motif, is not conserved, resulting in secondary structure conservation among Yfr10 homologs being very low  or a NucleoSpin Gel and PCR-Clean-up kit (Macherey-Nagel) according to manufacturer’s instructions. For biotinylation, two volumes of KIO4 (6 mM) were added to 1.5 nmol of RNA and was incubated at room temperature, in the dark for one hour. Subsequently, one volume of ethylene glycol/H2O (1:1) was added, and the RNA was precipitated by adding 2.9 volumes of ethanol (100%) and 0.1 volumes of NaCl (3.3 M) at −20 °C for a maximum of 60 min. The RNA was centrifuged at 13,000 g for 30 min at 4 °C, the supernatant was removed, and the pellet was washed with 70% ethanol. The air-dried pellet was then resuspended in 24 µl 10 × PBS (pH 7.4), 6 µl H2O, and 20 µl EZ-Link Alkoxyamine-PEG12-Biotin (50 mM in DMSO). The solution was incubated at 37 °C in the dark for 3 h and was periodically mixed, after which 50 µl of NaBH4 (20 mM) and 100 µl of Tris-HCl  were added and the mixture was incubated in the dark at 4 °C for 30 min. After the RNA was precipitated overnight as described above, free EZ-link-PEG12-alkoxyamine was removed by washing the RNA with water in Amicon ultracel 10-K columns (Merck).Transcript templates for 13 Table . The desProchlorococcus MED4 cultures  were pelleted at 15,000 g for 10 min at 22 °C. The cell pellets were washed with a solution containing 1.3 M betaine and 10 mM HEPES (pH 8) and were centrifuged again. The pellets were resuspended in lysis buffer (100 mM Tris-HCl (pH 7.5), 500 mM LiCl, 10 mM EDTA, 5 mM DTT, and 1% LiDS) and lysed using a OneShot constant pressure system (Constant Systems Limited) at 1.5 kBar. Cell debris was separated from the cell lysate by centrifugation for 30 min, 13,000 g, at 4 °C. For immobilization of the bait RNA, 250 µl of Dynabeads Streptavidin MyOne C1 (ThermoFisher Scientific) per sample were prepared following the manufacturer’s instructions. The beads were washed once with binding buffer (20 mM Tris-HCl (pH 7.5), 1 M LiCl, and 2 mM EDTA). Biotinylated RNA was then loaded onto the beads in binding buffer for 10 min at room temperature with gentle agitation. Subsequently, the beads were washed first with binding buffer then with lysis buffer. To reduce the binding of nonspecific RNA to the beads, the cell lysate was first incubated with unloaded beads, which were prepared as described above, for 10 min at room temperature with gentle agitation. The unbound cell lysate incubated with the empty beads was then incubated with beads charged with the sRNA of interest for 10 min at room temperature with gentle agitation. The beads were washed with 1 ml buffer A (10 mM Tris-HCl (pH 7.5), 0.3 M LiCl, 1 mM EDTA, and 0.1% LiDS), 1 ml buffer B (10 mM Tris-HCl (pH 7.5), 0.3 M LiCl, and 1 mM EDTA), and 0.5 ml buffer C (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, and 1 mM MgCl2). Bound RNA was eluted in 150 µl of elution buffer  by incubating the sample for 2 min at 65 °C, and the beads were immediately removed by placing the tube on a magnet and transferring the supernatant to a new tube.42. Annealing mixtures containing 0.2 pmol of in vitro-synthesized target RNA, 2 pmol of the 5′ end-labelled primer ppa_RT  were heated for 10 min at 70 °C and then chilled on ice for at least 5 min. cDNA synthesis was performed for 2 h at 30 °C using SuperScript III Reverse Transcriptase (ThermoFisher Scientific) according to the manufacturer’s instructions. The reaction was inactivated by incubation for 15 min at 70 °C, which was followed by RNase H treatment for 20 min at 37 °C and a final heat inactivation for 5 min at 95 °C in RNA loading buffer. DNA sequencing ladder reactions were performed with the same 5′ end-labelled primer used for cDNA synthesis and the same template DNA used for target RNA in vitro synthesis using a USB Thermo Sequenase Cycle Sequencing Kit (Affymetrix). Primer extension products and sequencing reactions were separated on 8.3 M urea-6% polyacrylamide sequencing gels, and the vacuum-dried gels were exposed to imaging plates. Signals were visualized using a Thyphoon FLA 9500 instrument  with Quantity One software (Bio-Rad).The primers were labelled as described previouslyRT Table  without In vitro-synthesized RNA was produced from annealed primers #4, 5, 23–34 (Table ppa in vitro-synthesized RNA was incubated with 20 units FastAP (Thermo Fischer Scientific) in a 200 µl reaction volume at 37 °C for one hour. Dephosphorylated in vitro-synthesized RNA was labeled with [γ-32P] ATP by T4 Polynucleotide Kinase (Thermo Fischer Scientific). RNA footprinting was performed