Woody tropical plants contain high levels of complex organic compounds that inhibit the chemical procedures needed to extract RNA or DNA, thus compromising downstream applications such as RNA sequencing and analysis of gene expression. To overcome this issue, researchers must use extraction protocols using CTAB/PVP buffer instead of commercially available DNA/RNA extraction kits. However, these protocols are time-consuming, use toxic chemicals like phenol and chloroform, and can only be used to process a small number of samples at a time. To overcome these issues, we developed a new CTAB/PVP based protocol for RNA or DNA extraction that eliminates the traditional phenol/chloroform step. Furthermore, the protocol was developed for 96-well plates to speed up processing.Our new protocol enabled us to successfully extract RNA from macadamia, avocado, and mango tissues that are traditionally difficult to work with. This RNA was then successfully used to synthesise cDNA for real-time quantitative PCR and to generate good quality RNA-Seq libraries. Our protocol can be easily converted for rapid DNA extraction from different tropical and sub-tropical tree species.This method enables safer and faster DNA and RNA extraction from recalcitrant species, thus facilitating future work on tropical trees.The online version of this article (10.1186/s13007-019-0447-3) contains supplementary material, which is available to authorized users. Arabidopsis thaliana L. (Heyn)) [Molecular experiments reveal the physio-genetic structure and function of plant species, enabling growers to improve productivity in changing environmental conditions. However, the protocols used to extract RNA or DNA from plants were developed using herbaceous species such as Arabidopsis ( (Heyn)) –4, and d (Heyn)) , 6. Extrth Report on Carcinogens from the U.S. Department of Health and Human Services [For herbaceous plants, the extraction of RNA or DNA can be easily achieved with silica membranes, however these methods do not work efficiently with tropical woody species. Furthermore, this technique only retains RNA strands longer than fifty nucleotides, eliminating the small RNA which are of emerging importance in molecular plant sciences . TherefoServices . Given tServices .Persea americana L.), Macadamia (Macadamia integrifolia L.) and Mango (Mangifera indica L.). Our method does not require phenol or cholorophorm and can be used to extract 96 samples in a 96-well plate in less than 1 day. The resultant RNA can be sequenced and used to produce good quality RNA-Seq reads. The DNA extraction method can also be used on Coffee tree (Coffea arabica L.) and Eucalyptus  samples. This protocol will facilitate novel molecular research on tropical trees that have previously been too difficult for large-scale genetic survey and experimentation.In this study, we developed and tested a new method for extracting DNA and total RNA, including small RNA, from diverse tissues of tropical/subtropical woody species, including Avocado . DNA was extracted from mature leaves whilst RNA extractions were performed on stem, leaf, root, flower and axillary bud tissues. Arabidopsis cv. Columbia-0 and garden pea cv. Torsdag were also used to assess the efficiency of the method on herbaceous plants.Various tissue samples were collected from field-grown Avocado cv. ®, SPEX). Leaf, bud, flower and root samples were ground in 2 ml tubes (up to 96 at a time) and stem tissues were ground in 15 ml vials (up to 12 at a time). Frozen samples were disrupted for 1 min at 1750 rpm and refrozen for 30 min at − 80 °C before being disrupted again for one or two cycles. The amount of powder required for the extraction was transferred in a new tube using a spatula. The estimated weight was assessed based on preliminary assessment volume/weight.Fresh material was flash frozen in liquid nitrogen or dry ice and stored at − 80 °C before being ground to powder (homogenised). Frozen samples were ground with an automated tissue grinder  0.5 mMSDS 10%70% ethanolDNase I + DNase bufferRNase ATransfer 0.5–50 mg (not more) of ground sample into a 2 ml tubeAdd 625 µl of CTAB buffer and 25 µl of DTT 0.5 mM (mix them prior to use)Vortex well and incubate at 60 °C for 15 min. Vortex every 5 minNote: At that stage, SDS precipitates with CTAB making the sample cloudyONLY FOR RECALCITRANT SAMPLES: Add 65 µl of SDS 10% and vortex well g for 15 min at room temperatureNote: Tissue debris will pellet in the bottom of the tube and SDS/CTAB will form a semi-solid layer at the surfaceCentrifuge at 20,000Note 1: In case too much top layer has been mistakenly taken up, put the samples into new 2 ml tubes and repeat this step .Note 2: Unclear precipitate can form depending on the tissue (this was the case with avocado buds). Transfer into a filter column (Qiagen QIAshredder or Macherey–Nagel NucleoSpin Filters) instead of the 2 ml tube and repeat this step.Remove the tubes carefully from the centrifuge and transfer 550–600 µl of the liquid phase into a new 2 ml tube or into a 2 ml cavity of a 96 well plate and proceed to the nucleic acid precipitationAdd 550 µl of pre-cooled (− 20 °C) isopropanol into each tube/well and vortex wellNote: At that stage you can leave the samples for days or weeks at − 20 °CLeave at − 20 °C for 15 ming for plates or 45 min at 20,000g for tubesCentrifuge the tubes/plate at 4 °C for 1 h at 3100Tip the tubes/plate to remove isopropanolAdd 1 ml 70% ethanolg for plates or 20,000g for tubesCentrifuge at 4 °C for 10–15 min at 3100Tip the tubes/plate to remove the ethanolCentrifuge the tubes/plate briefly and remove remaining ethanol with a pipetteLet the samples dry on the bench for 10 min.Add 175 µl of RNase-free water and pipette up and down to resuspend the pelletPlace the plate at 50–60 °C for 2–3 min, vortex and quick spin downAdd 20 µl of DNase buffer and 5 µl of DNase I per tube or well (mix them before)Vortex briefly, quick spin down and incubate at 37 °C for 20–30 minImmediately proceed to the next step.Add 200 µl of pre-cooled (− 20 °C) isopropanol into each tube/well and vortex wellNote: At that stage, you can leave the samples for several days at − 20 °CLeave for 15 min at − 20 °Cg for tubesCentrifuge at 4 °C for 1 h at 3100 g for plates or 45 min at 20,000Tip the tubes/plate to remove isopropanolAdd 1 ml of 70% ethanolg for tubesCentrifuge at 4 °C for 10–15 min at 3100 g for plates or 20,000Tip tubes/plate to remove ethanolCentrifuge the tubes/plate briefly and remove remaining ethanol with a pipetteLet the samples dry on the bench for 10 minAdd 40–100 µl of warm (60 °C) RNase-free water and pipette up and down vigorouslyVortex well and quick spin downDetermine RNA quantity and quality by gel electrophoresis and spectrophotometry, and store the samples at − 80 °C.Transfer up to 50 mg (no more) of ground sample to a 2 ml tubeAdd 400 µl of CTAB buffer and 4 µl of RNase A + RNase buffer (mix them together as before)Vortex well and incubate at 37 °C for 1 h. Vortex them and invert every 15 minNote: SDS precipitates with CTAB making the sample cloudyAdd 30 µl of SDS 10% and vortex well , add only 15 µl of SDS 10%)g for 15 min at room temperatureNote: Tissue debris will pellet in the bottom of the tube and SDS/CTAB will form a semi-solid layer at the surfaceCentrifuge at 20,000Note 1: In case too much top layer has been mistakenly taken up, put the samples into new 2 ml tubes and repeat this step .Note 2: In case of dirty/unclear precipitate can form depending on the tissue (this was the case with avocado buds), transfer into a filter column (Qiagen QIAshredder or Macherey–Nagel NucleoSpin Filters) instead of the 2 ml tube and repeat this step.Remove carefully from the centrifuge and transfer 350–400 µl of the liquid phase into a new 2 ml tube or into a 2 ml 96 well plate and proceed to the nucleic acid precipitationAdd 350–400 µl of pre-cooled (− 20 °C) isopropanol to each tube/well and vortex wellNote: At that stage, you can leave the samples for several days at − 20 °CLeave at − 20 °C for 15 ming for plates or 45 min at 20,000g for tubesCentrifuge the tubes/plate at 4 °C for 1 h at 3100Tip the tubes/plate to remove isopropanolAdd 1 ml of 70% ethanolg for plates or 20,000g for tubesCentrifuge at 4 °C for 10–15 min at 3100Tip the tubes/plate to remove ethanolCentrifuge the tubes/plate briefly and remove remaining ethanol with a pipetteLet the samples dry on the bench for 10 minAdd 90 µl of warm (60 °C) RNase-free water, pipette up and down and vortex well to resuspend the pellet.