Drosophila, that UPF1 constantly moves between the nucleus and cytoplasm by a mechanism that requires its RNA helicase activity. UPF1 is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary glands and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNAs, and cells depleted of UPF1 show defects in the release of mRNAs from transcription sites and their export from the nucleus.UPF1 is an RNA helicase that is required for nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3’UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from  Saccharomyces cerevisiae genetic screen for mutations that enhance up-frameshift tRNA suppression (NAM7 (S. cerevisiae) and SMG2 (Caenorhabditis elegans) – from other genetic screens (UPF1 (UP-Frameshift-1) is a universally conserved eukaryotic protein that was first identified in a pression , and gai screens . Cells t screens .These observations are generally interpreted as evidence that UPF1 and related proteins are primarily required for nonsense-mediated mRNA decay (NMD), a conserved eukaryotic mRNA surveillance mechanism that detects and destroys mRNAs on which translation terminates prematurely . NMD is The exact role of UPF1 in NMD is uncertain though. Standard models postulate that UPF1 monitors translation termination on ribosomes by interacting with a peptide release factor (eRF1 or eRF3). However, recent reports on mammalian translation systems have suggested, in contrast to earlier reports on other organisms , that UPUPF1 is an ATP-driven helicase that unwinds RNA secondary structures and so can displace RNA-bound proteins . UPF1 isUPF1 is most abundant in the cytoplasm where its roles discussed above depend on ribosomal translation and occur on cytoplasmic mRNAs. A fraction of UPF1 was expected in the nucleus though, as the protein traffics in and out of the nucleus in mammalian cells . Some stDrosophila. First, we demonstrate that UPF1 is a highly mobile protein that constantly shuttles between the nucleus and cytoplasm, and its distribution in the cell, with more in the cytoplasm than the nucleus, depends on its RNA binding properties and approximately reflects that of mRNA. UPF1 associates with nascent transcripts on chromosomes – mostly with Pol II transcribed, but also with some Pol III-transcribed genes. Relatively more of the transcript-associated UPF1 is bound with exons than with introns, suggesting that intron recognition might act as a roadblock to the 5’-to-3’ transit of UPF1 along the pre-mRNA. Most strikingly, UPF1 is needed for the efficient release of polyadenylated mRNAs from most chromosomal transcription sites and for their export from the nuclei. These observations indicate that UPF1 starts scanning pre-mRNA transcripts whilst they are still being assembled into ribonucleoprotein (RNP) complexes on chromosomes and suggest that UPF1 fulfils previously unrecognised role(s) in facilitating nuclear processes of gene expression and mRNA export. The broad and dynamic association of UPF1 with mRNAs redefines it from being primarily an NMD-inducing factor to being a global player in mRNA processing in the nucleus as well as in the cytoplasm, and might also explain why none of the prevailing models satisfactorily explain how UPF1 could target specific transcripts to NMD.In the present study, we show direct evidence that UPF1 is globally involved in nuclear processing of mRNAs in Drosophila UPF1 outside the RNA helicase domain: one epitope in the N-terminal flanking regions (antibody 1C13 against Pep2), and two near the C-terminus   see . Followiproteins . The antproteins .Drosophila salivary glands, which are made up of large secretory cells with polytene nuclei. UPF1 was most abundant in the cytoplasm and perinuclear region, and there was also distinct but less intense nuclear staining, mainly around the chromosomes and around the nucleolus  and Fluorescence Recovery after Photo-bleaching (FRAP)  – to exaDrosophila salivary glands; this (GFP-UPF1) showed a intracellular distribution similar to that of the endogenous protein, with an intense cytoplasmic signal and a weaker, but still obvious, signal in the regions occupied by the chromosomes  , a drug omosomes .Heat-shock caused a similar redistribution of much of UPF1 from the cytoplasm to the nucleus  that inhibit its RNA helicase activity due to the loss of ATP hydrolysis . Both thThe data indicate that wild-type UPF1 is freely mobile within cell compartments and that it constantly moves in and out of the nucleus by mechanisms that involve the CRM1-dependent nuclear export pathway and requires its RNA helicase activity.Drosophila salivary glands. These well-characterised giant interphase chromosomes are formed after multiple rounds of endoreplication without chromosomal segregation, and they provide a powerful system to visualise transcription and pre-mRNA processing at individual gene loci.To gain insight into the role(s) of UPF1 in the nucleus, we used immunostaining to examine whether it associates with the polytene chromosomes of UPF1 was present predominantly at interbands and puffs: cytologically distinct chromosome regions in which the chromatin is less condensed and that correspond to transcriptionally active sites . The immWe then undertook double immunostaining of chromosomes for UPF1 and for Ser2 Pol II – the form of Pol II that transcribes through the main body of genes which is characterised by having the C-terminal domain (CTD) of its largest subunit Ser2-phosphorylated . Much ofThe association of UPF1 with the chromosomes depends on transcription. This is illustrated by the changes in UPF1 immunostaining that followed heat-shock, which induces transcription at specific cytological puffs encoding heat-shock proteins and of hsrω lncRNAs at locus 93D . This reThese observations suggested that UPF1 associates with genes that are being transcribed. UPF1 was also recruited to other genes following transcription activation, such as to an ecdysone-inducible transgene , at normal temperature . No UPF1Actin5C provided a striking example of correspondence between the ChIP-seq and polytene immunostaining results: it was one of the most UPF1-enriched genes in the ChIP-seq data (Actin5C) and displayed one of the brightest UPF1 chromosomal signals at the gene locus corresponding to interband 5C on the X chromosome (We examined UPF1 and Ser2 Pol II association with multiple gene loci by chromatin immunoprecipitation (ChIP) of S2 cell extracts, followed by high-throughput DNA sequencing (ChIP-Seq). UPF1 was associated with many transcriptionally active genes, most of which are Pol II transcription sites. romosome . The ChIThe enrichment profile of UPF1 at Pol II loci closely followed that of Ser2 Pol II, and UPF1 enrichment being the greatest at highly expressed genes . Real-tiDrosophila and other organisms (NAT1 and Su(z)2 genes , and throughout their transcription units , whereasrganisms . Typicalrganisms . Strikin)2 genes  and the e.g. Adam TS-A, panel five in A comparison of the UPF1 loading of genes with different Ser2 Pol II loading profiles suggests that UPF1 association depends on transcription elongation: UPF1 did not associate with genes at which Ser2 Pol II was associated only with the TSS pausing site and which were not being actively transcribed  on salivary glands, a drug that blocks Pol II transcription by inhibiting Ser2 phosphorylation . In the S2 cells .UPF1 was found mainly at Pol II transcription sites, most of which are protein-coding genes, however, our ChIP-seq data revealed that UPF1 also binds at some Pol III genes. The latter included 7SK and both paralogous genes of 7SL snRNAs  – but noXrp1 ; and it is genome-wide, as demonstrated by comparing UPF1 association with introns and with their flanking exons in the ChIP-seq data from many genes .This exon-biased UPF1 enrichment was confirmed by real time PCR in multiple ChIP experiments ; Y14 and eIF4III are two EJC proteins that bind the nascent pre-mRNA but are not likely to play a direct splicing role in Drosophila; see . Expression of hUPF1 also suppressed the apparent smaller size of the salivary glands depleted of the endogenous UPF1; these glands were comparable in size to that of wild-type  RNA – which is referred to simply as poly(A) – by oligo(dT) FISH (fluorescence in situ hybridisation): this should detect mRNA that has been transcribed, spliced, released from Pol II and polyadenylated. In wild-type cells poly(A) was abundant and fairly evenly distributed throughout the cytoplasm, as would be expected for mature mRNA, and there was relatively little in the nuclei (An appreciable amount of poly(A) signal, which was not within clusters, was clearly at the chromosomes though, in the UPF1-depleted cells  – showedActin5C gene, in wild-type, but this band was obviously fluorescent in UPF1-depleted cells. Another example was site 2B – where constitutively expressed sta and rush are probably the most active genes at this larval stage – which showed a faint poly(A) signal in wild-type glands and a strong signal in UPF1-depleted cells. UPF1 was clearly associated with these transcription sites (2B and 5C) on polytene chromosomes (Actin5C).Both the number of transcriptional sites showing poly(A) accumulation and the amount of poly(A) RNA associated with these sites were strikingly increased in UPF1-depleted cells  in the release of mRNAs from transcription sites and in their transport out of the nucleus , the exon vs. intron bias in UPF1 binding is lost in cells depleted of the spliceosome component U1 snRNP. This suggests that, when U1 snRNP is bound to the 5’ss of an intron at the initial stage of splicing, it may hinder UPF1 translocation along the pre-mRNA and cause it to dissociate . Conversely, those few transcription sites at which poly(A) accumulates even in wild-type cells, may not normally be associated with UPF1, a striking example of which is the 2C transcription site on the polytene chromosomes, where poly(A) accumulation was most apparent, but no obvious UPF1 association was observed.The most striking effects of UPF1 depletion were retention of poly(A) RNA at transcription sites and then its failure to be exported effectively from the nucleus. Mature mRNAs that have been cleaved and polyadenylated are normally expected to be speedily released from transcription sites, but our data show that this is not always the case. We have both: a) identified some sites on polytene chromosomes that