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Sep 2020

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Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
基于荧光的锥虫体体外尿嘧啶插入或删除RNA编辑(FIDE)   

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Abstract

Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is catalyzed by a 0.8 MDa multiprotein complex termed the editosome. Here we describe an improved in vitro test to quantitatively explore the catalytic activity of the editosome. The assay uses synthetic, fluorophore-derivatized oligoribonucleotides as editing substrates, which enable the automated electrophoretic separation of the reaction products by capillary electrophoresis (CE) coupled to laser-induced fluorescence (LIF) detection systems. The assay is robust, it requires only nanogram amounts of materials and by using multicapillary CE/LIF-instruments it can be executed in a highly parallel layout. Further improvements include the usage of phosphorothioate-modified and thus RNase-resistant substrate RNAs as well as multiplex-type fluorophore labeling strategies to monitor the U-insertion and U-deletion reaction simultaneously. The assay is useful for investigating the mechanism and enzymology of the editosome. However, it can also be executed in high-throughput to screen for RNA editing-specific inhibitors.


Graphic abstract:



Characteristics of the fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE) assay


Keywords: RNA-editing (RNA编辑), U-insertion/U-deletion editing (尿嘧啶插入或删除), Editosome (编辑体), Guide RNA (向导RNA), RNA-processing (RNA加工), Mitochondrial gene expression (线粒体基因表达), African trypanosomes (非洲锥虫), Protozoan parasites (寄生性原生虫病)

Background

The RNA editing reaction in kinetoplastid protozoa such as African trypanosomes and Leishmania represents one the most striking posttranscriptional modifications of messenger (m)RNAs (reviewed in Göringer, 2012a). The reaction takes place within the single mitochondria of the organisms, which is similar to other eukaryotes and holds the genetic information for components of the mitochondrial translation apparatus and subunits of the electron transport and chemiosmosis systems. However, unlike in other organisms, more than 50% of these genes are “incompletely” encoded: substantial sequence information is missing – in some cases more than 50% – in addition to the absence of start and stop codons and the presence of frame-shifts. As a consequence, precursor (pre)-mRNAs from these genes must be corrected, i.e., "edited" to generate functional mRNAs. Biochemically, the editing reaction is characterized by the insertion and/or deletion of U-nucleotides. In African trypanosomes, more than 3000 U-residues are inserted and about 300 U’s deleted at hundreds of editing sites in 12 different pre-mRNAs. The process relies on small, non-coding RNAs, known as guide (g)RNAs (Blum et al., 1990; Hermann et al., 1995). Guide RNAs are trans-acting genetic elements. They initiate the reaction cycle by antiparallel base pairing to the pre-edited mRNAs, which results in the formation of a gRNA/pre-mRNA hybrid. The hybrid-RNA has a 3-helix junction-topology including a so-called “anchor” – helix that borders the sequence to be edited. Unpaired gRNA nucleotides next to the anchor-helix specify U-insertion events, non-base paired uridylates in the pre-mRNA become deleted. The mitochondrial machinery that executes the processing reaction is a high molecular mass protein complex. In analogy to the ribosome and spliceosome it has been named the editosome (reviewed in Göringer, 2012b). Editosomes function as a catalytic platform for all steps of the reaction cycle and execute the processing reaction in a cascade of enzyme-driven steps. This includes endo- and exonucleases, a terminal uridylyltransferase (TUTase), RNA-ligases as well as RNA-chaperone activities.


The basal steps of the editing reaction cycle have been unraveled with the help of an in vitro RNA-editing assay (Seiwert and Stuart, 1994; Kable et al., 1996; Igo et al., 2000; Igo et al., 2002). The protocol represents a chemically simplified experimental system in which both, the substrate pre-edited mRNAs as well as the trans-acting gRNAs are mimicked by short RNA-oligonucleotides. Using purified editosome preparations, the pre-mRNA oligonucleotides are edited in a gRNA-dependent manner either by inserting 3 U-nt or deleting 4 U's. Importantly, in order to sidestep the rate-limiting endonucleolytic cleavage of the pre-mRNAs, the corresponding oligoribonucleotides are "pre-cleaved", i.e., they are provided as 5'- and 3'-pre-mRNA cleavage (Cl)-fragments. The 5'-Cl oligoribonucleotides are typically 5'-(32P)-labeled to enable a radioactive readout of the in vitro reaction.