using Ambion RNase T1 according to manufacturer’s description. Briefly, 0.1 pmol of labeled ppa in vitro-synthesized RNA was mixed with 20, 40, or 80 pmol of unlabeled Yfr1, Yfr10, or mutant in vitro-synthesized RNA, denatured for 1 min at 95 °C and cooled to room temperature for 5 min. Subsequently, 1 µl of 1 µg/µl yeast RNA and 1 µl of 10 x structure buffer was added and samples were incubated at room temperature for 15 min. RNase T1 treatment was performed by adding 1 µl of 0.2 U/µl RNase T1 and the samples were incubated for 15 min at room temperature. Reactions were stopped by adding 10 µl of denaturing formamide loading dye. Alkaline ladder was obtained by incubating 0.1 pmol of labeled ppa in vitro-synthesized RNA at 95 °C for 5 min in 7.5 µl of alkaline hydrolysis buffer containing 1.5 µg of yeast RNA. Reactions were stopped by adding 10 µl of denaturing formamide loading buffer. RNase T1 G ladders was obtained by incubating 0.1 pmol of labeled ppa in vitro-synthesized RNA and 1 µl of 1 µg/µl yeast RNA in 9 µl sequencing buffer for 10 min at 50 °C, followed by the addition of 1 µl of 0.2 U/µl RNase T1 and incubation at room temperature for 15 min. Reactions were stopped by adding 12 µl of denaturing formamide loading dye. The samples were separated on 8.3 M urea-10% polyacrylamide sequencing gels, and the vacuum-dried gels were exposed to imaging plates. Signals were visualized using a Thyphoon FLA 9500 instrument  with Quantity One software (Bio-Rad).34 Table  using th18. In brief, 2 µg of affinity-purified RNA samples and 4 µg of control RNA samples (unbound cell lysate of empty beads) were treated with 4 units of TURBO DNase (Life Technologies) in 2 consecutive incubation steps, each at 37 °C for 15 min. Next, RNA clean-up and separation into small (17–200 nt) and large ( > 200 nt) RNA fractions was performed using RNA Clean & Concentrator columns (Zymo Research). The large RNA fraction was fragmented according to the protocol18 and was recombined with the small RNA fraction after RNA Clean & Concentrator column purification (Zymo Research). All subsequent follow-up steps were performed according to the protocol18 with the following modifications: the clean-up of RNA 5′-pholyphosphatase (Epicentre)-treated samples was performed using Clean & Concentrator column purification (Zymo Research). For RNA adapter ligation, the UGA linker , unmapped reads were removed with BAM filter  and that data was converted to the wig format using BAM to Wiggle . To merge different wig files, missing nucleotide positions were added and the read number was set 0. Read numbers were normalized to library size and merged grp files for each strand were generated. Grp files are available as supplementary files Libraries were prepared following the whole transcriptome protocol as previously describedhttps://github.com/SJLambrecht/GRP_Peakcall.The start and end of a peak were set if the difference between the average read coverage over a defined base range separated by a spacer exceeded the threshold Fig. . In addi42. Yfr2 transcripts (25 pmol) labelled with Cy3 and equal amounts of unlabelled Yfr2 D1, Yfr1, or Yfr10 were incubated with RNase E. Aliquots were withdrawn immediately after reaction assembly and after 20 min of incubation at 30 °C. Prior to analysis of RNA cleavage on 10% urea PAGE, cleavage reactions were purified using an RNA Clean and Concentrator Kit (Zymo).RNase E purification and cleavage assays were essentially performed as previously describedyfr1) or the control RNA under its own PLlacO promoter in the pZE12-luc-yfr2 plasmid, PCRs were performed with primers PromFw-AvrII and Yfr1PromRv-AvrII or primers PromFw-AvrII and pJV300PromRv-AvrII . Single-cell fluorescence was determined by flow cytometry using an Accuri C6 flow cytometer (BD Bioscience). Cell fluorescence was measured with an excitation wavelength of 488 nm and the emission was detected at 533/30 nm. The mean fluorescence per plasmid combination was calculated from 50,000 events (cells) of 12 individual clones.In general, GFP assays were performed as previously describedSupplementary InformationSupplementary InformationSupplementary InformationSupplementary InformationSupplementary InformationSupplementary InformationSupplementary Information