apparently accumulate poly(A) RNA even in wild-type glands; and b) shown that most of the active Pol II genes accumulate poly(A) RNA in UPF1-depleted salivary glands. Poly(A) RNA accumulation in UPF1-depleted cells is most prominent at genes with which UPF1 associates strongly in wild-type, such as Drosophila cells carrying mutation in the RNA helicase P68 (Evidence of retention of poly(A) and specific mRNAs in discrete nuclear foci or ‘dots’ has previously been reported in cells defective in RNA processing, initially in mRNA export and processing mutants in yeast ; and latcase P68 . WhetherDrosophila salivary gland cells. Possibly, in the absence of UPF1 function, mRNPs acquire or retain native conformations that hinder their release from the chromosome and make them prone to aggregation and, consequently, cause nuclear retention. Such a global role of UPF1 could explain, better than its involvement in NMD, why this protein is universally conserved in eukaryotes, why its depletion affects the expression of a large fraction of the genome, and possibly why expression of human UPF1 in rat models of amyotrophic lateral sclerosis (ALS) overcomes the pathology caused by over-expression or mutation of the RNA-binding protein TDP-43 , Tetramethylrhodamine Conjugate Wheat Germ Agglutinin . The antibodies used in western blotting: mouse anti-UPF1 , mouse anti-α-tubulin , rat anti-Ser2 Pol II , mouse anti-hnRNPA1 , rabbit anti-eIF4AIII (1:1000), rabbit anti-Y14 (1:1000); the last two antibodies were described previously , mouse anti-Pol II  and mouse anti-GFP .The following antibodies were used for immunostaining: mouse anti-UPF1 , mouse IgM anti-Ser2 Pol II , mouse anti-hnRNPA1  , mouse aeviously . The ant1118 strain was used as wild type (DGGR_108736). UAS-UPF1-RNAi (43144) and UAS-GFP-UPF1 (24623) were obtained from the Bloomington stock centre. The forkhead (Fkh) Gal4 has a salivary gland specific expression from early stage of development  so that it cannot be targeted by this RNAi construct, as demonstrated in Results.Flies were reared in standard corn meal fly food media at 24°C. The y welopment . The UPFescribed . The trad before . The trad before ; after sd before  into theTAATACGACTCACTATAGGGGAGAT-3’). The amplified PCR fragments were purified using MonarchPCR and DNA Cleanup Kit  and dsRNA was synthesised using the T7 RiboMAX express RNAi system . To induce RNAi, a six-well culture dish was seeded with 106 cells/well in serum-free media and mixed with 15 µg of dsRNA/well. Following 1 hr incubation at RT, 2 mL of complete media was added to each well and the cells were incubated for the next three days to knockdown the corresponding RNA and then harvested. The RNAi efficiency of UPF1, eIF4AIII and Y14 was measured by western blotting while snRNPU1-70K was measured by real time PCR.S2 cells (CVCL_Z232) were cultured in Insect–XPRESS media (Lonza) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin-Glutamine mix  at 27°C. These cells never tested mycoplasma positive. To make the RNAi constructs for UPF1, eIF4AIII, Y14 and snRNPU1-70K mRNA, the specific sequences were PCR amplified from S2 cell genomic DNA, using corresponding primer pairs . Along wDrosophila UPF1 tagged with GFP were generated by inserting the coding region of either wild-type UPF1 or UPF1(DE617AA) in either pAc  or pAGW  Gateway compatible vector carrying a Act5C promoter sequence, as previously described . The coding region was PCR amplified from a full length Drosophila UPF1 cDNA clone previously described ; and B310, pAc-C-term-GFP-UPF1(DE-AA) expressing escribed ; and theD. melanogaster UPF1 (TM technology). Hybridoma clones were generated and used to induce 18 ascites, which were then screened by western blotting of S2 cell protein extracts. Out of these, three that showed a single band of the expected size and minimal cross-reactivity were selected and more of the monoclonal antibodies were subsequently purified from the corresponding hybridoma cell culture in vitro. Unless otherwise specified, 7B12 was used as the anti-UPF1 antibody throughout this study.Antigens design, preparation, mice immunisation and hybridoma generation were carried out by Abmart (Shanghai). Twelve peptide sequences predicted to be highly immunogenic were selected from ter UPF1  and clonWhole-mount immunostaining was performed as previously described . In brieWandering third instar larvae were dissected in M3 media and tissues were incubated with or without Leptomycin B  for 1 hr at RT. To examine the real-time effect of LMB treatment in the living cell, salivary glands were dissected in M3 media and incubated with a hanging drop of 50 nM LMB in M3 media in a cavity slide . The fluFluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) methods have been previously described . SalivarApart from the changes detailed below, the procedure was mostly as previously described . BrieflyE. coli tRNA  for 12 hr at 42°C. Glands were then washed with freshly made wash buffer (50% Formamide in 2XSSC pH 7.2), followed by 2XSSC, 1XSSC and finally with 1XPBS three times each, with 5 min interval. Nuclei were counterstained with DAPI and tissues were mounted in PromoFluor Antifade mounting medium and examined under Leica TCS SP2-AOBS confocal microscope.Oligo (dT) FISH was done as previously described for mammalian cells with some modifications . SalivarFor polytene chromosomes oligo(dT) FISH, salivary glands were dissected in 1XPBS and incubated with fixing solution  for 5 min at RT. Chromosomes were squashed in the same solution and examined immediately under phase-contrast microscope to check if properly spread. Slides with good chromosomes were briefly dipped in liquid nitrogen and the coverslips were flipped off with a sharp blade. Slides were immediately dipped in 90% alcohol and stored at 4°C. Before hybridisation, slides were air dried and rehydrated in 1XPBS and then washed and hybridised, as described above for whole salivary glands. Chromosomes were counterstained with DAPI, mounted in PromoFluor Antifade mounting medium and examined under Nikon Eclipse Ti epifluorescence microscope.7) were harvested and washed with ice-cold 1X PBS containing 1X PhosSTOP  and 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail . Cells were incubated in the hypotonic AT buffer  for 20 min on ice and lysed with 2 mL Dounce homogenizer by 30 strokes with the tight pestle. Lysate was centrifuged at 5000 RPM for 5 min at 4°C in microcentrifuge and the nuclear pellet was resuspended in 500 μL IP buffer , 1X PhosSTOP, 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, 1 U/µL Ribolock RNase Inhibitor) for 20 min on ice. Nuclear lysates were sonicated using a Bioruptor sonicator (Diagenode) for 3 cycles of 30 s ON and 30 s OFF with maximum intensity. Following sonication, the lysates were centrifuged at 13000 RPM for 15 min at 4°C in a microfuge and the antibody (5 μg) was added to the clear supernatant, with or without addition of RNase A (100 µg/mL), and incubated overnight at 4°C on a rocker. Following incubation, 20 μL of prewashed paramagnetic Dynabeads  were added and incubated further for 2 hr at 4°C on a rocker. Beads were washed 5 times with IP buffer using a magnetic rack and proteins were extracted by adding 40 μL SDS-PAGE sample buffer.Immunoprecipitation was performed as previously described , with so7) were harvested and fixed with 1% formaldehyde  for 10 min at RT. Following fixation, cross-linking reaction was stopped by adding 125 mM Glycine for 5 min at RT. Cells were centrifuged at 2000 RPM for 5 min at 4°C, the pellet was washed twice with ice-cold 1X PBS containing 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail. The cell pellet was resuspended in 1 mL of cell lysis buffer  supplemented with 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail and 1X PhosStop and incubated for 10 min at 4°C. Cells were centrifuged and the pellet was resuspended in 1 mL nuclear lysis buffer  supplemented with 1X cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail and 1X PhosStop and incubated for 10 min at 4°C. The cell suspension was further diluted with 500 µL IP dilution buffer  and sonicated for 5 cycles at 30 s ON, 30 s OFF at maximum intensity using a Bioruptor sonicator (Diagenode); this produced an average fragment size of ~500 bp. Samples were centrifuged at 13000 RPM for 20 min in a microcentrifuge and the clear supernatant was transferred to a 15 mL tube. An aliquot of 100 µL supernatant was kept to extract input DNA. The supernatant was further diluted with 5 vol of IP dilution buffer. For each ChIP, typically we added 5 to 10 µg of antibody to this supernatant and incubated overnight at 4°C on a rocker. Prewashed 20 µL Dynabeads were added to the lysate-antibody mix and incubated further for 1 hr at 4°C on a rocker. Beads were washed 6 times with low salt buffer , once with high salt buffer  and once with 1X TE buffer . The beads were then incubated with 250 µL elution buffer  at RT for 15 min and eluted chromatin was reverse cross-linked by adding 38 µL de-crosslinking buffer  and then incubated at 65°C overnight on a rotator. Proteins were digested by adding 2 µL Proteinase K (50 mg/mL) and incubated at 50°C for 2 hr on a rocker. DNA was isolated using MonarchPCR and DNA Cleanup Kits. Real-time PCR quantification of DNA samples was carried out using the SensiFAST SYBR Hi-ROX Kit  in 96-well plates using an ABI PRISM 7000 system (Applied Biosystems). For NGS sequencing, ChIP and input DNA were further fragmented to 200 bp fragment size using a Bioruptor Pico (Diagenode). All ChIP-DNA libraries were produced using the NEBNext Ultra II DNA Library Prep Kit (New England Biolab E7645L) and NEBnext Multiplex Oligos for Illumina Dual Index Primers (New England Biolabs E7600S), using provided protocols with 10 ng of fragmented ChIP DNA. Constructed libraries were assessed for quality using the Tapestation 2200 (Agilent G2964AA) with High Sensitivity D1000 DNA ScreenTape (Agilent 5067–5584). Libraries were tagged with unique barcodes and sequenced simultaneously on a HiSeq4000 sequencer.S2 cells (2 × 107) were harvested and washed twice with ice-cold 1X PBS via centrifugation at 2000 g for 5 min each. Cells were resuspended in 1 mL ice-cold buffer AT and incubated on ice for 10 min. Cells were lysed using a 2 mL Dounce homogenizer by 30 strokes with the tight pestle. The lysate was divided into two aliquots and each aliquot of 500 