® to the gel. Gel visualisation was performed using the Gel Doc™ Gel Documentation System . The quality and purity of the samples were determined with a NanoVue™ Plus Spectrophotometer  measuring the absorbance at 280 nm for RNA and 260 for DNA.Quality control was performed by running RNA or DNA samples on a 1.1% agarose gel. The nucleic acids were stained by adding Red Sage® No-ROX Kit (Bioline) per reaction. Samples were amplified following the manufacturer’s instructions and fluorescence was monitored with a CFX384 Touch™ Real-Time PCR Detection System .For qRT-PCR, cDNA was obtained by reverse transcription using 250–500 ng of total RNA in 8 µl and 2 µl of 5 × iScript Supermix , following the manufacturer’s instructions. The cDNA was then diluted to 0.5 ng equivalent RNA in milliQ water per µl for a working template solution. Quantitative real-time PCR was then performed using 2 µl of 1 mM primer mix, 5 µl of cDNA and 3 µl SensiFAST™ SYBR® green mastermix, 2 µl of 1 × miScript universal primer, 2 µl 0.8 μM miRNA specific primer and 2 µl template cDNA. The qRT-PCR run was performed, and fluorescence was measured using a Rotor-Gene Q 6000 .For quantification of miRNA expression, low molecular weight cDNA was synthesised by ligation-mediated reverse transcription from 100 to 200 ng of total RNA using a miScript Plant RT Kit  kit per manufacturer’s protocol. The prepared cDNA was then diluted with milliQ water as per the manufacturer’s instructions for optimal amplification. The qRT-PCR reactions were performed as per manufacturer’s instructions  using: 10 µl of 1 × QuantiTect SYBRRNA libraries of pooled avocado, macadamia and mango stem, leaf, root, flower and axillary bud tissues were prepared as described by Kerr et al. . cDNA lihttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/).Trimmomtatic v. 0.35  was usedTo validate our RNA sequencing results, we mapped the reads to published draft transcriptome and genome sequences. Trimmed reads were mapped to published reference transcriptome and genome assemblies using HISAT v.2  using deIn the first couple of experiments described below, we developed and tested an RNA extraction method using 40–60 mg of frozen leaf tissue from avocado, macadamia and mango mature leaves. Most of the published RNA extraction procedures developed for recalcitrant tree species improved the RNA yield and quality by using a CTAB/PVP lysis buffer with high concentration of salt (NaCl) as described in the introduction , 17. We In order to improve the method, 1% SDS was added to the lysis buffer after the incubation step at 60°. SDS is a detergent which helps digest the cell membrane and release more nucleic acids during this step. SDS also has the property of disrupting the nucleic acid/protein interactions and facilitate the solubilisation of CTAB/nucleic acid complexes , leadingWe then tested whether the method was efficient with different tissue types. To test this, we performed the same procedure using 15–30 mg of four different macadamia tissue types . The quality/quantity control demonstrated that the method was efficient on these four tissue types from macadamia Fig. . Root tiWe wanted to develop the method in a way that allowed us to extract a high number of samples at the same time. To achieve this goal, we took advantage of the fact that after the lysis step, the method does not require pipetting under a fume hood with great precaution as is the case with methods using phenol and chloroform (different phases). We therefore performed the following steps in a 2 ml 96-well plate using the same procedure as with tubes. As with tubes, the extraction performed with plates generated satisfactory results as demonstrated by the quality control shown in the Fig. DORMANCY1 (DRM1) and miR172 in the three tree species demonstrates that the RNA can be used successfully for quantitative RT-PCR  for quality assessment after standard trimming and quality control using Trimmomatic [To further demonstrate the efficacy of our RNA extraction protocol, we used it to prepare normalised RNA-Seq libraries for each of avocado, macadamia and mango tree species and sequenced them using Illumina sequencing technology. The quality control of the libraries obtained using this method demonstrates that the RNA extracted with the method described in the paper could be successfully used for such a purpose Fig. : Fig. S2mmomatic , the datPersea americana var ‘drymifolia’ [Mangifera indica L. cv. ‘Shelly’ [Macadamia integrifolia cv. 741 ‘Mauka’ draft genome assembly [To verify our RNA sequencing, we mapped our reads after trimming to published transcriptome and genome assemblies using HISAT v.2  using demifolia’  and cv. mifolia’  transcri‘Shelly’  and ‘Zil‘Shelly’  transcriassembly . This quThe issues usually met with when performing DNA extraction methods for tree species are the same as with RNA extraction methods. We therefore decided to test whether this RNA extraction method could be converted to a genomic DNA extraction method. To achieve this, we modified the method by replacing the DTT from the CTAB buffer with RNase in order to remove the RNA during the lysis step. As for RNA extraction, 1% SDS was added at the end of the lysis step and a single precipitation/washing/elution round was performed to recover the purified DNA Fig. . We alsoSuccessful nucleic acid extraction from species recalcitrant in molecular biology requires the use of CTAB/PVP-based buffers. The results we described here clear demonstrated that this buffer was efficient to extract RNA and DNA from different tropical and subtropical species using challenging for this type of experiment. We also highlight that SDS-based buffer, used in some procedures , 37, canAlthough the CTAB/PVP-based methods are giving good results in term of RNA yield and quality, these methods not compatible with the increasing concern for health safety in the scientific community, due to the large amounts of phenol and chloroform used in these methods. Our results show that it is possible to precipitate RNA directly after the lysis step. However, RNA yields are low, especially for macadamia, a crop underrepresented in molecular studies. Although, CTAB and SDS react together to form micelles, adding 1% SDS at the end of the lysing step with a CTAB/PVP-based buffer could increase the RNA yields. This increase in final RNA yields was likely to be due to the property of SDS to separate proteins from RNA, since the effect of SDS on yield did not rely on the presence of CTAB or plant tissue in the reaction Fig. .In this experiment described in the Fig. Avocado, macadamia and mango can be classified as orphan crops as they are agriculturally important yet they are not well studied and therefore have limited genetic and genomic resources . The impWithin the last decade, the price of transcriptome sequencing has decreased considerably, prompting researchers to design larger scaled experiments. However, the toxicity and impracticality of the chemicals used for nucleic acids extraction for woody tropical species represent a major issue for these large-scale experiments. The method we described here constitutes a solution to alleviate these issues and will prompt for more large-scale experiments led on species considered recalcitrant in molecular biology.Additional file 1: Fig. S1. Melting curves for DRM1 and miR172 in avocado (A), mango (B) and macadamia (C). qRT-PCR reactions were performed using the RNA extracted by the CTAB/SDS-based method from the samples shown in Figs. Additional file 2: Fig. S2. Sequence quality of RNA extracted using the CTAB/SDS RNA extraction protocol. RNA was assessed by producing high-throughput RNA sequencing (RNA-seq) libraries of the avocado, macadamia and mango samples shown in Figs. A-Avocado; B-Mango; C-Macadamia, respectively). Sequence quality is based on phred scores, an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score, the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red). The graphs are representative of the reverse reads . Single pea buds are individual dormant axillary buds (less than 0.5 mg each).


Hepatitis E virus (HEV) is a leading cause of hepatitis worldwide. However, its infection biology and pathogenesis remain largely elusive. Furthermore, no proven medication is available for treating hepatitis E. Robust experimental models are urgently required to advance the research of HEV infection. Because of the lacking of a sophisticated small animal model, this study aimed to establish a mouse model of HEV infection.We constructed a full-length swine HEV cDNA clone of genotype 4 (named as pGEM-HEV) by reverse genetics approach. And we inoculated with HEV RNA in BALB/c mice to establish small animal model for HEV infection and pathogenesis studies.The capped RNA transcripts of pGEM-HEV prepared in vitro were replication-competent in HepG2 cells. Importantly, BALB/c mice intravenously inoculated with RNA transcripts of pGEM-HEV developed an active infection as shown by shedding viruses in feces, detectable negative strand of HEV in the liver, spleen and kidney, and causing liver inflammation.In this study, we successfully established of BALB/c mice-based small animal model for HEV provides an opportunity to further understand HEV pathogenesis and to develop effective antiviral medications. Orthohepevirus of the family Hepeviridae [Hepatitis E virus (HEV) is classified in the genus eviridae . It is aeviridae –5. A unieviridae –10.HEV is considered the most common cause of hepatitis worldwide . It causRobust experiment models are the most important tools for advancing fundamental and translational research of hepatitis E infection. Fortunately, several cell culture systems for propagating HEV have been recently developed –18. HoweWe previously have attempted to establish BALB/c nude mice-based HEV model . HoweverXba I restriction enzyme site and a T7 RNA polymerase core promoter were introduced at the extreme 5′ terminus. Twenty-four adenosines (A) was engineered at the 3′ end of viral genome, followed by a Cla I restriction enzyme site for plasmid linearization  was amplified with specific primers shown in Table Cla I, then digested with proteinase K, and purified by phenol extraction followed by ethanol precipitation. Capped RNA transcripts from pGEM-HEV were synthesized in vitro with the T7 RiboMAX™ Express Large Scale RNA Production System  according to the direction. The RNA transcripts from the cDNA clone was quantitated (1 μg /μL) and stored in − 80 °C until use.The full-length cDNA clones of pGEM-HEV was linearized by 2 in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS). To evaluate the infectivity of the pGEM-HEV clone, HepG2 (60~70% confluence) cells were transfected with the capped RNA transcripts as described previously [The human hepatoma HepG2 cells and human lung carcinoma A549 cells were incubated at 37 °C with 5% COeviously . Twenty-The protocol of animal experiments were approved by the Animal Care and Use Committee (ACUC) of Kunming University of Science and Technology. Six female, 8-week-old, SPF (specific pathogen free) BALB/c mouse purchased from SLAC laboratory animal (China), and maintained in a pathogen-free animal facility. Mice were negative to anti-HEV IgG and IgM antibodies, and HEV RNA. Six mice were randomly divided into 2 groups and each mouse was caged separately.Group A containing