In this study, we demonstrate automated in vitro evolution of a translation-coupled RNA system in a droplet flow reactor for the first time. This reactor contains approximately 1010 micro-scale droplets (average diameter is approximately 0.8 μm), which continuously fuse and divide among each other at a controllable rate. In the droplets, an RNA  replicate through the translation of self-encoded RNA replicase with spontaneously appearing parasitic RNA. We performed two automated replication experiments for more than 400 hours with different mixing intensities. We found that several mutations displayed increased frequencies in the genomic RNA populations and the dominant RNA mutants acquired the ability to replicate faster or acquired resistance to the parasitic RNA, demonstrating that Darwinian evolution occurred during the long-term replication. The droplet flow reactor we developed can be a useful tool to perform in vitro evolutionary experiments of translation-coupled systems.Automation is a useful strategy to make laborious evolutionary experiments faster and easier. To date, several types of continuous flow reactors have been developed for the automated evolutionary experiments of viruses and bacteria. However, the development of a flow reactor applicable to compartmentalized  In vitro reconstitution allows us to understand the design principles for the target biological phenomena and may also give rise to new technologies10. To date, various basic biological functions have been reconstituted, including genome replication, gene expression, membrane-associated functions, and others as recently reviewed13. Recently, higher-order biological phenomena, such as cell division machinery14, predator-prey oscillation15, DNA replication-cell division coupling16, and translation-coupled replication and evolution17 have being reconstituted from the combination of basic biological functions.One useful method to understand complicated biological phenomena is to reconstitute these systems from a set of molecules in vitro reconstitution. Natural organisms evolve through spontaneous mutations and natural selection during a long term process of self-reproduction. A similar evolutionary process was simulated in vitro by Spiegelman’s group in 196718. Indeed, they repeated the replication of a genomic RNA of the bacteriophage Qbeta in the presence of RNA replicase through a long serial transfer experiment and observed the evolution of smaller RNAs. Later, other self-replication systems of RNA or DNA were constructed and evolution was observed in some cases21.Evolution is another complex biological phenomenon that could be understood by 22. In this system, an artificial genomic RNA replicates via the translation of a self-encoded RNA replicase. In contrast to the previous system that did not include translation, this system requires cell-like compartments for evolution to ensure the linkage between genotype (RNA) with phenotype (replicase)17 and to repress spontaneously appearing parasitic RNAs23. In the previous study, we used water-in-oil droplets for cell-like compartments, which had been used to compartmentalize various biochemical reactions25.Recently, we constructed a translation-coupled RNA replication system by combining a cell-free translation system with Spiegelman’s RNA replication system28, bacteria31, fungi32, and artificial self-replicating DNAs33. However, an automated system applicable to a compartmentalized in vitro system has yet to be established.One of the largest hurdles in performing evolution experiments is the requirement of laborious experimental processes for long durations. To overcome this obstacle, automated systems, such as continuous flow stirred tank reactors (CSTRs) or chemostats, have been developed to examine the experimental evolution of bacteriophages35. In this system, the contents of the droplets were continuously diluted with the supplied water phases through fusion and division of the droplets induced by mixing. This type of the exchange of contents of water-in-oil droplets has been utilized to induce internal biochemical reactions in previous studies38. In our previous report, we succeeded in performing translation-coupled RNA replication in this droplet flow reactor for several hours35. However, the duration of the RNA replication was not sufficient to observe evolution.Recently, we developed a continuous droplet flow reactor in which oil and water phases are continuously supplied into a chamberin vitro RNA evolution in the droplet flow reactor. We carefully adjusted several parameters in the droplet flow reactor and successfully performed two long-term replication experiments for more than 400 hours each. Mutations accumulated in the RNAs during replication and the RNAs in the final populations acquired the ability to replicate faster or developed a tolerance to the parasitic RNA.In this study, we attempted