Here we describe a new RNA-editing assay (Del Campo et al. 2020). The test system is a revised version of the established in vitro RNA-editing assays (Stuart et al., 1998), however, instead of using radioactively labeled substrate-RNAs it utilizes fluorophore-derivatized RNA-oligonucleotides. This allows the usage of automated, highly parallelized capillary electrophoresis (CE)-instruments coupled to laser-induced fluorescence (LIF) detection systems. It also enables multiplex-type fluorophore-labeling experiments to monitor multiple RNAs in one experiment. Furthermore, we designed new sets of pre-mRNA and gRNA-mimicking oligonucleotides. The different RNAs represent synthetic, non-natural sequences not found in the mitochondrial genomes of kinetoplastid organisms and they are, aside from the editing site, identical for both the U-insertion and the U-deletion reaction. This avoids potential sequence context issues and enables the side-by-side monitoring of both editing reactions in one reaction vial. Furthermore, we designed phosphorothioate (PS)-modified versions of the substrate RNAs. Phosphorothioate internucleotide linkages increase the RNase-stability of the pre-mRNA and gRNA-mimicking oligonucleotides, which permits the usage of editosome-enriched mitochondrial protein extracts instead of highly purified editosome preparations. Lastly, we optimized the work flow of the assay by adjusting the material quantities and reaction volume to multiwell-plate formats. The assay satisfies all signal-to-noise criteria for high-throughput screening methods (Del Campo et al., 2020) and is able to derive quantitative data for every step of the catalytic reaction cycle. As such the protocol should be helpful in the search for RNA-editing specific inhibitory compounds. The general workflow of the Fluorescence-based Insertion/Deletion Editing (FIDE)-assay is summarized in Figure 1.



Figure 1. Workflow of the Fluorescence-based Insertion/Deletion Editing = FIDE-assay. Central steps of the protocol are on the left and boxed. Quality assessment steps and machine read-outs are illustrated on the right.


Materials and Reagents

All reagents, consumables and equipment are to be viewed as possible options. Unless stated otherwise, chemicals and consumables are stored at room temperature.

  1. Solvents and Reagents

    1. MeOH ≥99% p.a. (CARL ROTH, catalog number: 8388)

    2. EtOH ROTIPURAN® ≥99.8%, p.a. (CARL ROTH, catalog number: 9065)

    3. CHCl3 ≥99%, p.a (CARL ROTH, catalog number: 3313)

    4. Roti®-Phenol (TE-buffer equilibrated, pH 7.5-8.0) (CARL ROTH, catalog number: 0038, store at 4 °C)

    5. Isoamyl alcohol (3-Methyl-1-butanol), p.a. (Merck, catalog number: 100979)

    6. Glycerol ≥99.7%, p.a. (CARL ROTH, catalog number: 6962, store at 4 °C)

    7. Bradford assay reagent – detergent compatible (ThermoFisher Scientific, catalog number: 23246S, store at 4°C)

    8. Phenol/Chloroform/Isoamylalcohol (PCI) (see Recipes, store at 4 °C)


  2. Acids and Bases

    1. KOH ≥85%, p.a. (CARL ROTH, catalog number: 6751)

    2. HOAc 100%, p.a. (CARL ROTH, catalog number: 3738)

    3. HCl ≥25%, p.a. (CARL ROTH, catalog number: 6331)

    4. B(OH)3 ≥99.8%, p.a. (PanReac AppliChem, catalog number: 131015)


  3. Buffer Compounds and Salts

    1. Tris-(hydroxymethyl)-amino methane (TRIS) PUFFERAN® (CARL ROTH, catalog number: 4855)

    2. N-2-Hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES) PUFFERAN® ≥99.5%, p.a. (CARL ROTH, catalog number: 9105)

    3. Imidazole PUFFERAN® ≥99%, p.a. (CARL ROTH, catalog number: X998)

    4. KCl (CARL ROTH, catalog number: 6781)

    5. MgCl2·6H2O (CARL ROTH, catalog number: 2189)

    6. NaCl (CARL ROTH, catalog number: 3957)


  4. Reducing and Chelating reagents

    1. 1,4-Dithiothreitol (DTT) ≥99%, p.a. (CARL ROTH, catalog number: 6908, store at 4 °C)

    2. Tris (2-carboxyethyl)phosphine hydrochloride (TCEP) ≥98% (Sigma-Aldrich, catalog number: C4706, store at 4 °C)

    3. Na2-ethylenediaminetetraacetate (Na2EDTA)·2H2O (CARL ROTH, catalog number: 8043)

    4. Ethylene glycol bis (β-aminoethylether) tetraacetic acid (EGTA) ≥99%, p.a. (CARL ROTH, catalog number: 3054)


  5. Ribonucleotides

    1. Adenosine-5'-triphosphate (ATP), 100 mM, pH 8.0 (CARL ROTH, catalog number: K045, store at -20 °C)

    2. Uridine-5'-triphosphate (UTP), 100 mM, pH 8.0 (CARL ROTH, catalog number: K046, store at -20 °C)


  6. Gel Electrophoresis Consumables

    1. Acrylamide/Bisacrylamide (19:1) Rotiphorese®Gel 40 (CARL ROTH, catalog number: 3030, store at 4 °C)

    2. Urea (CARL ROTH, catalog number: 2317)

    3. Ammonium peroxydisulphate (CARL ROTH, catalog number: 9592)

    4. N,N,N',N'-Tetramethylethylendiamine (CARL ROTH, catalog number: 2367)

    5. Bromophenol Blue, Na+-salt (Merck, catalog number: 108122)

    6. Xylene cyanol FF, Na+-salt (Sigma-Aldrich, catalog number: X4126)

    7. Toluidine Blue O (Sigma-Aldrich, catalog number: T3260)

    8. 6× Native PAGE gel-loading buffer (see Recipes)