Mouse 1, 2 and 3 were inoculated with RNA transcripts of the swine HEV clone pGEM-HEV (10 μg /mouse), and Group B containing mouse 4, 5 and 6 were inoculated with equal volume PBS. Feces and serum were collected every week from each mouse after inoculation. Mice were anaesthetized with pentobarbital (80 mg/kg) and euthanized by cervical dislocation at 28 days post-inoculation (dpi). Tissues, including liver, spleen, kidney and colon were collected. Animal carcasses were safely disposed by the ACUC of the Kunming University of Science and Technology (WH17037).Total RNA in feces, serum and tissues were extracted by Trizol (Invitrogen), according to the manufacturer’s instructions. The isolated RNA was used to synthesize first-strand cDNA. The cDNA was added as the template to perform the reverse transcription nested PCR (RT-nPCR). Both positive strand and (or) negative strands of HEV in feces, serum and tissues were detected with strand-specific primers , 24 as dThe viral titer of HEV in the feces, serum and tissues were quantified using SYBR green-based quantitative RT-PCR (qRT-PCR) with HEV-specific primers . qRT-PCRThe HEV IgG and IgM antibodies in serum were tested by ELISA  according to the manufacturer’s instructions. The cutoff values for the IgG and IgM assay were determined based on 0.22 (or 0.24 for IgM) plus the mean OD450/630 values of serum from uninfected mouse .The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum were measured with an automated biochemistry analyzer .The livers, spleens, kidneys and colons were collected and HEV capsid protein were detected in these tissues by Western blotting. Briefly, the tissues were broken in liquid nitrogen by grinding, and were lysed in RIPA buffer. Equal volumes of tissue lysates from each condition were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of protein was visualized by direct Western blotting using antibodies directed against the indicated antigens.The mouse feces were suspended into 0.1% DEPC (diethyl pyrocarbonate)-PBS. The supernatant was collected by centrifuge and microorganisms were removed by filter. A549 cells were infected with the viruses recovered from mice experimentally infected with feces supernatant. Cells were harvested at 72 h post-infection, and the HEV ORF2 protein was be detected by Western blotting.The liver tissues were fixed in 10% neutral buffered formalin, sectioned at a thickness of 3 μm, and stained with hematoxylin and eosin. All sections were examined using a Nikon Ti-E microscope (Japan).Prism software (GraphPad Software) was used for statistical analysis. Data were presented as mean ± S. D..To assess the infectivity of the pGEM-HEV cDNA clone, capped RNA transcripts were transfected into HepG2 cells. Twenty-four hours post-transfection, HEV antigens were detected by immunofluorescence assay (IFA) with an anti-HEV ORF2 monoclonal antibody. The intense fluorescent signals of HEV antigens indicating that the transfected viral RNA was replication-competent in HepG2 cells  and PBS (Control), respectively. HEV RNA was first detected in feces at 3 dpi in group A inoculated with RNA transcripts, and it was all detected in feces from three mice feces after 7 dpi, and lasted to the end of the experiment Table . The vir5 ± 5.8 × 104 copies/g), spleen (1.3 × 104 ± 3.7 × 104 copies/g), kidney (7.1 × 104 ± 3.1 × 104 copies/g) and colon (1.9 × 104 ± 5.8 × 103 copies/g) of mice inoculated with HEV RNA transcripts at 28 dpi . To investigate whether BALB/c mice provoke similar response to RNA transcripts of HEV infection, we measured serum anti-HEV IgM and IgG. An increased anti-HEV IgM and IgG levels were observed in all the mice inoculated with HEV RNA transcripts compared with the control group Fig. . In the Hepatitis is swelling and inflammation of the liver, and most commonly caused by hepatitis virus infections. To determine whether infection of capped HEV RNA transcripts can also cause liver injury/inflammation in BALB/c mice, we first tested the widely used liver enzymes for detecting liver damage. The levels/activities were characterized using an automated biochemistry analyzer. To our surprise, the elevated ALT, AST and ALP levels/activities in BALB/c inoculated with HEV RNA transcripts was very similar to the clinical feature of HEV infected patients  inoculated with genotype 4 swine HEV RNA transcripts [In fact, we have previously attempted to establish HEV mouse model using BALB/c nude mice . Becausenscripts . HEV pronscripts . More inAlthough HEV infects BALB/c mice has been successfully established, key parameters should be determined in the future, such as identification of infected cell populations, observation of inflammatory manifestation, and then explicit comparisons with HEV infected patients to assess potential for clinical relevance. The establishment of HEV infection based on BALB/c mice is benefit to screen anti-HEV drugs and develop vaccines in vivo.In this study, we successfully established BALB/c mice-based animal model with an infectious cDNA clone of genotype 4 swine HEV constructed by reverse genetics approach. This swine HEV clone strain is capable of modeling HEV infection, and studying host response and pathogenesis. Furthermore, this model also bears importantly implications for future anti-HEV drug development.


Liver samples were collected from male C57BL/6 mice at several ages from prenatal through neonatal, infant, child to young adult. Results showed that most of the RNA modifying proteins were highly expressed in prenatal mouse liver with a dramatic drop at birth. After birth, most of the RNA modifying proteins showed a downregulation trend during liver maturation. Moreover, the RNA modifying proteins that belong to the same enzyme family were expressed at different abundances at the same ages in mouse liver. In conclusion, this study unveils that the mRNA expression of RNA modifying proteins follows specific ontogenic expression patterns in mice liver during maturation. These data indicated that the changes in expression of RNA modifying proteins might have a potential role to regulate gene expression in liver through alteration of RNA modification status.RNA modifications are recently emerged epigenetic modifications. These diverse RNA modifications have been shown to regulate multiple biological processes, including development. RNA modifications are dynamically controlled by the “writers, erasers, and readers”, where RNA modifying proteins are able to add, remove, and recognize specific chemical modification groups on RNAs. However, little is known about the ontogenic expression of these RNA modifying proteins in various organs, such as liver. In the present study, the hepatic mRNA expression of selected RNA modifying proteins involve in m Epigenetic regulation refers to the molecular events where gene expression is regulated without alterations in the DNA sequence . EpigeneThe diverse post-transcriptional modifications of RNAs, or called epitranscriptomics, have been recognized as another key player in epigenetic regulation mechanisms . The pioPreviously, the RNA modifications and epitranscriptomics were regarded as a relatively static status for a specific RNA structure. Whereas, recent studies have shown that the modifications of RNAs are a dynamic and reversible process . SimilarOntogenic development is a complicated process involving the buildup of genetic and epigenetic signatures . This ph6-methyladenosine (m6A), N1-methyladenosine (m1A), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), N7-methylguanosine (m7G), and pseudouridine (Ψ), was studied in mouse liver at different ages during postnatal maturation using RNA sequencing.In the current study, the ontogenic mRNA expression of RNA modification proteins involved in several frequent RNA modifications, including NThe procedures including animal experiments, RNA extraction, cDNA library construction, and RNA sequencing were described in previous publication .The RNA-Seq reads from the FASTQ files were mapped to the mouse reference genome (NCBI37/mm9) and the splice junctions were identified by TopHat 1.2. The output files in BAM  format were analyzed by Cufflinks 1.0.3 to estimate the transcript abundance.The selected RNA modification enzyme genes were retrieved from Cufflinks output for further analysis. Significant gene expression was determined by the drop-in-deviance F test of the fitted FPKM data to a generalized linear model with a Poisson link function, a statistic designed to measure the significance of a gene’s measured FPKM value relative to a zero FPKM value. Data were presented as the mean ± S.D. Ontogenic expression patterns were presented using the GraphPad Prism 7 software program .6-methyladenosine involves the addition of a methyl group at the N-6 position of an adenosine base. High-throughput sequencing following m6A-specific antibody immunoprecipitation has revealed a high prevalence of this modification in multiple RNA types across different species. The m6A is believed to be the most abundant RNA modification in mRNAs, but it also exists in other RNA types, including miRNAs, lncRNAs, tRNAs, rRNAs, and other small RNAs [6A modification has been shown to play a role in multiple biological processes, including development, metabolism, and circadian rhythm [Nall RNAs –23. The n rhythm , 25, thrn rhythm –28.6A is accomplished by a methyltransferase complex (writers) consisting of several components, including methyltransferase Like 3 (METTL3), METTL14, Wilms tumor 1 associated protein (WTAP), Vir like m6A methyltransferase associated (VIRMA or KIAA1429), RNA binding motif protein 15 (RBM15), and zinc finger CCCH domain-containing protein 13 (ZC3H13). Each component in the complex has specific roles in the installment of m6A modification at a precise location [6A writers followed specific trends. The expressions of METTL14 and ZC3H13 were the lowest among all tested components in the m6A methyltransferase complex. Compared to the prenatal expression, both METTL14 and ZC3H13 mRNA levels dropped ~50% at birth . So far, two enzymes have been identified as demethylases for the m6A modification, including fat mass obesity-associated protein (FTO) and AlkB Homolog 5 RNA Demethylase (ALKBH5) [6A status in health. As shown in The dynamic regulation of m(ALKBH5) , 35–37. 