µL was layered over a 1 mL cushion of buffer B , and centrifuged at 8000 RPM for 15 min at 4°C in a microcentrifuge. The supernatant was removed and the pellet was resuspended in 5 volumes of nuclear lysis buffer  and resuspended using a 2 mL Dounce homogenizer by three strokes with loose pestle and two strokes with tight pestle. Equal volume of 2X NUN buffer  was added to the nuclear suspension drop by drop while vortexing and the suspension was placed on ice for 20 min prior to spinning at 13,000 RPM for 30 min at 4°C. The supernatant was removed and TRI Reagent  was added to the histone–DNA-Pol II-RNA pellet. The TRI Reagent–pellet suspension was incubated at 65°C with intermittent vortexing to dissolve the pellet, and RNA was extracted following the manufacturer’s protocol. Poly(A) depletion was performed with Dynabeads Oligo(dT)25 (ThermoFisher Scientific). The purification of nascent RNA was assessed by RT-PCR of CG12030, CG5059 and CG10802 genes which have slow rates of co-transcriptional splicing  to assess quality and to determine concentrations. Aliquots of each sample were diluted to ~5 ng/µl, and tested with an Agilent Tapestation 2200 (Agilent G2964AA) using High Sensitivity RNA ScreenTapes kit (Agilent 5067–5579) to determine the RNA Integrity Number.Total-RNA (1 µg) was first poly(A) selected using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs E7490L) prior to library construction. Nascent RNA samples (100 ng) were processed without poly(A) selection. RNA libraries were prepared using a NEBNext Ultra Directional RNA Library Prep Kit (New England Biolab E7420L) and NEBnext Multiplex Oligos for Illumina Dual Index Primers (New England Biolabs E7600S), following standard protocols. RNA libraries were checked for quality using the Tapestation 2200 (Agilent G2964AA) with High Sensitivity D1000 DNA ScreenTape (Agilent 5067–5584). Multiplexed libraries were sequenced (50 bp single-end reads) on a HiSeq4000 sequencer.D. melanogaster Dm6 genome release and accompanying annotations. Assembly and alignment output files for each genome contig were then analysed with the ArrayStar and GenVision Pro software (from the same package) to view and compare read profiles on the genome. Profiles at selected regions were saved as high-resolution images.ChIP-seq and RNA-seq data were initially viewed and analysed using the Lasergene Genomics Suite version 14 (DNASTAR). Pre-processing, assembly and mapping of the sequencing reads in the FASTQ files were performed by the SeqMan NGen software of this package automatically after selecting the NCBI To perform the metagene analyses, an index for Dm6 was downloaded from the HISAT2 website. HISAT2 v2.1.0 was then used to align the FASTQ files on it. The resulting SAM files were converted to BAM format, sorted, and indexed with Samtools 1.6. For the cytoplasmic RNA-seq data, the NCBI RefSeq gene annotations for Dm6 were downloaded as a refGene table from UCSC Table Browser (genome.ucsc.edu). The LiBiNorm tool was then used to produce read counts per gene in an HTSeq-count compatible format based on the refGene file . Transcrwww.r-project.org). This analysis was done using either all introns annotated in Dm6 (151623) or those longer than 100 bp (76708). In either case flanking exons are significantly more enriched than introns. Here we have shown the result of analysis using just the longer introns as these were considered to be more informative because of the predicted lower resolution of ChIP at discriminating between closely adjacent sequences and because of the lower sequencing coverage of shorter introns compared to longer introns. All ChIP-seq and RNA-seq raw sequencing data and Bedgraph files were deposited in the GEO repository (Accession No GSE116808). All custom scripts used in this study are provided in Source Code File 1.A custom script was used to extract the sequencing read coverage from the Bedgraph files for each exon/intron/exon region in the dm6 annotation file xon_fly_gene (downloaded from UCSC Table Browser). To normalise for any bias in the sequencing, the UPF1 signal of each exon or intron was divided by the average coverage in the input sample. The fold change of UPF1 signal/input signal in introns was compared to that of their flanking exons using Wilcox.test (two sided and unpaired) in R  and UPF1-GFP to examine the subcellular distribution of Upf1 in a very large number of experiments. Experimental approaches include immunofluorescence (IF) analyses of salivary glands and polytene chromosomes, ChIP-seq studies in S2 cells, RNAi knockdowns, and nuclear vs. cytoplasmic distributions in response to LMB treatment. Based predominantly on correlative data, the authors conclude that Upf1 appears to be associated with "most of the active Pol II transcription sites and some Pol III-transcribed genes…," i.e., their conclusions represent a large leap from the conventional notion of UPF1 (and the NMD pathway) as a cytoplasmic mRNA surveillance system.Singh et al. describe the isolation of monoclonal antibodies that recognize Essential revisions:The reviewers agree that the manuscript should be "streamlined" to eliminate the less compelling data represented in the last two figures. In addition, the manuscript relies heavily on the monoclonal antibodies, and these need to be characterized better. The key concerns are:1) Uncertainty about the reliability of the antibody used for immunolocalization of UPF1.2) Absence of any information about the features of nascent transcripts that provide mechanistic insight into their putative interaction with UPF.Experiments are recommended that would address mechanistic depth are those in which the "binding" ability of mutated UPF1 is assessed. More precisely, the authors should test mutations in UPF1 known to affect the protein's ability to bind RNA. Such mutational analyses would be critical to determine whether there is bona fide UPF1:RNA interaction, or whether some other interaction  has led to the observed result. If these can be addressed to the satisfaction of the reviewers, we would consider a revised manuscript. However, you may prefer to submit to another journal if you feel these issues, including validation of the antibodies, could not be addressed in a few months.Reviewer #1:Drosophila salivary gland chromosome spreads and in S2 cells, combined with ChIP-seq data, the authors confirm previous evidence that UPF1 is a shuttling protein that exchanges rapidly between cytoplasm and nucleus, with the majority of UPF1 residing in the cytoplasm. They further show that inside the nucleus, UPF1 localizes at sites of active transcription (primarily Pol II transcription sites), suggesting that UPF1 binds RNA co-transcriptionally. While the data for these findings is compelling and in my opinion would justify publication on its own , the authors went on with additional experiments and further analysis of their ChIP-seq data that led them to conclude (i) that intron recognition interferes with UPF1 association, (ii) that Pol II tends to stall near transcription start sites in UPF1-depleted cells and (iii) that UPF1 depletion leads to nuclear mRNA retention. While the effect of UPF1 depletion on poly(A)+ RNA is striking, the conclusions of points (i) and (ii) are much less compelling and would need further investigation to gain more mechanistic insight, as detailed below. I therefore suggest removing these two parts from the manuscript. If confirmed by additional data, these findings can be published later in separate papers.Singh and colleagues present here a large amount of interesting data revolving around UPF1 functions in the nucleus at the site of transcription and during mRNA export. Using immunostainings on Reviewer #2:Drosophila UPF1 and then utilize those reagents (primarily a single antibody) and UPF1-GFP to examine the subcellular distribution of UPF1 in a very large number of experiments. Experimental approaches include immunofluorescence (IF) analyses of salivary glands and polytene chromosomes, ChIP-seq studies in S2 cells, RNAi knockdowns, and nuclear vs. cytoplasmic distributions in response to LMB treatment. Based predominantly on correlative data, the authors conclude that UPF1 appears to be associated with "most of the active Pol II transcription sites and some Pol III-transcribed genes…," i.e., their conclusions represent a large leap from the conventional notion of UPF1 (and the NMD pathway) as a cytoplasmic mRNA surveillance system. Although the authors' conclusions raise the possibility of unforeseen functions for UPF1 they are contingent on two key questions: (1) how reliable are the antibody-based experiments? and (2) what feature of nascent RNA transcripts might be responsible for their apparent interactions with UPF1? Unfortunately, neither question is addressed adequately.Singh et al. describe the isolation of monoclonal antibodies that recognize Specificity of the anti-UPF1 monoclonal antibodies was considered by comparisons of IF results with those obtained with UPF1-GFP, and examination of the consequences of RNAi knockdown of UPF1. UPF1-GFP results were markedly different from those obtained in IF analyses , with the UPF1-GFP results largely showing cytoplasmic and (interesting) perinuclear staining without any significant nuclear staining, whereas the IF experiments seem to indicate that UPF1 can be found in all three locations. RNAi knockdown of UPF1 was only examined in salivary glands, where it depresses but does not eliminate anti-UPF1 staining of polytene chromosomes. Importantly, the authors did not present an experiment examining IF of S2 cells or mosaic tissues in which the UPF1 gene has been deleted. These critical experiments are a fairly commonplace approach to demonstrating unambiguous specificity of an antibody reagent. Other trivial controls that are lacking are results from an experiment using only the secondary antibody and IF localization of a protein definitively known to be cytoplasmic (using the same secondary antibody used for the UPF1 experiments).Drosophila ribosomal proteins harboring fluorescent tags manifest cytological distributions that are substantially similar to the putative UPF1 distributions seen in the current manuscript . Clearly, one possibility is that the distributions seen in the present work are an indirect consequence of UPF1 interactions with chromosome-associated ribosomes or ribosomal proteins. Further, in light of UPF1's