    9. 2× Denaturing PAGE gel-loading buffer (see Recipes)

    10. TBE buffer, pH 8.3 (see Recipes)

    11. Gel staining solution (see Recipes)

    12. Gel destaining solution (see Recipes)


  7. Capillary Electrophoresis Consumables (see Note 1)

    1. Glass capillary (Polymicro Technologies, catalog number: 1068150017)

    2. Performance optimized Polymer 6 (POP-6TM) (ThermoFisher Scientific, catalog number: 402837, store at 4 °C)

    3. 10× CE/LIF-buffer (Sigma-Aldrich, catalog number: B4930, store at 4 °C)

    4. H2O LiChrosolv® (Merck Millipore, catalog number: 115333)

    5. Highly deionized formamide (Hi-DiTM Formamide) (ThermoFisher Scientific, catalog number: 4311320, store at -20 °C)


  8. Editosomes and RNA-editing reaction mix

    1. Affinity-purified editosomes in 10 mM HEPES/KOH, pH 7.5, 1 mM Mg(OAc)2, 75 mM NaCl, 0.1% (w/v) NP-40, 0.1 mM TCEP, 1 mM imidazole and 10 mM EGTA, 20% (v/v) glycerol (see Note 2, store at -20 °C)

      Alternatively for HTS-applications: Editosome-enriched Trypanosoma brucei mitochondrial protein extracts in 20 mM HEPES/KOH, pH 7.5, 10 mM Mg(OAc)2, 30 mM KCl, 0.5 mM DTT, 20% (v/v) glycerol (see Note 2, store at -20 °C)

    2. 3x RNA-editing reaction mix (see Recipes, store at -20 °C)


  9. Oligoribonucleotides (see Notes 3 and 4)

    Standard-FIDE assay:

    U-insertion

    1. 5’-FAM-synCl14ins: FAM-(CH2)6-AAAGGAAAUAUAGU

    2. 3'-synCl16: pAGGUGAUUCCAUUGAG-(CH2)6-NH2

    3. syn-gRNAins: CUCAAUGGAAUCACCUAAAACUAUAUUUCCUUU

    U-deletion

    1. 5'-FAM-synCl17del: FAM-(CH2)6-AAAGGAAAUAUAGUUUU

    2. 3'-synCl16: pAGGUGAUUCCAUUGAG-(CH2)6-NH2

    3. syn-gRNAdel: CUCAAUGGAAUCACCUACUAUAUUUCCUUU


    Standard-FIDE assay – phosphorothiate (PS)-modified (*) RNAs:

    U-insertion

    1. 5’-FAM_modCl14ins: FAM-(CH2)6-AAAGGAAAU*A*U*A*GU

    2. 3’-modCl16: pAG*G*U*GAUUCCAUUGAG-(CH2)6-NH2

    3. modgRNAins: C*U*C*AAUGGAAUCACCUAAAACUAUAUUUCC*U*U*U

    U-deletion

    1. 5’-FAM_modCl17del: FAM-(CH2)6-AAAGGAAAU*A*U*A*GUUUU

    2. 3’-modCl16: pAG*G*U*GAUUCCAUUGAG-(CH2)6-NH2

    3. modgRNAdel: C*U*C*AAUGGAAUCACCUACUAUAUUUCC*U*U*U


    Multiplex ("one-pot") U-insertion/U-deletion assay:

    U-insertion

    1. 5'-TAMRA_Cl18: TAMRA-(CH2)6-GGAAGUAUGAGACGUAGG

    2. 3'-Cl13: pAUUGGAGUUAUAG-(CH2)6-NH2

    3. gRNAins: CUAUAACUCCGAUAAACCUACGUCUCAUACUUCC

    U-deletion

    1. 5’-FAM_Cl22: FAM-(CH2)6-GGAAAGGGAAAGUUGUGAUUUU

    2. 3'-Cl15: pGCGAGUUAUAGAAUA-(CH2)6-NH2

    3. gRNAdel: GGUUCUAUAACUCGCUCACAACUUUCCCUUUCC


    Dual fluorophore assay:

    U-insertion

    1. 5'-TAMRA_Cl18: TAMRA-(CH2)6-GGAAGUAUGAGACGUAGG

    2. 3'-Cl13_FAM: pAUUGGAGUUAUAG-(CH2)6-FAM

    3. gRNAins: CUAUAACUCCGAUAAACCUACGUCUCAUACUUCC

    U-deletion

    1. 5’-FAM_Cl22: FAM-(CH2)6-GGAAAGGGAAAGUUGUGAUUUU

    2. 3’-Cl15_TAMRA: pGCGAGUUAUAGAAUA-(CH2)6-TAMRA

    3. gRNAdel: GGUUCUAUAACUCGCUCACAACUUUCCCUUUCC


    RNA-size standards:

    1. 3'-FAM_13: AUUGGAGUUAUAG-(CH2)6-FAM

    2. 5'-FAM_22: FAM-(CH2)6-ggaaagggaaaguugugauuuu

    3. 5'-FAM_37: FAM-(CH2)6-ggaaagggaaaguugugauuuugcgaguuauagaaua

    4. 5’-FAM_44: FAM-(CH2)6-CUAGUACUCUCAUCAACAUAAGUCUCAUACUUCCGACAUGCA-CG

    Reaction vials:

    1. Thin-walled Polypropylene Reaction Tubes 0.2 ml (VWR peqlab, catalog number: 732-3215)

    2. Thin-walled Polypropylene Reaction Tubes 0.5 ml (VWR peqlab, catalog number: 732-3207)

    3. Polypropylene Reaction Tubes 1.5 ml (Sarstedt, catalog number: 72.706)

Equipment

  1. Spectrophotometer (Thermo Scientific, model: NanoDrop 2000C)

  2. Thermocycler (Biometra, T3)

  3. CE/LIF-based DNA-sequencer (Applied Biosystems, model: ABI PRISM 310 Genetic analyzer)

  4. Benchtop Centrifuge (Eppendorf, model: 5417 R)

  5. SpeedVac Vacuum Concentrator (Savant Instruments, model: SVC-100)

  6. Dry-Block Heating System (Grant, model: QBD2)

  7. Vortex Mixer (Scientific Instruments, model: Vortex Genie 2)

  8. DC-High Voltage Power Supply (Biometra, model: P30)

  9. Gel-Electrophoresis Chamber (BIO-RAD, model: Mini-PROTEAN® Tetra Cell)

  10. Micropipettes (Gilson, models: Pipetman P10, P20, P200, P1000)

  11. Gel Documentation System (INTAS, model: Gel iX20 Imager)

Software

  1. R (The R Project for Statistical Computing, https://www.r-project.org/)

  2. Programmer-type text editor (such as Vim, https://www.vim.org or Atom, https://atom.io)

Procedure

  1. gRNA/pre-mRNA substrate formation

    Two thin-walled 0.5 ml polypropylene reaction tubes are used to separately assemble the trimeric U-insertion and U-deletion gRNA/pre-mRNA substrate RNAs. The final gRNA/pre-mRNA concentration is 10 nM.

    1. Combine the 3'-pre-mRNA Cl-fragment, the fluorescently labeled 5'-pre-mRNA Cl-fragment and the corresponding gRNA oligoribonucleotide at a final concentration of 30 nM each in TE buffer (pH 7.5). You will need 10 µl of gRNA/pre-mRNA substrate for a 30 µl editing reaction.

    2. Mix by vortexing (5 s) and collect the content of the tubes by brief centrifugation (10 s quick-spin).

    3. Thermally unfold the oligoribonucleotides for 2 min at 75 °C and anneal the 3 RNAs by cooling to 27 °C with a rate of 0.08 °C/s.

    Note: The formation of the gRNA/pre-mRNA hybrid must be verified in a separate analysis (see Note 5). The yield of the trimeric RNA-hybrid should be ≥90%.


  2. RNA-editing reaction and sample clean-up

    1. Add an equal volume of 3× editing reaction-mix to the annealed gRNA/pre-mRNA substrate from Step 1c.

    2. Aliquot 20 µl of this pre-mix into thin-walled 0.2 ml polypropylene reaction tubes.

    3. Adjust the volume with ddH2O, so that the final volume, including the volume of editosomes (to be added later), will be 30 µl.

    4. Mix by vortexing (5 s) and collect the content of the tubes by brief centrifugation (10 s quick-spin).

    5. Add editosomes and mix by gently pipetting up and down (see Note 6).

    6. Incubate at 27 °C for 30 min to 3 h (see Note 6).

    7. Transfer samples to 1.5 ml reaction tubes and terminate the reaction by adding 17 µl ddH2O, 3 µl 125 mM Na2EDTA and 50 µl PCI. Mix vigorously.

    8. Spin for 5 min at >15,000 × g at 4 °C.

    9. Transfer the H2O-phase to a new 1.5 ml reaction tube. Add 5 µl 3 M NaOAc/HOAc (pH 4.8) and 121 µl 100% (v/v) cold (-20 °C) EtOH. Mix and immediately spin at 4 °C for 30 min at >15,000 × g to pellet the RNA.

    10. Aspirate the supernatant and add 0.75 ml 70% (v/v) cold (-20 °C) EtOH.

    11. Wash RNA-pellets by vortexing and spin for 5 min at 4 °C at >15,000 × g.

    12. Repeat Steps 2j-2k.

    13. Remove any liquid by aspiration and dry RNA-pellets for 2 min in vacuo.

    14. Dissolve RNA-pellets in 15 µl Hi-DiTM formamide. Collect the contents of the tube by brief centrifugation (10 s quick-spin).