to establish automated E. coli. The genomic RNA encodes the catalytic subunit of an RNA replicase (Qbeta replicase), which is translated and forms an active replicase with EF-Tu and EF-Ts in the translation system. The replicase catalyzes the replication of the genomic RNA. During replication, parasitic RNAs, which have lost the replicase gene, appeared spontaneously, possibly through recombination40. These RNAs replicate using the replicase translated from the genomic RNA .In previous report, we have succeeded in replication for only few hours in the droplet flow reactor. We believe that this was caused by a high mixing intensity in the second tank, which was uncontrollable in the previous system. In this study, we modified the control program to make the mixing intensity controllable. After several trials with different mixing intensities and dilution rates, we have successfully performed long-term replication experiments for 412–421 hours at two different (low or high) mixing rates in the second tank. The key factors that are important for sustainable replication was described in the Methods section. The low and high mixing rates were conducted by changing the working period of the stirrer in the second tank (duty ratio) at 5% or 20%, respectively. The size of droplets was not significantly changed with these duty ratios Fig. . During 23. Parasitic RNAs was also detected just after the large peak of the genomic RNA. A similar result was also observed at the high mixing rate  at a low concentration of parasitic RNA, the host genomic RNA replicates and the parasitic RNA follows; (2) the parasitic RNA finally overwhelms the host genomic RNA and inhibits the replication of the genomic RNA; (3) then, both the host genomic and parasitic RNAs are decreased by dilution; and (4) after sufficient dilution of parasitic RNA, the genomic RNA can restart replication. By repeating these (1–4) processes, the oscillation dynamics in RNA populations manifest. The factors that affect the dynamics were reported in our previous theoretical study41.The oscillation dynamics observed in the RNA populations can be explained by the interaction between host genomic and parasitic RNAs as described in a previous studyE. coli strain. The plasmids were extracted from 5–11 independent colonies at each time point and the inserted sequences were analyzed.We analyzed the host RNA sequence at several time points during the long-term experiments. We collected the host genomic RNAs from the time point corresponding to the peaks of oscillation . After reverse transcription using specific primers, the cDNA fragments were ligated to a vector and transformed into an Common mutations that were found in more than 20% of the clones at each time point are listed and the frequencies are represented as a heat map Fig. . Under lTo analyze the properties of the genomic RNAs after evolution, we picked one mutant RNA at 357 h under low mixing conditions and 421 h under high mixing conditions as evolved RNAs, referred to as L357 and H421, respectively. These mutant RNAs contained all of the fixed mutations in the population, .To compare the replication of each evolved genomic RNA (L357 or H421) with that of the original genomic RNA (Ori), we performed the translation-coupled replication experiments with each genomic RNA in the absence or presence of a parasitic RNA. L357 RNA replicated faster than the original RNA in the absence of the parasitic RNA Fig. , left, aE. coli42 or phi29 phages43, or an artificial replication system composed of rolling-circle replication and recombination44. Our automated droplet flow reactor would be a useful tool to allow faster and easier in vitro evolution experiments involving various self-replicating systems.The automation of experimental procedures is important step to make long and laborious evolution experiments faster and easier, thus, accelerating scientific findings. However, automation has not been successfully applied to translation-coupled replication systems because these systems require cell-like compartments to link genotype and phenotype. In this study, we demonstrated automated replication and the evolution of genomic RNA in a droplet flow reactor for the first time. In previous evolutionary experiments, we manually performed a 1000 h replication in approximately 200 days. Here, we performed 400 h replications twice in a total of approximately 34 days, a much shorter duration than previous experiments. This system can be used to evaluate other self-replication systems that replicate through translation, such as the reconstituted