known role as an RNA-binding protein I would have expected that the authors would test whether UPF1 mutations known to impair the protein's RNA-binding capacity altered any of the cytological or ChIP-seq results seen by the authors.Little insight is presented with regard to possible salient features of nascent transcripts that might mediate their putative interactions with UPF1. In light of known interactions between UPF1 and ribosomes it is surprising that the authors did not consider their own published work demonstrating that eLife. Your article is being evaluated by James Manley as the Senior Editor, a Reviewing Editor, and two reviewers.Thank you for sending your article entitled "The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from gene loci" for peer review at Given the list of essential revisions, including new experiments, the editors and reviewers invite you to respond with an action plan and timetable for the completion of the additional work. We plan to share your responses with the reviewers and then issue a binding recommendation.Both reviewers have concerns regarding the work, although to different degrees. They have discussed this extensively between them. Reviewer 1 is concerned about the interpretation of the ChIP data against the suggestion that it be removed or at least qualified that UPF1 could bind after intron removal. In any case, this observation does not seem to shed much light on the process.Reviewer 2 judges that, since the interpretation of the data hinges on the specificity of the antibodies, there needs to be more rigorous demonstration that the antibodies have been well characterized and indirect effects have been ruled out. The reviewer states, "The current version of the manuscript continues to rely on RNAi knockdowns to substantiate MAb reliability, an approach that is less convincing than mosaics." and further "Most importantly, the paper is comprised of a series of observations without definitive experiments that rule out indirect interactions as explanations for the observed results."In order to satisfy the reviewers, it appears that you will need to deliver a more definitive manuscript that addresses these concerns. Please let us know how you would plan to respond to the mentioned concerns.Reviewer #1:The authors have provided additional data to demonstrate the specificity of their antibodies, as requested.As suggested be the reviewers, they also have streamlined the manuscript by removing the less compelling parts on a suggested role of UPF1 in Pol II pausing and at the nuclear envelope. Against the reviewer's suggestion, they decided to retain their ChIP-seq experiments showing that more UPF1 is associated with exons than with introns. They argue that my alternative – and less exciting – interpretation of their data, namely that this might simply result from a substantial fraction of UPF1 binding only after intron removal, was unlikely to be valid. However, I do not understand why the fact that UPF1 was also found associated with intronless genes would challenge my interpretation of the data. With ChIP, one has no information at which stage during the transcription of an intronless gene UPF1 binds the nascent transcript. The same is true for intron-containing genes and if a fraction of UPF1 would bind after an intron was spliced out, the net result would be more UPF1 bound to exons than to introns.The newly added rescue of the poly(A)+ RNA accumulation in the nucleus in the absence of UPF1 demonstrates the specificity of this observation and strengthens the conclusion.Thus, even though I do not agree with the author's interpretation of the ChIP data, all my major concerns have been satisfactorily addressed and I support accepting the manuscript for publication.Reviewer #2:Drosophila UPF1 and then utilize those antibodies  and UPF1/GFP constructs to examine the subcellular distribution of UPF1 in S2 cells and salivary glands. Additional experiments evaluated ChIP-seq in S2 cells, and nuclear vs. cytoplasmic distributions in response to LMB treatment. Based predominantly on correlative data, the authors conclude that UPF1 appears to be associated with most of the active Pol II transcription sites and some Pol III-transcribed genes. As such, their conclusions represent a large leap from the conventional notion of UPF1 (and the NMD pathway) as a cytoplasmic mRNA surveillance system. Although the authors' conclusions raise the possibility of unforeseen functions for UPF1, they are contingent on two key questions: (1) how reliable are the IF, ChIP-seq, and GFP-UPF1 experiments? and (2) is it possible that the observed UPF1:RNA interactions are indirect?Singh et al. have submitted a revised version of their manuscript that bears a close resemblance to the original, both in experimental approach and in the concerns raised by the respective experiments and conclusions. As before, the authors begin by describing the isolation of monoclonal antibodies that recognize Previously, I was concerned about the 7B12 antibody, particularly: (i) the very strong additional band that showed up in the westerns of Figure 1—figure supplement 1B and (ii) the lack of any tests of the antibody using UPF1 deletion/mosaic tissues. The current version of the manuscript continues to rely on RNAi knockdowns to substantiate MAb reliability, an approach that is less convincing than mosaics. Further, I find the vast overexpression of UPF1-GFP (~10x the level of endogenous UPF1), and its distribution differences relative to endogenous UPF1 to be additional concerns. In the ChIP-seq experiments, far too much attention is paid to hypothetical explanations for the differences in exon vs. intron signals  while little attention was paid to the ~10x lower recovery of UPF1 ChIP-seq vs. Pol II ChIP-seq.Most importantly, the paper is comprised of a series of observations without definitive experiments that rule out indirect interactions as explanations for the observed results. The authors only make that concern worse by filling the discussion with numerous unsubstantiated explanations for the apparent shuttling, the ChIP-seq results, and the experiments with the UPF1 helicase mutant.eLife's standards.In short, this manuscript just isn't up to  [Editors' note: the authors’ plan for revisions was approved and the authors made a formal revised submission.]Essential revisions:The reviewers agree that the manuscript should be "streamlined" to eliminate the less compelling data represented in the last two figures. In addition, the manuscript relies heavily on the monoclonal antibodies, and these need to be characterized better. The key concerns are:1) Uncertainty about the reliability of the antibody used for immunolocalization of UPF1.As explained in detail in our responses to reviewer 1 below, we are very confident that the three UPF1 antibodies detect specific signals in all the assays we have performed. Prompted by this concern though, we have redone all the controls and most of the additional experiments suggested. The results of these confirm that the antibodies are specific and we show the additional data in new supplementary figures/panels in the revised manuscript: .2) Absence of any information about the features of nascent transcripts that provide mechanistic insight into their putative interaction with UPF.Experiments are recommended that would address mechanistic depth are those in which the "binding" ability of mutated UPF1 is assessed. More precisely, the authors should test mutations in UPF1 known to affect the protein's ability to bind RNA. Such mutational analyses would be critical to determine whether there is bona fide UPF1:RNA interaction, or whether some other interaction  has led to the observed result. If these can be addressed to the satisfaction of the reviewers, we would consider a revised manuscript. However, you may prefer to submit to another journal if you feel these issues, including validation of the antibodies, could not be addressed in a few months.We have done more experiments as suggested, to assess whether UPF1 RNA “binding” ability affects its nuclear localization. We examined this in S2 cells transfected with GFP tagged constructs expressing either wild-type UPF1 or a mutant version which carries a well characterized mutation in the ATPase domain that inhibits its RNA helicase activity, which is required for UPF1 dynamic binding and dissociation from mRNAs. The results showed the ATPase mutant version is not retained in the nucleus following incubation with LMB, unlike wild-type which readily accumulates in the nucleus . In the revised Discussion section we therefore proposed that this rapid shuttling of UPF1 between nucleus and cytoplasm primarily depends on UPF1 binding to RNP cargos that are being continuously exported out of the nucleus, rather than on a direct or protein adapter-mediated interaction with CRM1, as previously suggested (referred in the manuscript). A more detailed molecular analysis of how UPF1 interacts with nascent mRNPs will need to be done in future, but we feel that this would go beyond what we aimed to demonstrate with this initial study – see our more detailed response to reviewer 1’s criticism about this point below.We agree that it is plausible that a substantial fraction of UPF1 might associate indirectly with nascent transcripts through interactions with RNPs loaded on them. Following the reviewer suggestion, we have speculated what these RNPs and mechanisms might be in the revised Discussion section (see our detailed response to the reviewer below).As advised, we have “streamlined” the manuscript. All the data that suggested a role of UPF1 in Pol II pausing were omitted ; these data remain public on the bioRxiv preprint and we hope to investigate this further and publish them at a later stage separately, as advised by reviewer 1. We have also omitted the data suggesting a role of UPF1 at the nuclear envelope .We have kept the data showing that more UPF1 is associated with exons than with introns . Although we agree that more data will be required to gain insight in to the mechanism, this exon vs. intron bias in the ChIP-seq data is genome-wide and apparent at several genes that we have verified by qPCR (see our detailed response to reviewer 1 about this point). However, we simplified the figure by moving three of the panels to a supplementary figure  and hope this change will make the manuscript both more concise