6A-mediated cellular functions depend on reader proteins, which can recognize and bind to m6A-modified RNAs. The YTH domain containing proteins are the best characterized m6A readers [6A reader protein, involved in glial development in mice [6A readers in mouse liver. The expression of PRRC2A had a constant decrease during liver development. At day 60, the PRRC2A expression was only 20% compared to prenatal expression. YTHDF1 was the most abundant YTH domain containing reader, followed by YTHDF2, YTHDC1, YTHDF3, and YTHDC2. Regardless of the differences in expression levels, all these YTH domain containing readers showed a sharp decrease in expression at birth, ranging from 70% decrease (YTHDC2) to 32% decrease (YTHDF2)  .1-methyladenosine consists of addition of a methyl group at the N1 position of adenosine. The m1A modification was first identified in tRNAs and rRNAs decades ago [1A modification in mRNAs [1A modification in tRNAs was important for tRNA folding, stability, and tRNA-protein interaction [1A modification was also observed at several sites in rRNAs, which affect the tertiary structure of the ribosome and downstream gene translation [1A modification has a relatively low abundance in mRNAs, with a potential function in regulating translation process [The Nades ago . Recentlin mRNAs , 48. Theeraction –52. The nslation , 54. In  process .1A methyltransferases responsible for tRNA modification [1A modification at multiple rRNA sites in both human and mouse cells [1A writers remain unknown. As shown in 1A writers showed a decrease trend during development. The expression of ribosomal RAN-processing protein (RRP8) was the highest among all tested m1A writers and it decreased constantly in mouse liver with the highest level at the prenatal age. The expression of TRM6 decreased from prenatal day 2 to postnatal day 10 to about 40%, followed by a relatively stable expression until adulthood. The expression of TRM61A dropped constantly, but the dropping rate was reduced after postnatal day 10.TRM6 and TRM61A are two identified mfication . Nucleomse cells . These k1A demethylases, ALKBH3 and ALKBH1 [Two proteins in the AlkB family have been identified to be md ALKBH1 , 57. Ove1A reader proteins, which have a role in translation regulation [1A readers  modification was first identified in DNA molecules, but also found later in RNAs [5C modification has been identified to be widely distributed in multiple types of RNAs, both coding and non-coding [5C modification includes regulation of translation, RNA metabolism, and RNA trafficking [The 5-methylcytosine , which contain S-adenosyl-L-methionine (SAM)-dependent MTase domains, are the major writers of m5C modification. Several members of the NOP2/Sun RNA methyltransferase family (NSUN) have been identified to methylate different RNA molecules [5C modification in tRNAs [The RNA molecules , 67. Amoolecules –70. The olecules , 72. NSUolecules , 74. Thrin tRNAs , 76. How5C is still very limited. There are currently no identified proteins, which can remove the methyl group at m5C modified sites. But it has been reported that m5C modification can be converted to other types of modifications, for example hm5C [5C might be accomplished by further modification of the methyl group instead of removing it.The study of demethylase of mple hm5C . This in5C reader protein, which regulates the exportation of m5C modified mRNAs [5C reader protein, which was reported to promote the pathogenesis of human urothelial carcinoma of the bladder in an m5C-modification-dependent mechanism, where the binding of YBX1 maintained the stability of target mRNAs [The Aly/ REF export factor (ALYREF) is the first identified med mRNAs . As showet mRNAs . From Fi5C, the hm5C was first identified in DNAs, which was later identified in mammalian RNAs [5C are still focused on its role in DNA modifications with very limited knowledge on RNA modifications. Studies have shown that the hm5C in the coding sequences of mRNAs increases translation efficiency through unknown mechanisms [Similar to mian RNAs . Howeverchanisms .5C to hm5C in both DNAs and RNAs [5C in RNAs, which indicates the existence of hm5C writers other than TETs [5C erasers in RNAs. Future studies are still needed to cover this knowledge gap.The ten-eleven translocation (TET) family proteins, including TET1, TET2, and TET3, were reported to mediate the oxidation of mand RNAs . As showhan TETs . Current5C modified RNAs. The UHRF2 was reported to bind to hm5C modified DNAs [The ubiquitin-like with PHD and ring finger domains 2 (UHRF2) proteins might have the potential to bind to hmied DNAs , 82. Fig7-methylguanosine (m7G) is another most prevalent modifications existing in multiple types of RNAs, including tRNA, rRNA, and mRNA [7G was reported to regulate the stability and decay pathways of tRNAs, which further involved in the regulation of physiological processes including cellular development and human disease [7G is also conserved in rRNAs in multiples species. Catalyzed by the WBSCR22/TRMT112 complex, where the TRMT112 protein acts as a co-activator of the methyltransferases WBSCR22, m7G modification was reported to regulated the processing, export, and maturation of several pre-rRNAs and rRNAs [7G cap modification was first identified, which is mediated by the RNA (guanine-7-) methyltransferase (RNMT)/RAM methyltransferase complex, and regulate the export, stability, splicing, transcription, translation, and decay of mRNA [7G in a transcriptome wide manner and found the existence of m7G in internal mRNAs and long non-coding RNAs, with a potential role in translational regulation [Nand mRNA –86. In t disease –89. The nd rRNAs , 91. In  of mRNA –95. Utilgulation , 86.7G modification. METTL1 was reported to catalyze the formation of m7G on both tRNAs and mRNAs. As showed in 7G methyltransferase catalyzing the formation of m7G on pre-rRNAs and rRNAs. The expression of WBSCR22 dropped at birth and rebounded to its prenatal level at day 5, followed by another drop , a C-C glycosidic isomer of uridine (U), is derived from the incorporation of C5 into the glycosidic bond . Recent In RNAs, uridine is transformed into pseudouridine by a class of enzymes known as pseudouridine synthases (PUSes). Different PUSes have shown preferences for different residues in tRNAs or rRNAs. Several PUSes were identified in mouse liver by RNA-sequencing, as shown in The erasers and readers of the Ψ modification are still largely unknown. Future studies are needed to fill in these knowledge gaps.Even though RNA modifications have been known for decades, the biological significance of these modifications has only been recognized in recent years, owning to the development of genome-wide and high resolution sequencing techniques. Recent studies have identified more than 150 types of RNA modifications covering almost all RNA types in the genomes of different species . AlthougMettl3, the m6A writer gene, has been shown to be embryonically lethal in mice with dysregulation of pluripotency in embryonic stem cells [6A modifiers also affected neuronal development and reproductive function  [6A modification, some other types of RNA modifications are also involved in development regulation. Knockout of m5C modification writer Nsun2 or Dnmt2 has been reported to affect stem cell differentiation and endochondral ossification, respectively [Development involves dynamic regulation of gene expression in cells, where RNAs play a non-negligible role in this process. Chemical modifications of RNAs have emerged as promising mechanisms in modulation of development process. Previous studies have shown the importance of RNA modifying proteins in development. Deletion of em cells . Knockougenesis) . Aside fectively , 107. Al6A, m1A, m5C, hm5C, m7G, and Ψ, were studied in mouse liver at different ages during development. Most of these RNA modifying proteins showed ontogenic changes in mRNA levels along with liver development, where the majority of them showed dramatic drops at birth followed by downregulations during postnatal development. These data indicate that RNA modification status might also have such ontogenetic changes, which is regulated by the alterations in RNA modifying proteins.In the present study, the mRNA expression of genes involved in several of the most common RNA modifications, including mThe data also showed that most RNA modifying enzymes are high expressed in prenatal compared to postnatal mouse liver, at which stage the cells are more stem-like. The studies mentioned above have suggested the important role of RNA modifications in stem cell functions. So it may be interesting to study how the RNA modifying proteins are expressed in the early stages of prenatal development. However, in the current research, only prenatal day two liver samples were collected and future experiments are needed to analyze the prenatal expression of RNA modifying proteins.Many other liver-specific genes have been reported to show specific ontogenic expression patterns during development, including phase I and phase II drug metabolizing enzymes , 108. HoIn summary, the present study provides new knowledge about the ontogenic mRNA expression of multiple RNA modifying proteins during development of mouse liver. Such knowledge can serve as foundation for future studies on the impact of RNA modifications in gene regulation during liver development. Understanding the biological significance of RNA modifications in liver development or functions will also benefit the study of drug metabolism or liver diseases in the future.