genomic DNA replication of In this study, we found that distinct genomic RNA mutants dominated the populations in the two evolutionary experiments. Under a low mixing condition, a mutant RNA that replicates faster dominated the population; however, under a high mixing condition, a mutant that was resistant to parasitic RNA was dominant. Since we performed only one experiment under each condition, we cannot conclude that this evolutionary result was caused by the different mixing intensities. However, the mixing intensity likely changed the evolutionary outcome since different intensities alter the propagation rates of RNAs among the droplets, which can affect the extent of the interaction between genomic and parasitic RNAs. It would be interesting to repeat these experiments to understand the relationship between the evolutionary consequences and the propagation rates of RNA.Another interesting phenomenon that we had not previously observed was the temporal domination of mutations. Some mutations were almost fixed in the population at 186 h, undetected at 306 h, and then reappeared at 357 h. This dynamic change in mutation frequency cannot be explained by sampling error since some mutations (A850G) were also found in all of the sequenced clones 51. As the original genomic RNA, we used the clone after the previous evolutionary experiment at Round 128, named N96(+)17. The host genomic RNAs were prepared by in vitro transcription with T7 RNA polymerase as described previously52. In the competition experiment , the inside solutions of which were mixed by magnetic stirrers . The mixing intensities were controlled by changing the spinning period of the magnetic stirrers in the tanks. The details of the droplet flow reactor are shown in Fig. The construction of the droplet flow reactor has been previously reported in detailers Fig. . The tanIn this study, we performed two long-term replication experiments in the droplet flow reactor with different mixing intensities in the second tank (high or low). To change the mixing intensity, we set the mixing period (duty ratio) to 20% (5 sec mixing in 25 sec) or 5% (3 sec mixing in 60 sec) for high or low mixing conditions, respectively. The rotation frequency of the stirrers was constant (4800 rpm). The dilution rate was set at 0.2 /h (100 uL/h oil and 1 uL/h aqueous phases) during most periods for both experiments. These parameters were changed slightly at the beginning and last stage of the experiments  duty ratio in the second tank, we succeeded in performing all three independent long-term replication experiments for 400 h. The results of two of the experiments are shown in this study.34.The RNA replication solution contained a fluorescent protein  and did not contain protein factors or genomic RNA was used as the aqueous phase. After operating the droplet flow reactor for 48 h, the size distribution of the droplets in the second tank was measured by fluorescent microscopy according to the previous study (n = 531 or 738)35. The average parasitic RNA concentrations were measured from the band intensities after polyacrylamide-gel electrophoresis (PAGE) and staining with SYBR green II using s222 RNA standard. PAGE was performed with 8% gel according to the previous study40. Gel images were shown in Fig. The average host RNA concentrations in the droplets were measured at the indicated time by quantitative PCR after reverse transcription according to a previous study23 (Fwd: 5′-GTCCCCCCCTTCCGGGGGGGTCCCCGGGGATCCTCTAGAG-3′ and Rev: 5′-TGCGCGAGGTGACCC-3′) and transformed into E. coli JM109 strain. Plasmids were extracted from 5–11 colonies at each time and sequenced.At the time indicated in Fig. The original N96(+) RNA, and each of the evolved RNAs L357 or H421) (100 pM) was mixed with the reconstituted translation system with or without 10 nM S222 RNA (parasitic RNA) and incubated at 37 °C for 6 h. At the times indicated in Fig. 21 .Supplementary Information