and clear.With regards to our conclusion that UPF1 plays a role in the release of mRNAs from transcription sites and export to the cytoplasm, as suggested by reviewer 1, we have tested whether the striking poly(A) accumulation phenotype observed in UPF1 depleted cells can be rescued by expression of a UPF1 transgene, which is not targeted by UPF1-RNAi. The result was a clear-cut rescue of this phenotype, which, hence, provides further indication that nuclear poly(A) accumulation is a direct effect of UPF1 depletion (discussed further in response to this reviewer).Reviewer #1:Singh and colleagues present here a large amount of interesting data revolving around UPF1 functions in the nucleus at the site of transcription and during mRNA export. Using immunostainings on Drosophila salivary gland chromosome spreads and in S2 cells, combined with ChIP-seq data, the authors confirm previous evidence that UPF1 is a shuttling protein that exchanges rapidly between cytoplasm and nucleus, with the majority of UPF1 residing in the cytoplasm. They further show that inside the nucleus, UPF1 localizes at sites of active transcription (primarily Pol II transcription sites), suggesting that UPF1 binds RNA co-transcriptionally. While the data for these findings is compelling and in my opinion would justify publication on its own , the authors went on with additional experiments and further analysis of their ChIP-seq data that led them to conclude (i) that intron recognition interferes with UPF1 association, (ii) that Pol II tends to stall near transcription start sites in UPF1-depleted cells and (iii) that UPF1 depletion leads to nuclear mRNA retention. While the effect of UPF1 depletion on poly(A)+ RNA is striking, the conclusions of points (i) and (ii) are much less compelling and would need further investigation to gain more mechanistic insight, as detailed below. I therefore suggest removing these two parts from the manuscript. If confirmed by additional data, these findings can be published later in separate papers.As suggested, we have removed all these data from the revised manuscript; these remain public in the initial bioRxiv preprint (395863).Xrp1, new Figure 5B). Irrespective of what the mechanism responsible might be, we think this striking exon-biased enrichment needs to be discussed in the manuscript. As introns and exons are defined co-transcriptionally, such an enrichment profile is consistent with the rest of the data and our conclusion that UPF1 association with nascent transcripts/RNPs is what primarily drives recruitment to transcription sites. Although the road-block model that we proposed  is largely a working model, as we made it clear in the manuscript, it is the best explanation we can give presently for why less UPF1 is associated with the intron than the upstream flanking exon. The reviewer argues instead that a more likely interpretation for our results is that a substantial fraction of UPF1 binds the RNA after intron removal. We considered this possibility, but several of our observations argue against it. First, UPF1 is also associated with intronless genes, for example at the heat-shock genes such as 87A and 87C on the polytene chromosomes , and Gapdh1 in S2 cell as detected by ChIP . Second, UPF1 was also associated with our intronless transgene upon ecdysone induction . Third, a ChIP enrichment profile such as that of Xrp1  strongly suggests that UPF1 should be able to get released from exons as transcription progresses. Contrary to the reviewer’s prediction, the overall UPF1 ChIP-seq signal does not visibly change between the last three exons of Xrp1 , and more significantly, UPF1 association does not, on average, increase toward the 3’ end of genes .As for the data indicating a link between intron recognition and UPF1 association with nascent transcripts , we think that this is perhaps one of the most exciting parts of our observations. This was indeed a completely unexpected result, and, as the reviewer commented, difficult to believe, initially also by us. However, our UPF1 ChIP-seq data clearly indicate that more UPF1 is associated with exons than with introns genome wide, and this observation was validated with multiple ChIP experiments and by real-time PCR of different genes  and UPF1-GFP to examine the subcellular distribution of UPF1 in a very large number of experiments. Experimental approaches include immunofluorescence (IF) analyses of salivary glands and polytene chromosomes, ChIP-seq studies in S2 cells, RNAi knockdowns, and nuclear vs. cytoplasmic distributions in response to LMB treatment. Based predominantly on correlative data, the authors conclude that UPF1 appears to be associated with "most of the active Pol II transcription sites and some Pol III-transcribed genes…," i.e., their conclusions represent a large leap from the conventional notion of UPF1 (and the NMD pathway) as a cytoplasmic mRNA surveillance system. Although the authors' conclusions raise the possibility of unforeseen functions for UPF1 they are contingent on two key questions: (1) how reliable are the antibody-based experiments? and (2) what feature of nascent RNA transcripts might be responsible for their apparent interactions with UPF1? Unfortunately, neither question is addressed adequately.Specificity of the anti-UPF1 monoclonal antibodies was considered by comparisons of IF results with those obtained with UPF1-GFP, and examination of the consequences of RNAi knockdown of UPF1. UPF1-GFP results were markedly different from those obtained in IF analyses , with the UPF1-GFP results largely showing cytoplasmic and (interesting) perinuclear staining without any significant nuclear staining, whereas the IF experiments seem to indicate that UPF1 can be found in all three locations. RNAi knockdown of UPF1 was only examined in salivary glands, where it depresses but does not eliminate anti-UPF1 staining of polytene chromosomes. Importantly, the authors did not present an experiment examining IF of S2 cells or mosaic tissues in which the UPF1 gene has been deleted. These critical experiments are a fairly commonplace approach to demonstrating unambiguous specificity of an antibody reagent. Other trivial controls that are lacking are results from an experiment using only the secondary antibody and IF localization of a protein definitively known to be cytoplasmic (using the same secondary antibody used for the UPF1 experiments).Although the relative nuclear level of GFP-UPF1 (in the earlier version we wrongly referred to as UPF1-GFP) is not as high as endogenous UPF1 in salivary glands (this is probably a consequence of the fact that GFP-UPF1 is highly overexpressed) its overall pattern is similar to that of the endogenous protein: the signal is mostly cytoplasmic but also apparent in the region of the nucleus occupied by the chromosomes, and absent from the centre of the nucleolus. Moreover, as demonstrated by the LMB treatment, UPF1-GFP is constantly shuttling between nucleus and cytoplasm. As for the authenticity of the perinuclear signal, though this is most obvious with GFP-UPF1, it was also detected by immunostaining of the endogenous UPF1. This is apparent in some of the other cell types shown . In the revised manuscript, to address the concern about the antibody specificity, we have shown additional immunostainings of normal and UPF1-depleted salivary glands that demonstrates the veracity of the perinuclear signal of endogenous UPF1, with any of the three antibodies and, consistent with it being specific, depleted by UPF1-RNAi . Please also see our response to reviewer 1 about the specificity of the antibody.Little insight is presented with regard to possible salient features of nascent transcripts that might mediate their putative interactions with UPF1. In light of known interactions between UPF1 and ribosomes it is surprising that the authors did not consider their own published work demonstrating that Drosophila ribosomal proteins harboring fluorescent tags manifest cytological distributions that are substantially similar to the putative UPF1 distributions seen in the current manuscript . Clearly, one possibility is that the distributions seen in the present work are an indirect consequence of UPF1 interactions with chromosome-associated ribosomes or ribosomal proteins. Further, in light of UPF1's known role as an RNA-binding protein I would have expected that the authors would test whether UPF1 mutations known to impair the protein's RNA-binding capacity altered any of the cytological or ChIP-seq results seen by the authors.We agree that a substantial fraction of UPF1 might be recruited indirectly on the nascent transcripts, by, for example, an interaction with associated RNPs. We agree with the reviewer that, as controversial as this explanation may be, these could be ribosomal subunits loaded on the nascent transcript . Prompted by the reviewer’s comment, we discussed this possibility in the revised manuscript and explained our reasoning for thinking that simple affinity of UPF1 for RNA is not likely to be the primary reason why UPF1 associates with nascent transcripts. For example, UPF1 does not associate with rRNA genes, spliceosomal snRNAs, and most highly active Pol III genes. What is/are the putative mechanisms linking ribosome/ribosomal subunits association and UPF1 association with nascent transcripts will need to be addressed in future .As for the suggestion to test UPF1 mutants, we performed additional experiments in S2 cells transfected with different variants of UPF1, including mutations (DE-AA) in its RNA helicase domain. The results of these experiments strongly indicate that UPF1 RNA helicase activity is in fact required for its nucleocytoplasmic shuttling, and that it is possibly the primary determinant of its localisation in the cell where UPF1 distribution mirrors that of the mRNA, which is characteristically more abundant in the cytoplasm than in the nucleus. This new data have been added as a new figure , described in the revised manuscript and their implications discussed in detail. Notably, as we have referred to in the new Discussion section, the DE-AA mutation that inactivates the ATPase domain resides within the region previously thought to function as a non-canonical “nuclear localization signal” in mammalian cells, as UPF1 lacks a clear NLS, as mentioned by the reviewer below.