However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for  The RNA coliphage MS2 is one of the oldest model systems of modern molecular biology and its detailed investigation has led to numerous fundamental findings and applications. For example, its genome was the first to be completely sequenced, revealing for the first time the genetic organization of a biological entity . The smaPrimer-independent replication of MS2 (+) strand genomes proceeds via complementary (−) strand intermediates, which serve as templates for efficient transcription of viral progeny (+) strands Figure  8,9). T,9. T8,9)in vitro gene expression is the general difficulty of designing RNA constructs that are still suitable as efficient replication templates . We finThe preparation of all DNA and RNA constructs is described in the Supplementary Methods. Primers used in this study are listed in In vitro protein synthesis was performed using the PURExpress® system (NEB), which utilizes two solutions: solution A  and solution B (ribosomes and proteins including T7 DNA-dependent RNA polymerase) (ymerase) . The totLys tRNA . Template DNA was omitted in the negative control reaction. Samples were incubated for 2  h at 37 °C in a nuclease-free PCR tube (Thermo Fisher Scientific) using a ProFlex PCR System (Thermo Fisher Scientific) and subsequently treated with 0.6 μl RNase Cocktail™ Enzyme Mix  for 15 min at 37°C to degrade non-incorporated GreenLys tRNA. 7.5 μl sample were mixed with an equal volume 2× Laemmli sample loading buffer (incl. 200 mM DTT) and denatured for 2.5 min at 65°C. Samples were analysed by conventional discontinuous SDS-PAGE (10% gel) run at 4°C  on a Midi-format electrophoresis system (Atto). The fluorescent signal of the de novo expressed rep β subunit was imaged on a fluorescence laser scanner  at either 473 nm (blue LD laser/510LP filter) or at 532 nm (green SHG laser/575LP filter). Total protein and the molecular-weight size marker  were visualized after SYPRO Ruby (Bio-Rad) staining using the same instrument .A standard PURE reaction programmed with 17.5 nM (250 ng) of a linear Rep DNA template (under control of a T7 promoter) was supplemented with 0.6 µl FluoroTect™ Greengene]MS2 (−) RNA). For real-time detection, standard PURE reactions were supplemented with the following final concentrations of DNA or RNA templates and reagents/additives:Design and synthesis of individual readout constructs  are descMS2 (−) RNA, 6% (w/v) PEG 4000 and 10 μM DFHBI-1T fluorophore. β-gal α-complementation by MS2 RdTT: 70 nM MS2-rep/MS2-ddrep (+) RNA, 70 nM [α]MS2 (−) RNA, 70 nM ω-protein and 50 μM FDG substrate. sfGFP expression by MS2 RdTT: 70 nM MS2-rep/MS2-ddrep (+) RNA, 70 nM [sfGFP]MS2 (−) RNA and 6% (w/v) PEG 4000. Full-length β-gal expression by MS2 RdTT: 70 nM MS2-rep/MS2-ddrep (+) RNA, 70 nM [lacZ]MS2 (−) RNA, 6% (w/v) PEG and 50 μM FDG substrate. T for F30-Broccoli transcription by MS2 replicaseComparison +/– cUTR: 70 nM MS2-rep/MS2-ddrep (+) RNA, 350 nM standard [F30-Bro]MS2 (−) RNA/truncated 350 nM [F30-Bro]MS2 (−) RNA, 6% (w/v) PEG 4000 and 10 μM DFHBI-1T fluorophoreT, for sfGFP expression by MS2 RdTT. Comparison +/– cUTR: 70 nM MS2-rep/MS2-ddrep (+) RNA, 70 nM standard [sfGFP-RBS2]MS2 (−) RNA/70 nM truncated [sfGFP-RBS2]MS2 (−) RNA and 6% (w/v) PEG 4000. SP6 pol expression by MS2 RdTT coupled with sfGFP expression by SP6 DdTT: 70 nM MS2-rep/MS2-ddrep (+) RNA, 35 nM [SP6 pol]MS2 (−) RNA, 16 nM (125 ng) SP6-sfGFP DNA, 5,5% (w/v) PEG 4000 and ∼1 U RNase inhibitor (moloX) per μl reaction. Note that the latter reaction was slightly diluted (13.5 μl versus 12.5 μl) due to the high amount of additives required to perform the reaction.F30-Broccoli transcription by MS2 replicase: 70 nM MS2-rep/MS2-ddrep (+) RNA, 350 nM [F30-Bro]All reactions were prepared in MicroAmp Fast 8-Tube Strips (Thermo Fisher Scientific) and incubated at 37°C in a StepOne Real-Time PCR System (Thermo Fisher Scientific). Fluorescence signals were recorded every 60 s . If not stated otherwise, all experiments were performed in technical triplicates from single master mixes.MS2 (−) RNA, 6% (w/v) PEG 4000 and ∼1 U RNase inhibitor (NEB) per μl reaction. Reactions were incubated at 37 °C in a nuclease-free PCR tube (Thermo Fisher Scientific) using a ProFlex PCR System (Thermo Fisher Scientific). Sample aliquots (5 μl) were taken at different time points , mixed with 5× native RNA loading buffer  glycerol, 0.05% (w/v) bromophenol blue), shock-frozen in liquid nitrogen and stored at −80°C until further use. To create standard curves for in-gel F30-Broccoli fluorescence detection, in vitro transcribed [F30-Bro]MS2 (+) RNA (Supplementary Methods) was diluted in nuclease-free H2O (supplemented with 0.02% (w/v) PEG 4000 to prevent RNA adhesion to the tube surface at low concentrations) (MS2 (+) RNA standards that were diluted in H2O. Thus, to enable a more precise quantification of the de novo transcribed [F30-Bro]MS2 (+) RNA concentrations in MS2 transcription reactions, all fluorescence values were therefore multiplied with a correction factor of 1.3 (see Supplementary Files), which was determined by quantifying and comparing equivalent amounts of [F30-Bro]MS2 (+) RNA diluted either in H2O or PURE reaction buffer.An appropriately up-scaled standard PURE reaction (∼10-fold) was programmed with 70 nM MS2-rep (+) RNA, 70 nM [F30-Bro]rations)  to final® TBE Gel, Bio-Rad) run at 100 V in 1× TBE on a Mini-format electrophoresis system (Bio-Rad). RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific) was used as molecular weight standard. Following electrophoresis, gels were washed 3 × 5 min with ultrapure H2O and then stained for 20 min in 20 ml DFHBI-1T staining solution . The fluorescent signal of the de novo transcribed [F30-Bro]MS2 (+) RNA was imaged on a fluorescence laser scanner  at 473 nm (blue LD laser/510LP filter). Gel band intensities were quantified using ImageQuant TL 1D 7.0 . Brightness and contrast adjustments of the displayed gel image were applied homogenously.All samples and the standards were analysed by native TBE-PAGE (5% Mini-ProteanlacZ-RBS2]MS2 (−) RNA (containing a weaker ribosome binding site) at a concentration of 60 nM. For compartmentalization, samples were aspirated from the PCR tubes into the PTFE tubing by operating syringe pumps  in withdrawal mode at 4000 μl/h. Subsequently, the filled sample tubes were connected to the inlets of the PDMS chip and injected at a flow-rate of 400 μl/h for the aqueous phase and the oil phase each. The resulting emulsion droplet creams were loaded into rectangular 50 × 500 μm glass capillaries (VitroTubes) by capillary action. For imaging, the filled glass capillaries were placed on a glass microscope slip and capillary inlets were sealed with 5 Minute-Epoxy to prevent evaporation. All images were taken on a LSM 780 confocal laser scanning microscope equipped with a custom environmental chamber pre-heated to 37°C and a Plan-Apochromat 10×/0.45 M27 objective (Carl Zeiss). Green fluorescence of the droplets was excited using the 488 nm Argon laser on the first channel (Fluorescence) with the corresponding filter (λem = 559 nm), while transmission bright-field images were also collected to access overall droplet shapes throughout the experiment. Time series for both reactions were acquired with 3 min imaging intervals for 80 cycles. The images in Figure Droplet nozzles were fabricated through soft lithography with final chips cast from SU8 masters as 10:1 base:curing agent poly-(dimethylsiloxane) , bonded to glass using oxygen plasma and surface treated with cytop 809M  to be fluorophilic, as previously described . Monodisin vivo presumably from the association of the catalytic rep β subunit with E. coli host factors similar to the Qβ replicase  of the rep β subunit in a commercial PURE system, which contains the T7 DNA-dependent RNA polymerase (T7 pol). To this end, we engineered a linear DNA construct encoding a minimized version of the MS2 genome devoid of all coding sequences except for the rep β subunit with an upstream T7 promotor (Rep DNA). Using this construct, we could indeed detect de novo synthesis of the 60.8 kD rep β subunit in the PURE system by the GreenLys labelling approach and conventional SYPRO Ruby staining in yields matching the concentration of the translation factors present in the PURE system  is located at the 5′ end of the (+) strand genome and the UTR trailer sequence  overlaps with the end of the rep β subunit gene at the 3′ end  and the UTRT (cUTRT) found in the genomic (−) strand are sufficient for general template recognition and transcription initiation by the MS2 replicase.Next, we set out to probe if the expressed rep β subunit can form an active replicase through complex formation with the proposed E system . RNA repE system  to preveE system ,40. TherE system . In partE system . The UTRTo test this hypothesis, we created two types of RNA modules: Readout modules for the detection of RNA-dependent RNA transcription and a replicase module (MS2-rep (+) RNA) encoding the open reading frame of the rep β subunit. The readout modules were designed as such that they should serve as synthetic (−) RNA templates for the MS2 replicase and were created by inserting the antisense strands of genes between both cUTRs Figure . The expde novo synthesized MS2 replicase and, if applicable, their translation into functional proteins. As first readout, we co-incubated the PURE components with the replicase module MS2-rep (+) RNA, the fluorophore DFHBI-1T and the readout module [F30-Bro]MS2 (−) RNA, in which the antisense strand of the F30-Broccoli aptamer (F30-Bro)  in the PURE system Figure . In detaF30-Bro) ,47 was em Figure . In cont mutated ,48.E. coli β-galactosidase (lacZ). We programmed PURE reactions with [α]MS2 (−) RNA (544 nt), MS2-rep (+) RNA, recombinant ω-protein and the fluorogenic β-galactosidase substrate Fluorescein di-β-D-galactopyranoside (FDG). As expected, we observed strong FDG turnover suggestive of successful α-complementation of the active β-gal tetramer  RNA, FDG and either MS2-rep (+) RNA or MS2-ddrep (+) RNA. Minor differences between the two samples were indeed observable  RNA templates.In conclusion, we demonstrated