The human immunodeficiency virus type 1 (HIV-1) is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome (AIDS). The aim of this study was to construct an RNA-positive control based on armored (AR) RNA technology, using HIV-1 RNA as a model.pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography.The MS2 maturase, a coat protein gene (at positions 1765 to 1787) and HIV-1 1 to 105 was prepared. Real-time PCR assays had a range of detection between 101 and 105. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33.The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 10Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory. Human immunodeficiency virus (HIV) infection is one of the most critical challenges to global public health,2. The aPCR-based assays are common methods for the evaluation of viruses and the related transcripts in biological samples. However, controlling the reverse transcription step in PCR reaction and especially applying a control similar to the template sequence are critical requirements of this method.E. coli and enters the bacteria using maturase protein. After entering the bacteria, the viral RNA acts as messenger RNA and translates into structural proteins. In the MS2 assembling stage, one molecule of viral +ssRNA, one copy of the maturase protein (A-protein), and 180 copies of coat (14 kDa) protein are required[Researchers have recently reported the encapsidation of target RNA in another viral capsid such as MS2, which improved the RNA stability. The MS2 required.The phage packaging starts by binding 180 copies of coat protein to a specific region in a hairpin- like structure consisting of 19 nucleotides, which is located near 5’ side of the phage genome. Among these nucleotides, residues A-4, U-5, and A-7 act as recognition sites in the hairpin and play a role in the phage packaging.According to the “armored (AR) RNA’’ technology, almost any exogenous RNA in an appropriate size can be integrated into downstream of the Pac site of MS2 phage genome and non-specifically assembled into expressed MS2 phage capsid, leading to the formation of target AR RNA by phage-like particles (PLPs). Once quantified, the AR RNA can be used as substitute controls and standards for the clinical diagnosis of virus RNA by RT-PCR and qRT-PCR. The aimE. coli strain BL21 (DE3) was used as the host strain for the expression of our target . The pET32a plasmid containing ampicillin resistance gene and T7 promoter was used as expression vector in this study.pol (336 bp) gene were amplified by specific primers , while the HIV-1 pol primer sequences contained HindIII and NotI restriction sites.Commercial MS2 phage was purchased from Sigma-Aldrich (Germany). MS2 maturase, coat protein gene (1751 bp), and HIV-1  primers  using SuBamHI/HindIII site of pET32a plasmid using standard protocols. The resulting plasmid, pET32a-MS2, was propagated in E. coli BL21 (DE3) and extracted with Qiagen miniprep plasmid extraction kit , as per the manufacturer’s instruction.The amplified MS2 sequence was cleaned up and cloned into HindIII  and NotI  restriction enzymes. The digested product was then cleaned with Qiagen PCR clean-up kit and cloned into the HindIII/ NotI sites downstream of the MS2 pac site on pET32a. Each DNA fragment described above was verified by direct DNA sequencing before digestion with restriction enzyme and prior to insertion into linearized pET32a.To generate the recombinant plasmid of pET32a-MS2-HIV, the HIV PCR product was first digested by E. coli strain BL21 (DE3) harboring pET32a-MS2-HIV was cultured in 1 L LB broth medium containing 50 μg/ml of ampicillin and grown in a shaking incubator at 200 ×g at 37 °C for 12 h. The protein expression was then induced by adding 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at the same conditions for 16 h. Subsequently, the bacterial pellet was collected by centrifugation and disrupted by sonication. The cell debris was removed by centrifugation at 16,000 ×g at 4 °C for 10 min. The supernatant contained AR RNA particles, E. coli genome, and DNA plasmid. RNA and DNA contaminations were removed by treatment with 10 mM CaCl2, RNase A, and DNase I .The The supernatant was collected and transferred into a 50-ml conical tube, followed by the slow addition of NaCl up to a final concentration of 2%, with constant and gentle stirring. Next, polyethylene glycol (PEG) 6000 was added to a final concentration of 10%; the mixture was stirred for about 1 h to ensure complete solubilization of the PEG. The beaker was transferred to a refrigerator, and the virus and other proteins were allowed to precipitate at 4 °C for 72 h. The precipitate was collected by centrifuging at 15,000 ×g at 4 °C for 20 minutes. The precipitate was suspended in a small volume of TES buffer . Finally, the AR was purified by a Sephacryl S-200 resin in TES buffer .The PLPs stability was analyzed by agarose gel electrophoresis (1%) and staining with gel red dye. The intact AR was confirmed by transmission electron microscopy (TEM), followed by negative staining with 1% phosphotungstic acid.6, as previously described [The number of MS2 PLP was determined by UV spectrophotometry using the Avogadro’s constant, extinction coefficient