    15. Denature the RNA-samples by incubation at 95 °C for 2 min. Snap-cool on ice.


  3. CE/LIF-separation

    1. Transfer 3 µl of the samples to 0.5 ml reaction tubes (see Note 13).

    2. Add 4 fmol of each of the 4 size-standard RNA oligonucleotides.

    3. Add Hi-DiTM formamide to a final volume of 20 µl. Mix and spin down.

    4. Cut off the lids and place the tubes into the autosampler of the CE/LIF-instrument.

    5. Run capillary electrophoresis

      On an CE/LIF-ABI PRISM® 310 DNA-Sequencer with a 50 cm glass capillary use the following settings: ModuleSeq POP6 (1 ml); laser power: 7 mW; injection voltage: 2.5 kV; run voltage: 12.2 kV; sample injection time: 10 s (see Note 7); run time: 25 min.

    6. After completion of the CE/LIF-run, inspect the raw electropherograms. Since only non-saturated CE/LIF-traces can be quantitatively analyzed it may be necessary to re-run the samples at a higher dilution.

Data analysis

Output of the CE/LIF-separation is an unprocessed electropherogram, which is retrieved as a binary data file. For extracting the electropherogram data points and for downstream processing, we use the programming language R. R is a free software for statistical computing and graphics and it compiles and runs on UNIX, macOS and Windows operating systems.

  1. Open R.

    Commands can directly be typed into the R-console. By pressing ENTER/RETURN, the command will be executed. However, for more elaborate procedures, it is more convenient to type all commands into a text editor or the in-built editor of R (see Note 8) followed by copying the code to the R-console.

    OS X: File → New document

    Windows: File → New script

  2. Install Bioconductor and the R packages “pracma” and “sangerseqR”.

    Bioconductor provides the package “sangerseqR”, which is used to extract the fluorescence signal intensities (FI) in relative fluorescence units (RFU) from the binary sequencing files. The package “pracma” provides the functions “peakfind”. “pracma” is not included in the basic R-package collection.


    if (!requireNamespace("BiocManager", quietly = TRUE))

        install.packages("BiocManager")

    BiocManager::install(version = "3.11")

    install.packages("pracma")

    install.packages("sangerseqR")


  3. Create a folder for your FIDE-experiment as a working directory.

  4. Move the CE/LIF-binary data files to this folder.

  5. Set your experiment folder as working directory for the current R-session. All files generated during the active session will then be saved there.


    setwd("/folder1/
    folder2/…/myExperimentFolder")

  6. Upload all packages required for the downstream analysis.


    library("pracma")

    library("sangerseqR")


  7. The binary CE/LIF-files are uploaded to the R-workspace using the function “read.abif” from the “sangerseqR” package provided within Bioconductor. They are converted to an abif class object, from which the CE/LIF-raw data (i.e., relative fluorescence units (RFU)) are extracted.


    datafile <- read.abif("myAbiFile")


    ceData <- data.frame(datafile@data$DATA.1, datafile@data$DATA.2,

    datafile@data$DATA.3, datafile@data$DATA.4)


  8. Analyze and process the CE/LIF-raw data.

    1. Visualize the electropherogram.


      plot(ceData[,1], type = "l", col = "blue", xlab = "EM", ylab = "FI/RFU", main = "raw data")

      lines(ceData[,2], col = "green")

      lines(ceData[,3], col = "orange")

      lines(ceData[,4], col = "darkred")

      legend("topleft", c("1", "2", "3", "4"), fill=c("blue", "green", "orange", "darkred"))


    2. Select the appropriate trace and pass the channel number to the R-environment.


      trace <- traceNumberForAnalysis (Note: The argument is a true integer)


    3. Perform baseline correction (see Note 9).


      dataBg <- ceData[,trace] - median(ceData [500:2000,trace])


    4. Identify peaks (see Note 10).


      peaks <- data.frame(findpeaks(dataBg, threshold = 50, minpeakheight = 100, zero = "-"))

      colnames(peaks) <-c("height", "max", "start", "end")


    5. Integrate peak areas.


      area <-c()

      for(i in 1 : nrow(peaks)) {

      start <- peaks [i,3]

      stop <- peaks [i,4]-1

      sum <- sum(dataBg [start:stop])

      area <-round(c(area,sum),0)

      }

      results <-data.frame(peaks, area)


    6. View the results in the R-console.


      results


    7. Write results to file.


      myOutName <- "myNameForResults"

      write.table(data.frame(num=rownames(results), results), file = paste (myOutName, ".txt", sep = ""), append = F, sep = "\t", dec = ".", row.names = F, col.names = T, quote = F)


    8. Visualize the peaks in the CE-profile (Figure 2).


      plot(peaks[,2],peaks[,1],type="h",col="red", xlim = c(1000, 1700),

      ylim = c(0,max(dataBg)+200), xlab = "EM", ylab="FI/RFU")

      text(peaks[,2],peaks[,1]+150, rownames(peaks), cex = 1)

      lines(dataBg,type="l",col="black")