that the de novo synthesized (+) strand would also serve as template for more (−) strand synthesis, thereby initiating an RNA amplification cycle. To quantify the amount and kinetics of the de novo transcribed (+) strand, we monitored the fluorescence increase upon [F30-Bro]MS2 (+) RNA transcription in MS2 RdTT reactions programmed with [F30-Bro]MS2 (−) RNA and MS2-rep (+) at different time points of the reaction by non-denaturing PAGE  RNA . When PURE reactions were programmed with 70 nM [F30-Bro]MS2 (−) RNA and 70 nM MS2-rep (+), we observed a near-stoichiometric formation of full-length [F30-Bro]MS2 (+) RNA during the first hour of incubation  template RNA into inert RNA duplexes (After demonstrating successful (−) to (+) ssRNA conversion by the MS2 replicase, we sought to determine the amount of (+) strand that is synthesized during a typical RdTT reaction and whether the E Figure . The natn Figure . No furtduplexes .in vitro transcribed [F30-Bro]MS2 (+) RNA. If both (−) and (+) strand could serve as a template, a further increase in F30-Bro levels would have been expected. However, no significant fluorescence increase compared to a negative control was observed during 3 h of incubation, implying that (+) strand readout modules do not serve as templates for (−) strand synthesis under our tested conditions (To further probe whether MS2 replicase is capable of synthesizing (−) strands from engineered (+) strands, we initiated RdTT reactions directly with nditions .MS2 (+) RNA could be increased by adding more (−) strand RNA template. To this end, we titrated different concentrations of [F30-Bro]MS2 (−) RNA into MS2 RdTT reactions containing 70 nM MS2-rep (+) and quantified the reactions after 2 h of incubation (de novo synthesized [F30-Bro]MS2 (+) RNA increased until 210 nM input [F30-Bro]MS2 (−) RNA and plateaued at a maximal yield of ∼90 nM (MS2 (+) RNA.In the following, we tested if the amount of [F30-Bro]cubation . Under tf ∼90 nM . Higher In conclusion, the new MS2 RdTT system enables unidirectional conversion from (−) to (+) strand of various RNAs without further (unregulated) amplification as in most current Qβ systems ,27. The In an attempt to further minimize the UTRs required for MS2 RdTT, we compared MS2 RdTT-based α-complementation with the α-peptide mRNA embedded between either full-length cUTRs  or minimMS2 (−) RNA (containing both full-length cUTRs) with transcription from a truncated [F30-Bro]MS2 (−) RNA lacking the cUTRT domain  RNA) had barely any impact on sfGFP expression levels  strand templates are rather suggestive of a context-dependent influence of the 129 nt segment on RNA template structure and/or stability rather than of a direct role of the RNA domain in transcription. For example, deleting the cUTRT from the 410 nt F30-Broccoli construct decreases the overall RNA length and, thus, the synthetic burden for transcription by ∼43%, which may explain the overall increase in synthesis yields by MS2 replicase.Next, we set out to test if both cUTRs of (−) strand templates are required for MS2 RdTT. For the Qβ replicase, it has been suggested that both 5′ and 3′ termini of genuine RNA templates cooperate during and after the initiation step, presumably via a circular configuration through a terminal helix . Howevern Figure , we founn Figure . On the s Figure . Both fiin vitro translation systems is a well-established method in synthetic biology and useful in applications such as high-throughput screening, molecular evolution of enzymes, or bottom-up synthesis of artificial cells (MS2 (−) RNA and MS2-rep (+) RNA (or MS2-ddrep (+) RNA as control) using a custom microfluidic setup  and monitored the fluorescence of individual droplets by fluorescence microscopy during incubation at 37°C. We observed robust fluorogenic transcription of the F30-Broccoli aptamer in the droplets during the first 90 min . Such a ‘cross-talk’ between DNA- and RNA-dependent in vitro transcription-translation would largely expand the repertoire for the generation of synthetic circuits and switches used in synthetic biology. As a proof of concept, we designed an experiment such that the synthesis of active MS2 replicase would lead to MS2 RdTT of SP6 DNA-dependent RNA polymerase (SP6 pol) using [SP6 pol]MS2 (−) RNA as the template. The de novo expressed SP6 pol should then catalyse SP6 DdTT of sfGFP from a linear DNA template with an SP6 promoter (SP6-sfGFP DNA). We chose the SP6 pol for DdTT to bypass the T7 pol, which is already present in the PURE system, because both polymerases use different promotors. Altogether, the system coupling both MS2 RdTT and SP6 DdTT combines 5 steps of macromolecular synthesis in a one-pot reaction: Three translation events  and two transcription events ([SP6 pol]MS2 (+) RNA and sfGFP mRNA)  Figure . To our ) Figure . Thus, o) Figure . Such le) Figure ,34,59–61in situ in a recombinant in vitro transcription-translation system. The in situ produced replicase can directly initiate transcription of various RNA templates if its 3′ terminus is fused with cUTRL—a short 3′ terminal domain of the viral (−) strand. While low levels of RNA transcription can already be achieved with a minimalistic 43 nt version of cUTRL, maximal transcription levels require fusion to the full-length cUTRL  strand) and internal RNA-sites (In contrast to the transcription of (+) from (−) strands, we observed no (−) strand synthesis from synthetic (+) strand RNAs even if both UTRs were present. This finding implies either that the 3′-end of the tested (+) strand construct is only poorly accessible for replicase initiation or that additional sequence elements are required for a complete replication cycle. In agreement with the latter explanation, replication of the Qβ (+) strand by the Qβ replicase is crucially dependent on long-distance interactions between UTRNA-sites ,62–64 anNA-sites , which aNA-sites .in vitro transcription-translation systems described. In these systems, target genes are typically embedded in the (+) strands of small, non-genomic ‘parasitic’ RNA-scaffolds such as RQ135  strand synthesis from an RNA template without further amplification distinguishes the MS2-based RdTT system from similar Qβ-based systems, which are currently the only other bacterial RNA-only as RQ135 ,66 or MDas RQ135 ,24,26. Tas RQ135 ,27, it cRNA pool ,26. The he ideal . Furtherswitches , oscillaswitches ,70, biosswitches , or artiswitches .gkz817_Supplemental_FilesClick here for additional data file.


RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA–protein complexes using silica beads. In a variant approach (PAR‐TRAPP), RNAs were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR‐TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR‐TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA–peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.The  However, the identification of protein–RNA interaction sites has long been challenging, particularly in living cells. Individual protein–RNA interactions can be characterized, if known, by mutagenic and biochemical approaches, but this has always been labour‐intensive. The difficulty is compounded by the fact that many interactions do not fall within characterized interaction domains, and even apparently well‐characterized RNA binding domains can show multiple modes of RNA interaction Escherichia coli following crosslinking in actively growing cells. These approaches should allow characterization of the protein–RNA interactome at steady state, as well as dynamic changes following stress exposure, in almost any system.The TRAPP techniques for identification of RNA–protein interaction are based on recovery of denatured RNA–protein complexes on silica beads, followed by mass spectrometry. They additionally permit identification of the peptide, and indeed the amino acid, that is crosslinked to RNA during UV irradiation in living cells. We applied TRAPP to identify RNA binding proteins from yeast and In all TRAPP techniques, we initially covalently linked all RNAs to associated proteins by UV crosslinking in actively growing cells. Detailed protocols for each of the TRAPP workflows are given in −2, since similar doses have previously been used in many publications mapping protein‐binding sites on RNA in yeast and E. coli  were grown in the presence of lysine and arginine or [13C6]‐lysine plus [13C6]‐arginine. Isotope‐labelled and isotope‐unlabelled cells were mixed after irradiation but prior to cell lysis. In all experiments, label swaps between crosslinked and non‐crosslinked samples were included to confirm that the labelling did not affect the outcome of the analyses . We initially irradiated with 1.4 J cmyses Fig A–C.Briefly, irradiated cells were harvested by centrifugation, resuspended in buffer containing 2 M guanidine thiocyanate plus 50% phenol, and lysed by beating with zirconia beads. The lysate was cleared by centrifugation and adjusted to pH 4 by adding 3 M sodium acetate. The cleared lysate was incubated with silica beads in batch for 60 min. The beads were extensively washed in denaturing buffer: first in buffer containing 4 M guanidine thiocyanate, 1 M sodium acetate pH 4, then in low salt buffer with 80% ethanol. The ethanol wash is expected to reduce recovery of bound DNA Avison, . Nucleic−1 cm−2), raising concerns about changes in RNA–protein interactions during this extended period of irradiation. We therefore constructed a crosslinking device  to the culture  for 3 h prior to irradiation, in addition to SILAC labelling. 