of 0.125 mg/ml of MS2 bacteriophage per absorbance unit at 1 OD 260 nm, and a molecular weight of 3.0 × 10pol gene of the HIV-1 genome. The latter sequence was downloaded from HIV database (https://www.hiv.lanl.gov/content/index), truncated and inserted at down-stream of MS2 genome. The primers and probe sequences, as shown in 5 to 101 copies/µl of AR particles were assayed. To do so, RNA molecule was first released by incubation at 80 °C for 5 min. After that, real-time PCR detection was performed using step one real-time PCR instrument , as well as QuantiTect Probe RT-PCR Kit  and TaqMan Master Mix. Reaction was prepared and performed with 12.5 µl buffer, 300 nm forward and reverse primers, 150 nm probe, and 12 µl AR RNA. The RT reaction lasted for 35 min at 50 °C, followed by a denaturation step for 15 min at 95 °C and 12 cycles of 15 s at 95 °C, 35 s at 62 °C, and 35 s at 72 °C without fluorescent collection, followed by 40 cycles of 10 s at 94 °C, and annealing and extension for 40 s at 60 °C. The fluorescent signal was acquired at the annealing-extension step at 60 °C.The primers and probe were designed by AlelleID ver. 6 and Beacon Designer ver. 8  and confirmed in the most conserved region of the pol gene (336 bp), which were obtained by RT-PCR from MS2 RNA  using Superscript® III One-Step RT-PCR System with Platinum® Taq High-Fidelity DNA Polymerase kit .Figures AR particles were precipitated with PEG 6000 and negatively stained with 1% phosphotungstic acid. The TEM image confirmed that AR particles had icosahedral shape and a 30-nm diameter .2 (TSM) at -4 °C over six months. The AR RNA was stable in +4 °C, although naked RNA was rapidly degraded .SDS-PAGE analysis of induced BL21 (DE3)/pET-32a MS2-HIV confirmed the expression of an ~14-kDa protein . This 145 to 101 was prepared. The RT-PCR was first performed on standard to obtain the limit of detection (LoD) of the assay. As shown in 5 with R2 value and standard curve slope of 0.998 and -3.33, respectively, suggesting constantly high amplification efficiency (greater than 99%).A tenfold serial dilution of AR-MS2 + HIV-1 from 10in vitro transcription, for producing positive RNA as an internal or external control in PCR assay[E. coli by transformed plasmid and assembled along with the phage genome in the MS2 capsid; these PLPs contain the target sequence and are resistant to ribonuclease enzyme. The aim of current study is the constructing the positive-control RNA based on AR RNA technology, using HIV-1 RNA as a model.Polymerase chain assays are commonly used, both quantitatively and qualitatively, for the diagnosis and evaluation of viral load in HIV-infected patients,16. TherPCR assay. RNA molPCR assay. Therefopol conserved region of the HIV-1 genome was ligated downstream of MS2 gens in pET32a. The recombinant plasmid was transferred into E. coli strain BL2 (DE3), and the AR RNA was assembled following to IPTG induction. We also have shown the durability of AR RNA in the presence of ribonuclease enzyme. The sequence of coat gene that has an overlap region with lysis gene of the phage was mplified with PCR to prepare the plasmid that could produce large numbers of PLPs without bacterial lysis. Therefore, the produced PLPs were not infectious in accordance to biosafety concerns.To construct the positive-control AR RNA, 1751 bp of MS2 genome containing maturase, coat, and packaging sequence was cloned into pET32a plasmid. Moreover, 336 bp of E. coli DE3 (BL-21) strain.In a previous study, HIV-1 subtype B RNA was packaged in AR RNA,14. Howe2 affinity chromatography, RNA-protein complex, and electroelution from an agarose gel with a GeBAflex-tube[et al.[1 copies/µl of AR RNA, indicating a constantly high amplification efficiency (>99%).The current PLP purification method involves the combined use of gradient ultracentrifugation, column chromatography, COflex-tube,23. In tflex-tube,24. In abe[et al., the cybbe[et al., MS2 phabe[et al.,28. In tThere are several commercial RNA controls for various infectious agents based on AR technology, but due to the high cost of these controls, their usage is limited in developed countries.The results obtained in our study could be a basis for the stabilization of other infectious agents. Moreover, we have developed a virus-based control that requires an accurate analysis and evaluation with a panel of other infectious agents, as well as HIV-1 positives sample. This positive control can be used as a basis for launching an in-house HIV-1 virus assay and can be made it readily available to laboratories and HIV research centers.