    9. Save plot to file.


      pdf(file = paste(myOutName, ".pdf", sep=""))

      plot(peaks[,2],peaks[,1],type="h",col="red", xlim = c(1000, 1700),

      ylim = c(0,max(dataBg)+200), xlab = "EM", ylab="FI/RFU")

      text(peaks[,2],peaks[,1]+150, rownames(peaks), cex = 1)

      lines(dataBg,type="l",col="black")

      dev.off()



    Figure 2. R-based data analysis output. A. Table representation of all peaks of the processed data specifying height, maximum (max), start, end and peak areas. B. Plot of the electropherogram (FI=f(EM)). FI=Fluorescence Intensity; RFU=Relative Fluorescence Unit; EM=Electrophoretic Migration (see Note 11).


  9. Peak assignment.

    The assignment of the different peaks of the electropherogram is performed with the help of fluorophore-modified size-standard oligoribonucleotides (see Materials and Reagents, subitem I. Oligoribonucleotides). We recommend to use four FAM-derivatized RNA-oligonucleotides with a nucleotide-length of 13 nt, 22 nt, 37 nt and 44 nt. Small amounts (4 fmol each) of the four RNAs can be spiked into the samples and are used to extrapolate the nt-length of every RNA-species. As shown in Figure 3A, the electrophoretic migration of marker oligoribonucleotides is linear over a wide separation range. Figure 3B shows a typical example of a "spiked-in" FIDE U-deletion assay and Figure 3C shows representative FIDE-results for both, the U-insertion and the U-deletion assay in which the identity and nt-length of every RNA-species is specified.


  10. Calculate editing activity.

    Several possibilities exist to define and calculate the RNA-editing activity in order to compare different samples (see Note 12):

    1. Ratio of the peak area of the fully edited RNA-product over the sum of all assigned peaks.

    2. Peak area ratio of all ligated RNA-products over all assigned peaks.

    3. Peak area ratio of all processed RNA-species compared to all assigned peaks.

    4. Peak area ratio of all processed RNA-species compared to the area of the RNA-input peak.



    Figure 3. Representative FIDE-results. A. Top: Electropherogram of a CE/LIF-separation of 10 size-standard oligoribonucleotides ranging from 13 nt to 44 nt. Bottom: Plot of the molecular weight (MW) of the marker RNA-oligonucleotides in relation to their electrophoretic mobility (EM). A linear fit of the data points results in an R-squared (r2) of 0.995. B. Representative electropherogram of a FIDE U-deletion assay (green trace) with "spiked-in" marker RNA-oligonucleotides 13 nt, 22 nt, 37 nt and 44 nt in length (black trace). C. Typical electropherograms of FIDE U-insertion (left) and U-deletion (right) assays. The individual peaks are assigned as: RNA-input, reaction intermediates, partially edited RNAs, fully edited RNA-product and by-products. Numbers specify the nt-length of the different RNA-species. FI=Fluorescence Intensity. RFU = Relative Fluorescence Unit.


Modifications to the standard FIDE-protocol

  1. High-throughput FIDE

    For high-throughput screening (HTS)-applications FIDE can be downscaled to adapt the assay to multiwell plates. Lower limit for both editing reactions are roughly 5-10 fmol of each, editosomes and fluorophore-modified pre-mRNA/gRNA-hybrid in a reaction volume ≤5 µl. Because of the small amounts of protein and RNA in the samples, reactions can be stopped by directly adding Hi-DiTM formamide. This conveniently eliminates the phenol extraction and EtOH-precipitation steps prior to the CE/LIF-separation. Furthermore, FIDE can be performed with only partially purified mitochondrial extracts as editosome source. Due to the presence of RNases this requires the usage of phosphorothioate (PS)-modified and thus RNase-resistant substrate oligoribonucleotides as specified in the Materials and Reagents section.

  2. "One-pot" U-insertion and U-deletion editing – "matrixing"

    A notable advantage of FIDE is the possibility to monitor the U-insertion and U-deletion reaction simultaneously. This can simply be done by using different fluorophores for the two substrate RNAs (see Materials and Reagents, I. Oligoribonucleotides). However, since the emission spectra of fluorescent dyes usually spread into multiple channels of the CE/LIF-detector an additional data processing-step is required. This procedure is known as "matrixing" (Giddings et al., 1993; Giddings et al., 1998). For that the two fluorescently labeled oligoribonucleotides must be analyzed in separate, i.e., singleplex CE/LIF-runs (see Note 13). After baseline correction of the raw data, the fluorescence intensity (FI) of both fluorophores in every detector channel (i-n) is calculated as: FIchannel (i)/ΣFIchannel (i-n). The matrix can conveniently be created from the binary CE/LIF-files using the R-script “matrix.fun” provided in the Supplementary Material section. The function is loaded into the workspace using the command:


    source("pathToScript/matrix.fun.R")


    The mandatory argument is a character vector with the names of the singleplex CE/LIF-files:


    myMatrix <- matrix.fun(c("myFileDye1", "myFileDye2"))


    The matrix is stored as a tab-delimited text and can be used for all multiplex FIDE-assays run on the same CE/LIF-device.