4tU is rapidly incorporated into RNA as 4‐thiouridine, thus sensitizing RNA to UVA crosslinking. However, 4tU strongly absorbs UV irradiation and confers considerable UV resistance on the culture Fig D. Cells vice Fig E that deet al, In principle, silica binding can enrich both RNA‐ and DNA‐bound proteins, although UV crosslinking to dsDNA is expected to be inefficient and bound DNA should be reduced during ethanol washing of the silica beads (Angelov In the absence of UV irradiation (‐UVC), a low level of protein recovery was also visible following RNA binding to silica Fig C. These et al, MaxQuant quantitation initially failed to return a value for many peptides in TRAPP MS/MS data, predominately due to the absence of a detectable peptide in the −UV samples for the SILAC pairs Fig A–F. Thes−2 UVC in Saccharomyces cerevisiae identified 1,434 significantly enriched proteins, of which 1,360 were enriched more than twofold  for abundant proteins.UVA crosslinking in PAR‐TRAPP recovered similar number of annotated, RNA‐related proteins as TRAPP with the highest dose of UVC, while recovery of other proteins was similar to TRAPP with the lowest exposure to UVC Fig C. We assFrom these analyses, the optimal UVC exposure was unclear. Lower doses are potentially less noisy, but at the cost of recovering fewer proteins annotated functions in RNA biology, underlining the need for titration of the UV dose. As discussed below see , TRAPP iet al, −2 UVA plus 4tU, as in PAR‐TRAPP. There was a substantial overlap between proteins reported by PAR‐TRAPP and poly(A) capture techniques, and this was increased by including the results of high UVC dose TRAPP  capture  RNA isolation. We used TRAPP, as we could not identify a 4tU concentration that allowed both growth and efficient RNA–protein crosslinking. Initial crosslinking with ~1.4 J cm−2 UVC in E. coli identified 1,106 significantly enriched proteins, of which 1,089 were enriched more than twofold , while the two other subunits of the eIF2 complex, eIF2A (Sui2) and eIF2B (Sui3), were unchanged. The role of eIF2 is to escort initiator methionine tRNA to 40S subunits during translation initiation. Similar enrichment upon sorbic acid exposure was observed for the translation elongation factor and GTPase, eEF1A (Tef1), which escorts tRNAs to elongating ribosomes. However, the results may reflect alternative functions for these GTPases during stress response, involving different interactions with RNA. For example, eEF1A was implicated in tRNA export from the nucleus  enrichment can be used to select RNA‐crosslinked peptides species due to the presence of phosphate groups on RNA  columns, and the site of crosslinking was identified by tandem MS . Better results were obtained with Xi , while nuclease P1, used for RNA degradation, generates 5′ monophosphate nucleotides. This may be a result of degradation of crosslinked dinucleotides during sample preparation or mass spectrometry.As previously observed , which is active against a broad range of phosphorylated protein substrates but has limited activity towards nucleotides . We speculate that phosphorylation of these sites will predominately be associated with the RNA unbound populations of these proteins, potentially regulating association.It might be anticipated that charge repulsion would tend to block interaction between RNA and amino acids with closely spaced phosphorylation sites. Comparison of iTRAPP crosslinks with published sites of protein phosphorylation from all datasets revealed substantial enrichment for colocalization within 20 amino acids , which was robustly identified by TRAPP, but showed enrichment of less than twofold in PAR‐TRAPP. Enolase was also confidently identified as RNA binding by TRAPP in E. coli, as expected due to its presence in the RNA degradosome complex . We therefore wanted to determine whether TRAPP‐enriched factors are ENO1 gene was fused to a tripartite C‐terminal tag, His6‐TEV protease cleavage site‐protein A (HTP), and the fusion protein was expressed from endogenous PENO1. The strain expressing only Eno1‐HTP showed wild‐type growth, indicating that the fusion protein is functional. This strain was then used in CRAC analyses, alongside the non‐tagged negative control strain. In CRAC, proteins are subject to multistep affinity purification that includes two fully denaturing steps. Eno1‐HTP was efficiently crosslinked to RNA in actively growing cells using 254 nm UVC. Figure 32P] labelling of the RNA‐associated with purified Eno1, relative to the background in a non‐tagged strain. High‐throughput sequencing of cDNAs generated from RNAs bound to Eno1  . In either case, no further crosslinking can occur. Our interpretation of the difference between the TRAPP and PAR‐TRAPP data is, therefore, that 254 nm UV allows multiple chances of crosslinking during the period of irradiation, increasing the probability of capturing weak, transient interactions. In contrast, 4‐thiouridine reacts with UV only once and is therefore biased towards the recovery of stable interactions.The basis of the difference between crosslinking with 254 nm UV compared to 4‐thiouracil with UVA may lie in the properties of the nucleotides. Native nucleotides show very rapid UV radiation decay with subpicosecond lifetimes . Applying lower UV doses decreased enrichment, both for proteins with annotated links to RNA biology and the less expected proteins of intermediary metabolism. For yeast, we identified no level of UVC irradiation that was clearly optimal. Lower doses recovered fewer proteins that are not clearly linked to RNA metabolism, but many known RNA‐interacting proteins were lost, with an increased bias towards translation‐related proteins. For E. coli, the lowest dose tested (400 mJ cm−2) recovered relatively few proteins that were not previously linked to RNA metabolism. This may represent a good starting point for future analyses, given that we were unable to identify suitable conditions for PAR‐TRAPP in E. coli.Notably, there was strong conservation of RNA binding between homologous enzymes of intermediary metabolism yeast and −2 is lower than used in previously reported analyses of RNA–protein crosslinking in liquid culture. It is also notable that this dose is delivered to cells suspended in a volume of media surrounding a UVC source. It must be anticipated that for irradiation of a monolayer of cells attached to plates the effective UV dose delivered to cells will be substantially higher. Dose–response curves may therefore be necessary for future analyses of the RNA‐bound proteome using UVC.We note that 400 mJ cmet al, et al, et al, et al, et al, et al, et al, There has been substantial recent interest in the phenomenon of liquid–liquid phase separation or demixing , the RNA helicase Ded1, and the poly(A) RNA binding proteins Pub1 and Pab1. Moreover, P‐body components directly implicated in mRNA degradation also showed reduced RNA interactions, including the CCR4/NOT complex subunit Not3, decapping cofactors Pat1 and Edc3, surveillance factors, Upf3, Nmd2 and Ebs1, and the 5′ exonuclease Xrn1. In contrast, increased RNA interactions were observed for other P‐body‐associated proteins: These included the translation repressor Ssd1 and the dissociable RNAPII component Rpb7, as well as seven enzymes of intermediary metabolism, previously identified as P‐body components. Increased RNA interaction upon sorbic acid exposure was also found for a further additional 29 proteins of intermediary metabolism, 7 of which were previously implicated as parts of P‐bodies and stress granules. We speculate that stress signalling induced by sorbic acid exposure allows abundant proteins of intermediary metabolism to form more stable interactions with RNA, allowing PAR‐TRAPP detection. The enhanced RNA interactions for intermediary metabolism enzymes with RNA could be a result of protein modifications accruing on these proteins under stress. Alternatively, the cytoplasmic acidification, proposed to be involved in stress signalling  were grown at 30°C under shaking in yeast nitrogen base media (Formedium CYN0410), containing 2% w/v of D‐Glucose (Fisher G/0500/60), supplemented with Complete Supplement Mixture without tryptophan, lysine, arginine and uracil (Formedium DCS1339) and with 20 mg l−1 uracil (Sigma U0750‐100g). SILAC light media were additionally supplemented with 30 mg l−1 lysine (Sigma L5626‐100g) and 5 mg l−1 arginine (A5131‐100g), while 30 mg l−1 13C6 lysine (Cambridge Isotope Laboratories CLM‐2247‐H‐0.25) and 5 mg l−1 13C6 arginine (Cambridge Isotope Laboratories CLM‐2265‐0.5) were added to SILAC heavy media. Equal cell mass (by OD measurement) of heavy‐labelled and equal cell mass of light‐labelled cells were combined per experiment.Yeast cells  as described  crosslinking, the culture was harvested at OD−2 of UVA. Cells were harvested by filtration and frozen for later processing.For PAR‐TRAPP, using UVA (350 nm) crosslinking, cells were grown to OD 0.15, 4‐thiouracil (4tU) (Sigma 440736‐1g) was added to 0.5 mM from 1 M DMSO stock solution, and cells were allowed to grow for 3 h. Cells were harvested by filtration and resuspended in 800 ml of growth media without 4tU and placed on the irradiation tray of the eBox irradiation apparatus. The UVA lamps were pre‐warmed for 1 min, and the cells were irradiated for 38 s delivering 7.33 J cmEqual cell mass of heavy‐labelled and equal cell mass of light‐labelled cells were combined per experiment before lysis step.Escherichia coli cells  , 1 mM MgSO4. SILAC light media were additionally supplemented with 40 mg l−1 lysine (Sigma L5626‐100g) and 40 mg l−1 arginine (A5131‐100g), while 40 mg l−1 [13C6]‐lysine (Cambridge Isotope Laboratories CLM‐2247‐H‐0.25) and 40 mg l−1 [13C6]‐arginine (Cambridge Isotope Laboratories CLM‐2265‐0.5) were added to SILAC heavy media. Cells were grown to OD 0.5, diluted to 700 ml with growth media and irradiated with UVC (254 nm) using a Minitron (UVO3) . The UVA output of the eBox stabilizes after 1 min of lamp warmup. The sample is prevented from UVA exposure with sliding shutters during the warmup period, followed by shutters’ extraction and sample irradiation for 38 s.The eBox is a chamber with internal dimensions of 49.5 × 25.5 × 48 cm (W × H × D) lined with aluminium foil. The sample tray made of borosilicate glass is positioned between 2 banks of 10 PL‐L 36W/09/4P PUVA 350‐nm bulbs (Philips 871150061410040). Each bank is driven by 5 PC236TCLPRO‐TR ballasts (Tridonic 22176170). The two banks together consume 720 W of electrical power. UVA measurements were performed with Sper Scientific UVA/B Light Meter (850009), equipped with UVFS Reflective ND Filter, OD: 2.0 (THORLABS NDUV20B). Based on the spectral sensitivity of the meter, we estimate that in 38 s of irradiation time, we administer from 7.3 J cmg for 2 min and decanting the supernatant, containing beads of the smallest size. After the last wash, beads are resuspended in volume of water to achieve 50% slurry suspension.Silica beads (Fluka S5631‐500g) were resuspended in 1 M HCl and left for 24 h at room temperature. The beads were then washed with water three times by spinning in a 50‐ml falcon tube at 3,000 Escherichia coli cells pellets (140 ODs) were resuspended in 8 ml of 1:1 mix between phenol (Sigma P4557), saturated with 10 mM Tris–HCl pH 8.0, 1 mM EDTA and GTC buffer (4 M guanidine thiocyanate (Fisher BP221‐1), 50 mM Tris–HCl pH 8.0 (Invitrogen 15504‐020), 10 mM EDTA (Fisher D/0700/53), 1% beta‐mercaptoethanol), the sample was split between 8 2‐ml screwcap tubes, 1 volume of zirconia beads (BioSpec 11079105z) was added, and cells were lysed with FastPrep‐24 5 g  twice for 40 s at 6 m s−1. Lysates were recovered into 50‐ml falcon tube.Yeast mixed cell pellets were resuspended in 2 ml of phenol‐GTC buffer in a 50‐ml falcon tube, 3 ml of zirconia beads was added, and cells were vortexed for 1 min 6 times, with 1‐min incubation on ice between vortexing. 