[Editors’ note: what follows is the authors’ plan to address the revisions. This plan for revisions was approved and the authors made a formal revised submission.]Both reviewers have concerns regarding the work, although to different degrees. They have discussed this extensively between them. Reviewer 1 is concerned about the interpretation of the ChIP data against the suggestion that it be removed or at least qualified that UPF1 could bind after intron removal. In any case, this observation does not seem to shed much light on the process.Reviewer 2 judges that, since the interpretation of the data hinges on the specificity of the antibodies, there needs to be more rigorous demonstration that the antibodies have been well characterized and indirect effects have been ruled out. The reviewer states, "The current version of the manuscript continues to rely on RNAi knockdowns to substantiate MAb reliability, an approach that is less convincing than mosaics." and further "Most importantly, the paper is comprised of a series of observations without definitive experiments that rule out indirect interactions as explanations for the observed results."In order to satisfy the reviewers, it appears that you will need to deliver a more definitive manuscript that addresses these concerns. Please let us know how you would plan to respond to the mentioned concerns.Reviewer #1:The authors have provided additional data to demonstrate the specificity of their antibodies, as requested.As suggested be the reviewers, they also have streamlined the manuscript by removing the less compelling parts on a suggested role of UPF1 in Pol II pausing and at the nuclear envelope. Against the reviewer's suggestion, they decided to retain their ChIP-seq experiments showing that more UPF1 is associated with exons than with introns. They argue that my alternative – and less exciting – interpretation of their data, namely that this might simply result from a substantial fraction of UPF1 binding only after intron removal, was unlikely to be valid. However, I do not understand why the fact that UPF1 was also found associated with intronless genes would challenge my interpretation of the data. With ChIP, one has no information at which stage during the transcription of an intronless gene UPF1 binds the nascent transcript. The same is true for intron-containing genes and if a fraction of UPF1 would bind after an intron was spliced out, the net result would be more UPF1 bound to exons than to introns.The newly added rescue of the poly(A)+ RNA accumulation in the nucleus in the absence of UPF1 demonstrates the specificity of this observation and strengthens the conclusion.Thus, even though I do not agree with the author's interpretation of the ChIP data, all my major concerns have been satisfactorily addressed and I support accepting the manuscript for publication.Reviewer #2:Singh et al. have submitted a revised version of their manuscript that bears a close resemblance to the original, both in experimental approach and in the concerns raised by the respective experiments and conclusions. As before, the authors begin by describing the isolation of monoclonal antibodies that recognize Drosophila UPF1 and then utilize those antibodies  and UPF1/GFP constructs to examine the subcellular distribution of UPF1 in S2 cells and salivary glands. Additional experiments evaluated ChIP-seq in S2 cells, and nuclear vs. cytoplasmic distributions in response to LMB treatment. Based predominantly on correlative data, the authors conclude that UPF1 appears to be associated with most of the active Pol II transcription sites and some Pol III-transcribed genes. As such, their conclusions represent a large leap from the conventional notion of UPF1 (and the NMD pathway) as a cytoplasmic mRNA surveillance system. Although the authors' conclusions raise the possibility of unforeseen functions for UPF1, they are contingent on two key questions: (1) how reliable are the IF, ChIP-seq, and GFP-UPF1 experiments? and (2) is it possible that the observed UPF1:RNA interactions are indirect?Previously, I was concerned about the 7B12 antibody, particularly: (i) the very strong additional band that showed up in the westerns of Figure 1—figure supplement 1B and (ii) the lack of any tests of the antibody using UPF1 deletion/mosaic tissues. The current version of the manuscript continues to rely on RNAi knockdowns to substantiate MAb reliability, an approach that is less convincing than mosaics. Further, I find the vast overexpression of UPF1-GFP (~10x the level of endogenous UPF1), and its distribution differences relative to endogenous UPF1 to be additional concerns. In the ChIP-seq experiments, far too much attention is paid to hypothetical explanations for the differences in exon vs. intron signals  while little attention was paid to the ~10x lower recovery of UPF1 ChIP-seq vs. Pol II ChIP-seq.Most importantly, the paper is comprised of a series of observations without definitive experiments that rule out indirect interactions as explanations for the observed results. The authors only make that concern worse by filling the discussion with numerous unsubstantiated explanations for the apparent shuttling, the ChIP-seq results, and the experiments with the UPF1 helicase mutant.In short, this manuscript just isn't up to eLife's standards.Thank you very much for handling the reviewing of our manuscript. We are very pleased that reviewer 1 is now satisfied and supports its publication. Although they favor an alternative explanation for why more of UPF1 is associated with exons, we still think that the most plausible explanation is that it mostly associates with an exon prior removal of the downstream intron. However, we agree that a portion of UPF1 might bind to exons only after intron removal. We will discuss this possibility in the next revision and expand/modify our model of how UPF1 associates with nascent transcripts accordingly.Reviewer 2’s remaining concern about the specificity of the antibodies puzzles us. Following the first review round, as requested we have performed many control experiments to address this concern, and we think that the results leave no doubt about the specificity of the antibodies. Reviewer 1 appears satisfied with this interpretation. Briefly, in the revision we have shown that all three monoclonal UPF1 antibodies produce similar immunostainings of both the polytene chromosomes  and the salivary glands , and UPF1-RNAi depletes all three antibodies signals in the cells which express the RNAi construct, but not in the adjacent fat body cells attached to the glands . Moreover, GFP-tagged UPF1 shows a similar subcellular distribution, and in particular a highly similar banding pattern on the polytene chromosomes .In the latest assessment reviewer 2 points out that the 7B12 antibody detects an additional strong band in one of the Western blots . Although this was not pointed out in their initial review, it was by reviewer 1 and we had addressed it in our previous response. In brief, this extra band was seen only in the initial screening where the blots were incubated with ascites produced with the 7B12 hybriodoma, this cross-reactivity is minimal when using purified IgG from hybridoma supernatants . As explained in the manuscript, we have used only purified antibody not ascites in all subsequent assays.This reviewer states that will be more convincing to test the specificity of the antibodies using genetic mosaics tissues. We don’t think that such experiments will be more informative or cleaner than what we have already performed. UPF1 is required for growth and survival of cell clones in imaginal discs , as a consequence homozygous Upf1(-) clones can’t be easily recovered and consist of just a few cells, as previously reported .As for whether a fraction of UPF1 might be recruited indirectly on the nascent transcripts, by, for example, an interaction with associated RNPs, we agree, and, as suggested by reviewer 2’s original criticism, we have discussed this possibility extensively in the revised manuscript. Strangely, now they criticise us for having done so. Moreover, irrespective of its loading mechanism, we think that the data fully supports the manuscript main conclusion that UPF1 associates with nascent transcripts.In summary, given that reviewer 1 appears to be satisfied with antibodies’ specificity and the rest of the manuscript, we would like you to consider whether doing the substantial more work requested by reviewer 2 is required. We are very excited about these findings and think the association of UPF1 with nascent transcripts is evolutionally conserved.References:Drosophila development. PLoS genetics 2, e180.Metzstein, M.M., and Krasnow, M.A. (2006). Functions of the nonsense-mediated mRNA decay pathway in


The CCAAT box-harboring proteins represent a family of heterotrimeric transcription factors which is highly conserved in eukaryotes. In fungi, one of the particularly important homologs of this family is the Hap complex that separates the DNA-binding domain from the activation domain and imposes essential impacts on regulation of a wide range of cellular functions. So far, a comprehensive summary of this complex has been described in filamentous fungi but not in the yeast. In this review, we summarize a number of studies related to the structure and assembly mode of the Hap complex in a list of representative yeasts. Furthermore, we emphasize recent advances in understanding the regulatory functions of this complex, with a special focus on its role in regulating respiration, production of reactive oxygen species (ROS) and iron homeostasis. S. cerevisiae, the CCAAT box is reported to be found at promoters of the genes encoding cytochromes and those who are specifically activated by non-fermentable carbon sources  and the CBF/NF-Y , respectively , CCAAT transcription factor/nuclear factor 1 (CTF/NF1), and CCAAT displacement protein (CDP), were found to selectively recognize the CCAAT homologous sequences . Among tectively . In thisS. cerevisiae represents the first identified factor in this family and its discovery was originated from the studies by Guarente and his colleagues. They found that this heteromeric complex contains three subunits  and was originally recognized as a transcriptional activator through its capacity of binding to the CCAAT box located in the UAS2 element of CYC1, a gene encoding iso-1-cytochrome C , and is necessary for DNA binding and transcriptional regulation . The Hapchrome