    Data analysis for multiplex FIDE-assays

    1. Matrixing

      1. Read the binary CE/LIF-files to R and extract the raw data as described in: Data analysis, 1-7.

      2. Upload the matrix file to the workspace, if the matrix has been created in another R-environment.


        myMatrix <- data.matrix(read.delim("pathAndNameOfMatrixFile", header=FALSE))


      3. Perform baseline correction.


        for (i in 1:ncol(ceData)) {

        ceData [,i] <- ceData[,i] - median(ceData[500:2000,i])

        }


      4. Matrixing and correction for negative values.


        for (i in 1:nrow(ceData)) {

        ceData [i,] = data.matrix(ceData)[i,]%*%t(solve(myMatrix))

        }

        ceData <- replace(ceData, ceData < 0, 0)


    2. Peak detection and quantification

      U-deletion and U-insertion assays are analyzed separately from the corresponding traces as described for the singleplex FIDE/CE-assay (see Data analysis 8, d-i) after passing the trace number to R.


      trace <- traceNumberForAnalysis Dye channel number for U-deletion or U-insertion assay, respectively. (Note: The argument is a true integer).


      dataBg <- ceData[,trace]


      Continue as described in: Data analysis, 8, d-i.


  3. Dual-fluorophore FIDE

    By using a 5'-fluorophore-derivatized 5'-mRNA oligonucleotide and at the same time a 3'-fluorophore labelled 3'-mRNA oligonucleotide, it is possible to monitor the reaction pathway of both pre-mRNA-fragments in one experiment. This can be done for the U-insertion and the U-deletion reaction alike and the corresponding oligoribonucleotides are listed in the Materials and Reagents section. In this case the sequences of the different RNA-oligonucleotides are derived from the pre-mRNA encoding subunit 6 of the mitochondrial ATP synthase (A6) from Trypanosoma brucei. Similarly, the reaction pathway of the gRNA can be traced by using a 5'-fluorophore-modified gRNA-oligoribonucleotide.

Notes

  1. For the CE/LIF-instrument we exclusively use reagents of the highest chemical grade. For instance, we use LiChrosolv®-H2O for cleaning purposes and for diluting the CE/LIF-buffer. Highly deionized formamide (Hi-DiTM formamide) is used for sample preparations.

  2. Editosomes cannot be purchased commercially. They need to be purified from insect-stage African trypanosomes (Trypanosoma brucei) grown in axenic cell culture (Brun and Schönenberger, 1979). The complexes are either purified by tandem-affinity purification (TAP) using transgenic T. brucei cell lines that conditionally express TAP-tagged variants of editosomal proteins (Golas et al., 2009; Gerace and Moazed, 2015) or from mitochondrial detergent extracts of wildtype trypanosomes (Böhm et al., 2012). Typically, 5 L of mid-log phase T. brucei cell culture (5 × 1010-8 × 1010 cells) yield about 50 µg of TAP-purified editosomes. The same number of cells yield about 0.5 mg of editing-active mitochondrial protein extract. Protein concentrations are determined by Coomassie Blue G-250 binding (Bradford assay [Bradford, 1976]).

  3. RNA-oligonucleotides are synthesized by solid-phase chemical synthesis on controlled pore glass (CPG)-beads using 2'-O-(tert-butyl)dimethylsilyl (TBDMS)-protected phosphoramidites. A 200 nmol synthesis scale typically yields ≥200 µg of pure oligoribonucleotide. RNA-oligonucleotides should be high-performance liquid chromatography (HPLC)-purified either by reversed phase or anion-exchange HPLC and verified by mass-spectrometry. We further recommend to scrutinize the purity of the synthesized RNAs in 8 M urea-containing polyacrylamide gels. Fluorophore modifications are introduced at the 5′- or 3’-termini of the different oligoribonucleotides. This can be done co- or post-synthetically. Internal fluorophore labelling strategies should be avoided to uphold the base pairing capacity of the RNA-molecules. We also recommend the introduction of linker sequences such as hexamethylene linkers between the fluorophore and the RNA to minimize sterical clashes with the nucleobases. Numerous fluorophores can be used as substituents. The specific choice depends on the wavelength specification of the laser-induced fluorescence (LIF)-detection system. Examples include 5-Carboxytetramethyl-rhodamine (TAMRA), 6-Hexachloro-fluorescein (HEX), 6-Carboxy-fluorescein (FAM), the Sulfo-cyanine dyes Cy5 and Cy5.5 as well as the Infrared fluorescent dye IRDye800 and WellRedD1. Additional modifications can be introduced such as phosphorothioate backbone modifications to increase the RNase-stability of the different RNAs. Exonucleolytic degradations from the 3'-end can be counteracted by introducing a 3'-terminal hexamethylene-amino-linker. Oligoribonucleotides representing 3'-pre-mRNA fragments require the introduction of a 5'-phosphate group using T4-polynucleotide kinase (T4-PNK) and ATP.