6 ml of phenol‐GTC mix was added, and lysates were vortexed for additional 1 min.g, 4°C for 5 min.Spin down samples in 50‐ml falcon tubes at 4,600 g for 10 min to clear the lysate.Recover the supernatant into 2‐ml tubes and spin at 13,000 Recover the cleared lysate into a 50‐ml falcon tube; avoid taking any pellet at this step.To the recovered lysate, add 0.1 volume of 3 M sodium acetate‐acetic acid pH 4.0, mix briefly and slowly add 1 volume of absolute ethanol, vortex to mix.Add 1 ml of 50% slurry suspension of silica in water (per 400 ODs of yeast cells), followed by 0.5 ml of ethanol to maintain 50% ethanol concentration in the mix.Load nucleic acids onto silica for 60 min on a rotating wheel at room temperature.g, 4°C for 2 min and resuspend the beads by vigorous vortexing in 15 ml of wash buffer I –acetic acid (Fisher A/0400/PB17) pH 4.0, 30% ethanol). At this point, samples can tolerate harsh mixing; therefore, the use of a FastPrep or a similar homogenizer with the corresponding 50 ml adapter is possible.Spin down the beads at 2,500 g, 4°C for 2 min, and discard the supernatant. One can use a vacuum system or pipette to remove as much of wash buffer as possible. Repeat the wash with buffer I 2 more times.Pellet the resuspended silica at 2,500 Wash the beads three times with 20 ml of wash buffer II  using the procedure described for wash buffer I.After the third wash, resuspend the beads in a small amount of wash buffer II using a vortex or a homogenizer. Transfer the resuspended silica to two 2‐ml tubes.g for 2 min at room temperature and discard the supernatant.Spin the tubes at 2,000 Dry the silica for 10 min in a SpeedVac at 45°C.g for 1 min to pellet the silica. Recover the supernatant into two new 2‐ml tubes. As the dissolved nucleic acids make the recovered supernatant viscous, some silica beads will be recovered together with the supernatant.Elute the nucleic acids three times with 500 μl per 2‐ml tube. Use vortex or a homogenizer to resuspend the silica and spin at 4,000 g for 1 min at room temperature. Transfer the supernatant into 2 fresh 2‐ml tubes and repeat the spin.Spin the recovered 2 × 1.5 ml supernatant at 14,000 Recover the supernatant into two 2‐ml protein LoBind tubes (Eppendorf 0030108132). Add 0.5 μl of RNaseA+T1 (Ambion AM2286) mix per tube and incubate the samples for 2 h in a SpeedVac at 45°C, followed by SpeedVac incubation at 65°C until the samples are fully dry. Alternatively, the sample can be left overnight in a SpeedVac at 45°C, which takes approximately 16 h to dry. If the sample is expected to have a large amount of DNA, consider adding a DNAse step or using a promiscuous nuclease such as benzonase or cyanase.The dried samples are resuspended in 75 μl Laemmli buffer per 2‐ml tube by vortexing, incubated at 100°C for 10 min. At this point, the two 2‐ml tubes are merged into one.Resolve approximately 10 μl on a gradient 4–20% Mini‐PROTEAN TGX gel (Biorad 4561095), and stain with Imperial protein stain (Thermo 24615) according to the manufacturer's protocol. The amount of sample to be loaded on a gel depends on the concentration of proteins in the sample. A good strategy is to run a test gel with a few varying loading to determine the optimal amount of sample to process.Each lane of the gel, corresponding to one SILAC mix was cut into several gel fractions. The extent of fractionation depends on the mass spectrometer. In our hands, even a single fraction can give up to 1,000 quantified proteins; however, this number grows with the number of fractions with diminishing returns.et al, −1 trypsin .De‐stain the excised fractions and digest the proteins with trypsin, as previously described  at 100 U mg−1 of DNA, supplemented with 200 U of RNAsin (Promega N211A). Treatment with Cyanase (Serva 18542.01) was performed in Tris pH 8.0 buffer supplemented with 6 mM MnSO4, using 0.1 U of enzyme per 1 μg of nucleic acids. Silver staining was performed as previously described in  was added 1:4,000 weight of RNA:weight of enzyme, and the sample was incubated at 37°C overnight to completely degrade bound RNA to nucleotide 5′ monophosphate products. Tris–HCl pH 8.0 buffer was added to 50 mM, DTT was added to 30 mM, and solid urea (Acros Organics 327380050) was added for 8 M final concentration to reduce and denature proteins. The solution was passed through a 30KDa MWCO Vivaspin Polyethersulfone concentrator , and flow through was discarded. Denaturation at this step is essential to prevent proteins smaller than 30 kDa from passing through the membrane . The pH was brought to 8.0 with 1 M Tris–HCl, and DTT was added to 2 mM, EDTA pH 8.0 to 1 mM and MnCl2 to 2 mM final concentration. To the “+phosphatase sample”, 800 units of λ phosphatase (NEB P0753S) was added and incubated for 1 h at 30°C, while the “−phosphatase sample” was mock treated.The sample was added to trypsin‐conjugated magnetic beads (Takara 635646) and incubated for 12 h with mixing at 37°C. The trypsin beads were removed from the supernatant, and the sample was diluted with water 1:1. Sodium acetate was added to 10 mM, ZnCl2 Mag Sepharose beads  according to the manufacturer's protocol. Peptides eluted from TiO2 sepharose were loaded onto C18‐StageTips as described above followed by LC‐MS analysis.TFA was added to 1%, ACN to 2.5% final concentration. Sample was loaded onto 4 C18 SPE cartridges (Empore 4215SD). Washed with 0.1% TFA and eluted in 0.1% TFA, 80% ACN. Eluates were dried in SpeedVac. Next, the sample was resuspended in 250 μl of binding buffer  with vortexing and brief sonication. Phospho‐group containing RNA‐crosslinked peptides were the captured with TiO−1 and eluted at a flow rate of 0.2 μl min−1 according to the following gradient: 2–40% mobile phase B in 136 min and then to 95% in 11 min. Mobile phase B was retained at 95% for 5 min and returned back to 2% a minute after until the end of the run (160 min). The iTRAPP analysis was performed on a QExactive mass spectrometer . The same LC conditions applied as described above.LC‐MS analyses were performed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer  coupled on‐line, to an Ultimate 3000 RSLCnano Systems . Peptides were separated on a 50 cm EASY‐Spray column , which was assembled on an EASY‐Spray source  and operated at 50°C. Mobile phase A consisted of 0.1% formic acid in LC‐MS grade water, and mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. Peptides were loaded onto the column at a flow rate of 0.3 μl minm/z) with an ion target of 4.0e5. MS2 in the ion trap at normal scan rates with ion target of 1.0E4 and HCD fragmentation , using the Andromeda search engine . Missing peptide intensity values were imputed using impute.minprob function of the imputeLCMD package version 2.0 . For S. cerevisiae PAR‐TRAPP and E. coli TRAPP data, we run impute.minprob function with the following parameters: q = 0.1 tune.sigma = 0.01. For S. cerevisiae TRAPP data, tune.sigma parameter was set to 0.0035. If in a given biological replicate a peptide was missing intensity in both + UV and −UV, the imputed values were removed. We then calculated +UV to −UV protein ratio in each experiment as median of peptides using “leading razor protein” as protein identifier. Proteins identified with at least two peptides in at least two experiments were considered for further analysis. The protein ratios were log2 transformed, and the statistical significance of the result was determined using the Limma package version 3.36.2   and the number of observed crosslinks outside the 20 residue window (Xobserved). In order to obtain the expected values, we shuffled the phosphosites using the MS excel “RANDBETWEEN” function in the crosslinked proteins and calculated the (X‐Piexpected) and (Xexpected). We performed the shuffling 100 times and calculated the average (X‐Piexpected) and (Xexpected) values, Finally, we performed a chi‐square test to identify if the observed values were significantly different from the averaged expected values.The modification sites for proteins recovered in iTRAPP were obtained from UniProt database on 12.07.2018 .We utilized The Yeast Metabolome Database  and parameters –s 1 –l 17 –r None (to ensure only uniquely mapping reads). The accompanying Novoindex file was generated using parameters –k 10 –s 1. To remove PCR duplicates that were not previously discarded during pre‐processing because of sequencing errors or differential trimming at the 3′ end, any reads with the same random nucleotides and with 5′ ends mapping to the same coordinates were collapsed into a single read (Tuck & Tollervey, et al, Downstream analyses were performed using the pyCRAC software (Webb VS and DT designed research; VS and EP performed biochemistry; CS and VS and performed and analysed MS data, CS, VS and LF performed bioinformatics; and VS, SB, CS, JR and DT analysed data and wrote the paper.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Dataset EV6Click here for additional data file.Dataset EV7Click here for additional data file.Dataset EV8Click here for additional data file.Dataset EV9Click here for additional data file.Dataset EV10Click here for additional data file.Review Process FileClick here for additional data file.