C . Intereschrome C  and a nochrome C , indicatchrome C . More imsubunits . The Hapsubunits .S. cerevisiae, this highly conserved factor was also discovered and analyzed in mammals including human and other vertebrates  identifies a total of 59 predicted genes coding for the three subunits of NF-Y in tomato, including 10 NF-YA, 29 NF-YB, and 20 NF-YC genes  of numerous cytochrome genes, acting as a master regulator of respiratory metabolism  in addition to the Hap2/Hap3/Hap5  are evolutionarily conserved . The arccleosome  but alsolexA-HAP2 fusion experiment revealed that the 265-amino-acid sequence of Hap2 was composed of an essential core of 65 amino acids, a signature sequence which could be further divided into a DNA recognition sequence containing 21 amino acids, although it is devoid of the classical DNA-binding motif, and a subunit association sequence containing 44 amino acids  is commonly recognized as the structural marker for all Hap3 homologs, and structurally, this motif is composed of a long central α-helix flanked by two smaller α-helices and is dimerized through the extensive hydrophobic contact in a head-to-tail assembly mode . The C. nditions .HAP5 depleting mutant, enforcing the role of Hap5 in gene activation  that are required for the assembly of the Hap2-Hap3-Hap5 heterotrimer  was identified in Hap5 (115–146 amino acids) and Php5 (71–102 amino acids). However, this small domain alone is not sufficient for recruitment of Hap4   located in A1A2 linker within the Hap/DNA complex was found to favor the correct preferred positioning of the A2 helix of the Hap2/HapB subunit toward the CCAAT box site, surprisingly, this amino acid somehow is lost in mammalian NF-YA  . This sean NF-YA .Aspergillus nidulans  and C. albicans (Hap43) (Aspergillus (HapX) and Fusarium (HapX)  domain encompassed by the CBC interaction domain was found to contribute to interactions between the recruitment domain of Hap5 and Hap4 binding   as well m (HapX) , revealem (HapX) . Similar of Hap5 . In yeas binding . S. cere regions . The lac regions .Arabidopsis revealed that the NF-YB/NF-YC heterodimer interacts with some specific transcription factors to form a complex (NF-YB-YC-TF) and regulates the expression of target genes through the binding of transcription factors to specific cis-elements located at the promoters  demonstrated that the CBC is assembled in the cytoplasm first, and then the non-conserved NLS2 of Hap2 interacts with the nuclear import machinery and transports the CBC to the nucleus through a “piggy-back” mechanism . As such, an interesting question was raised: how do the other subunits transport into the nucleus? It has been generally accepted that a two-step strategy is utilized in mammalian cells. The subunits NF-YB/CBF-A and NF-YC/CBF-C form a heterodimer, afterward they interact with the third subunit, NF-YA/CBF-B, to form a heterotrimeric complex . Studiesromoters . Howevercleotide . A comprechanism .S. cerevisiae indicate that the Hap complex, either directly or indirectly, controls the expression of hundreds of genes, highlighting its important role as a hierarchically highly placed transactivator. In S. cerevisiae, approximately 230 genes are positively regulated, while approximately 240 genes are negatively regulated by the Hap complex  were able to sense the cellular changes such as perturbed respiratory function and trigger the expression of a variety of genes involved in alteration of metabolic adaptations. One example is the glyoxylate cycle and fatty acid beta oxidation  or retrograde  signaling, is present under normal and pathophysiological conditions . Meanwhixidation . Evidencand Rtg3 . Deletiontenance .HAP4 gene appears to be dispensable in promoting the growth rate of either aerobiosis or anaerobiosis in a standard glucose medium. However, the growth pattern is changed in a low glucose condition where the cell is still able to respire in the absence of HAP4 gene, although its respiration capacity is significantly reduced. On the other hand, the growth rate is also related to the respiration regulation. Transcription of the respiration genes requires the Hap2/3/5 complex when the cells were grown below the intermediate specific growth rate, whereas the Hap4 protein is activated when they are above the intermediate specific growth rate  in response to iron availability  such as superoxides, peroxides, and hydroxyl radicals . Accordilability .Iron is an indispensable cofactor in various cellular processes including oxygen transport, amino acid metabolism, and DNA biosynthesis. However, excessive iron may catalyze the formation of reactive oxygen species that may destroy cells by damaging lipids, proteins, and nucleic acids, through a Fenton reaction . In factS. cerevisiae, iron regulation depends on the activation and cytoplasm-to-nucleus translocation of two transcriptional activators, Aft1 and Aft2. Under iron-depletion conditions, Aft1 and Aft2 directly bind to the conserved promoter sequences of iron uptake genes and activate the transcription, thus inducing iron acquisition from external sources and releasing free iron deposited in intracellular vacuoles  (Penicillium chrysogenum (Srep) (Neurospora crass (Sre) (Aspergillus fumigatus (SreA) (Histoplasma capsulatum (Sre1) (Cryptococcus neoformans (Cir1) (Unlike the iron-regulatory mode in  (Urbs1) , the ascm (Srep) , and Neuss (Sre) , and soms (SreA) , Histoplm (Sre1) , and Crys (Cir1) , is conts (Cir1)  The GATAS. pombe, iron homeostasis is controlled by the GATA-type transcription factor Fep1 and the CCAAT-binding factor Php4  and Hap43 (CCAAT-binding factor). Unlike the system of S. pombe, the opportunistic human fungal pathogen C. albicans has evolved a new transcription factor (namely Sef1) that could be integrated into the widely conserved regulatory circuit containing both Sfu1 and Hap43. Sfu1 was found to inhibit the expression of SEF1 and iron uptake genes in iron-replete conditions, however, under iron-depleting conditions, the expression of iron uptake genes and HAP43 who is responsible for the repression of iron utilization genes was specifically induced by Sef1  was identified and characterized previously  inhibits the expression of iron acquisition genes during iron adequacy, so it is important to accommodate iron excess. HapX (a CCAAT-binding factor) acts through interaction with CBC and the CBC/HapX complex is critical for adapting to both iron starvation and iron overdose. During iron starvation, the CBC/HapX complex inhibits genes expression involved in the iron-dependent pathway, such as the heme biosynthesis, to reserve iron and activate the iron carrier system to increase iron availability (Similar to the yeasts like lability . Howeverlability .In this review, we have described considerable progresses in understanding the structure and DNA-binding of the Hap complex in yeasts. Moreover, the Hap complex-dependent regulation network was evaluated. As stated, the Hap complex mainly regulates gene expression associated with the oxidative stress response, iron homeostasis, and virulence. However, our understanding of the HAP regulon is still incomplete and many important questions remain to be answered, such as specific mechanisms related to the DNA binding and iron sensing assigned by the Hap4/HapX, proteomic maps of the Hap complex and post-transcriptional/translational modification modes of Hap complex. Undoubtedly, deciphering these questions will greatly help our understanding of the Hap complex regulatory circuits in yeasts and other fungi.YM and CC conceived and designed this review, screened and selected the articles, analyzed and interpreted the information, drafted the manuscript, and read and approved the final version of the manuscript. YM designed the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ribosomes are ribonucleoprotein machines that decode the genetic information embedded in mRNAs into polypeptides. Ribosome biogenesis is tightly coordinated and controlled from the transcription of pre-rRNAs to the assembly of ribosomes. Defects or disorders in rRNA production result in a number of human ribosomopathy diseases. During the processes of rRNA synthesis, non-coding RNAs, especially snoRNAs, play important roles in pre-rRNA transcription, processing, and maturation. Recent research has started to reveal that other long and short non-coding RNAs, including risiRNA, LoNA, and SLERT (among others), are also involved in pre-rRNA transcription and rRNA production. Here, we summarize the current understanding of the mechanisms of non-coding RNA-mediated rRNA generation and regulation and their biological roles. In humans, there are approximately 300–400 copies of rDNA genes per haploid genome that are distributed over five chromosomes . Each rDNon-coding RNAs play essential roles in rRNA regulation. The small nucleolar RNA (snoRNA) is widely known to guide the nucleotide modifications and processing . RecentlLong non-coding RNAs comprise a fast-growing classes of RNA molecules with sizes greater than 200 nt. Most lncRNAs are first transcribed by polymerase II (Pol II), then capped, polyadenylated, and spliced after transcription . lncRNAsThere are several clusters of tandemly arrayed rDNA genes exist in each mammalian genome, yet not all of these repeats are transcribed. rDNA exists in two types of chromatin – a euchromatic conformation that is actively transcribed and a heterochromatic conformation that is transcriptionally inactive. Silent rDNA repeats are marked by heterochromatic histone modifications and CpG methylation at the rDNA promoter . SilenciInterestingly, by using mature pRNAs to tether heterochromatin at nucleoli in embryonic stem cells, O-methylation of rRNAs and participate in rRNA processing  data of SLERT-associated proteins identified DDX21, a DEAD-box RNA helicase that is involved in multiple steps of ribosome biogenesis . SLERT dUpon stress, cells utilize various strategies to suppress rDNA transcription to promote survival, for example, by inactivating certain transcription factors and inducing chromatin remodeling . FurtherIn density-arrested or serum-deprived cells, pre-rRNA synthesis is suppressed and rDNA is enriched with H4K20me3 marks, while PAPAS is upregulated. After starved cells are refed with serum, the levels of H4K20me3 modifications and PAPAS decrease. RNA immunoprecipitation (RIP) experiments revealed that quiescence-induced PAPAS recruits Suv4-20h2 to transcription-competent rRNA genes to trigger H4K20me3 modification and chromatin compaction. Furthermore, siRNA-mediated knockdown of endogenous PAPAS decreased the level of H4K20me3 modifications, but not the level of H3K9me3 modifications .Hypo-osmotic stress also upregulates PAPAS and inhibits rDNA transcription. However, unlike serum deprivation, hypo-osmotic shock does not increase the Suv4-20h2 occupancy and H4K20me3 abundance at rDNA loci, but rather induces the degradation of Suv4-20h2 . The Mi-Similar to hypotonicity, heat shock also increases PAPAS expression, induces the degradation of Suv4-20h2, recruits NuRD to rDNA, and turns off transcription of pre-rRNA . Recent Unlike other Pol I-transcribed rRNAs, 5S rRNAs are transcribed by Pol III. 5S rRNA genes are clustered as tandem repeats with intergenic sequences, and they are located on distinct chromosomes . A numbeFurthermore, human 5S-OT contains an antisense Alu element at its 3′ end . Alu is Therefore, the lncRNA 5S-OT modifies 5S rRNA and mRNAs via cis and trans mechanisms, respectively. Since 5S-OT is relatively conserved in eukaryotes from fission yeast to humans, it will be interesting to examine whether similar mechanisms are applicable in other organisms.Caenorhabditis elegans two decades ago  was first described in ades ago . siRNAs ades ago . In the ades ago . Here, wades ago .Schizosaccharomyces pombe lacking Cid14, rRNAs become substrates for the RNAi pathway, giving rise to siRNAs targeting rRNA  from the ribosomal DNA locus  are widely present in various organisms. In ing rRNA . In NeurNA locus .C. elegans, upon exposure to low temperature treatment or ultraviolet (UV) light, risiRNAs accumulate  complexes . The exoDownregulation of rRNA transcription is one of the major strategies to preserve cellular homeostasis upon encountering stress conditions and to limit energy consumption under unfavorable conditions . The risRibosome biogenesis is tightly coordinated and controlled from the transcription of pre-rRNAs to the assembly of ribosomes, a process that is influenced by many developmental programs and environmental stress challenges . Cells rMany questions still remain to be addressed to fully understand the mechanisms and roles of non-coding RNAs in anti-stress pathways and rRNA regulation. For example, how do these lncRNAs surveil distinct environmental stresses? Cold shock could induce untemplated addition of oligouridylation at the 3′ ends of 26S rRNAs to elicit the production of risiRNAs . Heat-shAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas aeruginosa uses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds to N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR to N-butanoyl-homoserine lactone (C4-HSL). There is a third P. aeruginosa acyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR in P. aeruginosa QS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked to qscR, PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a “brake” on QS autoinduction. Pseudomonas aeruginosa has a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors in P. aeruginosa. QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.Quorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacterium  Pseudomonas aeruginosa is an opportunistic pathogen of humans that uses a process called quorum sensing (QS) to regulate gene transcription in response to cell density (P. aeruginosa genome encodes two complete acyl-homoserine lactone (AHL) QS systems: the LasR-LasI system and the RhlR-RhlI system. LasI and RhlI are signal synthases that produce N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and N-butanoyl-homoserine lactone (C4-HSL), respectively  by QscR . This reruginosa , 14, witrs of QS . The antrs of QS , 15. UnlP. aeruginosa is not known. Several hypotheses have been advanced. A prior study suggested that QscR acts by modulating levels of lasI  gene, which is immediately adjacent to and transcribed divergently from qscR (PA1898)  , and a r(PA1898) . In QS t(PA1898) , 13, but(PA1898) .P. aeruginosa QS system is complex, with multiple inputs and transcription factors (P. aeruginosa QS systems and mechanisms that synchronize and stabilize QS in populations. We tested our hypothesis that QscR has a distinct regulon by performing chromatin immunoprecipitation sequencing (ChIP-seq) to identify direct targets of 3OC12-HSL-bound QscR. Surprisingly, we found that QscR bound only to the promoter of PA1897, the first gene of an operon containing the genes PA1895 to PA1897 (PA1895-1897), and we describe experiments showing that the products of these genes, not QscR itself, are the mediators of QS antiactivation.The  factors . We are qscR under the control of an arabinose-inducible promoter in a lasR rhlR qscR triple deletion (3R) mutant and compared DNA sequences captured by an anti-QscR antibody from this mutant  to those captured from a QscR-null mutant containing the vector only.To identify possible binding sites of QscR within the genome of PAO1, we performed a chromatin immunoprecipitation assay using a purified anti-QscR antibody, followed by next-generation sequencing of the DNA bound to QscR. QscR is produced at very low levels in strain PAO1, and we were unable to reliably detect QscR-DNA complexes from the wild type using the ChIP-seq approach. Therefore, we expressed qscR and the adjacent PA1897 gene   . This prlasI  and lasB (elastase). As expected and predicted by the ChIP-seq results, addition of QscR to the promoter of PA1897 resulted in a DNA gel shift, consistent with binding of QscR to this promoter target, as previously reported  with purified QscR and several promoters that have been shown to be repressed in a QscR transcriptomic analysis . We selereported  Fig. 2)lasI (whiThe ChIP-seq and EMSA results led us to hypothesize that the QscR counterregulation of QS occurs by activation of genes transcribed from the PA1897 promoter. Although transcription of QscR itself has been reported to be activated through this promoter (P. aeruginosa PA14)   Fig. 1)P. aerugisa PA14) . PA1895-qscR accelerates production of 3OC12-HSL and C4-HSL and the phenazine pyocyanin  signals . After 2 h of incubation, the 14C-AHLs were subjected to solvent extraction, separated by high-performance liquid chromatography (HPLC), and quantitated by scintillation counting. Even though cells expressing PA1895-1897 were delayed in pyocyanin production relative to the vector controls , there was no difference observed in 14C-AHL HPLC elution behavior  might together synthesize an antagonist to LasR. Other possibilities are that the products of PA1895-1897 interact with LasI, the 3OC12-HSL signal synthase, or that they catalyze reactions that reduce AHL substrate pools, so that cells simply produce less signal.We do not know how expression of the gene products encoded by PA1895-1897 alters signal production in P. aeruginosa QS circuitry might create a bistable system where, for a given range of signal concentrations, QS may be “on” or “off” depending on the prior state of the population . This possibility that QscR could detect signals produced by other bacteria might explain why it, uniquely among the P. aeruginosa LuxR homologues, has broad signal specificity . Unless otherwise indicated, Luria-Bertani (LB) broth buffered with 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) was used. Media were supplemented with antibiotics for selection as follows: for E. coli, ampicillin at 100 µg ml−1 and gentamicin at 10 µg ml−1; for P. aeruginosa, carbenicillin at 300 µg ml−1 and gentamicin at 100 µg ml−1. Deletion mutants of P. aeruginosa were constructed by homologous recombination as previously described  mutant. The qscR overexpression plasmid pJN105-QscR (qscR and 3R-pJN105-QscR were incubated with 2 µM 3OC12-HSL in LB-MOPS (pH 7.0) at 37°C with shaking. For the 3R strain containing pJN105-QscR, 20 µg/ml gentamicin, with or without 0.01% arabinose, was added for plasmid maintenance. Growth was monitored as optical density at 600 nm.Bacterial strains used in this experiment include a P. aeruginosa as previously described for LasR . The size of the fragments was confirmed to be in the 300-bp to 1,000-bp range by agarose gel electrophoresis. Fragmented DNA was immunoprecipitated using a 50% protein A Sepharose slurry  with purified anti-QscR antibody (see below). Cross-links between the protein and DNA were reversed by incubating the eluate at 65°C overnight. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation and was then further purified using a Monarch PCR and DNA cleanup kit (New England Biolabs). DNA was sequenced using an Illumina MiSeq system.A chromatin immunoprecipitation assay was performed for for LasR . BacteriNC_002516). Peaks were identified using MACS  peak detection  v 3.1.1 ChIP-seq pipeline. Single-end 150-bp reads were aligned to the PAO1 reference genome  to search for promoter sequences with a conserved CT-N12-AG binding motif for LuxR-type transcription factors , which was composed of a pancreatic digest of gelatin (20.0 g), magnesium chloride (1.4 g), and potassium sulfate (10.0 g) in 1 liter of purified water containing 10 g of glycerol per liter (520) by 17.072 , a portion of which is converted to S-adenosylmethionine, a substrate for AHL synthesis. To harvest 14C-AHLs, cells were removed by centrifugation and the cell-free supernatant was extracted twice with equal volumes of acidified ethyl acetate (EtAc). To test if the PA1895-1897 gene products modified 14C-AHLs, the ΔPA1895-1897 mutant harboring arabinose-inducible plasmid pJN105.PA1895-97 was inoculated into fresh PPM plus gentamicin and 0.05% arabinose and grown for 6 h (to an OD600 of between 0.7 and 0.8) at 37°C with shaking. A 5-ml culture aliquot was transferred to a 15-ml polypropylene tube containing 1/20 of the total 14C-AHL extract  and incubated at 37°C with shaking for 2 h. 14C-AHLs were reextracted twice with equal volumes of EtAc and evaporated to dryness. Approximately 30% of this extract was separated by C18 reverse-phase HPLC in a 10% to 100% methanol gradient with a flow rate of 0.75 ml/min as described previously (ΔPA1895-1897 mutant with pJN105 vector control and PPM broth.To obtain eviously , 40. P. glycerol  to mid-leviously . FractioSRP149739 and in BioProject under accession number PRJNA472067.Sequencing data from this study were deposited in the NCBI Sequence Read Archive under accession number