  4. RNA-concentration are calculated from UV-absorbency measurements at a wavelength of 260 nm (A260). Molar extinction coefficients (ϵ) for the different RNAs can be derived from nearest neighbor calculations using the OligoAnalyzer® tool at: www.idtna.com/scitools. Measurements can be performed with the help of NanoDrop-type UV-spectrophotometer instruments. Oligoribonucleotide concentrations ≥0.2 µg/µl and ≤2.5 µg/µl are well within the dynamic linear range of the instruments generating reliable measurements without the need for more sensitive, reporter fluorophore-based methods. Oligoribonucleotides are stored in TE buffer (pH 7.5) (see Recipes) at -20 °C at a concentration of 100 µM.

  5. In a separate set-up, it is necessary to confirm and quantify the formation/yield of the trimeric gRNA/pre-mRNA hybrid RNAs. For that combine 0.4 nmol of the fluorescently labeled 5‘-pre-mRNA Cl-oligoribonucleotides, the 3’-pre-mRNA Cl-oligoribonucleotides and the corresponding gRNAs. Thermally unfold and anneal the samples as described above (Procedure 1c). Add 6× Native PAGE gel-loading buffer containing 2 mM MgCl2 and separate the samples in 18% (w/v) polyacrylamide gels (gel dimensions: 7.2 cm × 8.3 cm × 0.1 cm). Run gels in TBE (pH 8.3), 2 mM MgCl2 at 4 °C and 8 W (constant) until the bromophenol blue marker has reached the bottom of the gel. As controls load all 3 oligoribonucleotides individually and optionally all bimolecular combinations of the RNA-oligonucleotides. Stain/destain the gels (recipes 2 and 3) and determine the degree of gRNA/pre-mRNA hybrid formation by densitometry. The majority of stained material (≥90%) should appear in the band with the lowest electrophoretic mobility, which represents the trimeric gRNA/pre-mRNA hybrid. Monomeric oligoribonucleotides should be negligible. A typical result is shown in Figure 4.

  6. The required amount of editosomes and the incubation time will depend on the specific editing activity (EA) (EA/protein mass) of the editosome preparation. In our hands, 50 ng of TAP-purified editosomes typically yield ≥20% of fully edited product within 60 min in the FIDE U-deletion assay.

  7. Prolonged injection times can result in peak anomalies/artifacts (Johansson et al., 2003).

  8. Use a code/programmer-type text editor and be aware that R is case-sensitive.

  9. The data region used for background correction is adapted to the CE/LIF-ABI PRISM® 310 DNA-Sequencer with a 50 cm glass capillary and may be adjusted for other CE/LIF-instruments.

  10. The settings for "threshold" and "minpeakheight" will influence the sensitivity of peak detection.

  11. The commands can be combined to enable the processing of multiple files in batch. A script is provided upon request from the authors.

  12. For methods I-III, the ratios correlate linearly with the editing activity, if less than 50% of substrate is processed. Method IV displays a higher dynamic range, but there is no linear correlation to the editing activity. Methods III and IV do not account for ligation activity, i.e., editosome preparation lacking ligase activity will be considered as active.

  13. It is of the essence that the peaks of the CE/LIF-electropherogram are below saturation. This can necessitate a rerun of the samples at higher dilution.



    Figure 4. Guide RNA/pre-mRNA substrate formation. Gel electrophoretic verification of the formation of gRNA/pre-mRNA hybrid RNAs for the U-insertion reaction (A) and the U-deletion reaction (B). The two PAA-gels show from left to right the fluorophore-labeled 5’-pre-mRNA cleavage fragment (5'-Cl), the 3’-pre-mRNA cleavage fragment (3'-Cl), the corresponding gRNA-oligoribonucleotide next to the two bimolecular complexes (5'-Cl/gRNA; 3'-Cl/gRNA) and the final trimolecular (5'-Cl/3'-Cl/gRNA) editing substrate (boxed, arrow). The yield of the trimeric RNA should exceed 90%. Fluorophore-subtituents are shown as chemical ring systems.

Recipes

  1. TBE buffer, pH 8.3

    89 mM Tris-(hydroxymethyl)-amino methane

    89 mM B(OH)3

    2 mM Na2EDTA

  2. Phenol/Chloroform/Isoamylacohol (PCI)

    Phenol/CHCl3/Isoamylacohol (25/24/1) [v/v/v]

  3. Gel staining solution

    7.5% (v/v) HOAc in H2O

    0.1% (w/v) Toluidine Blue O (C15H16ClN3S)

  4. Gel destaining solution

    10% (v/v) MeOH

    1% (v/v) HOAc

  5. TE buffer, pH 7.5

    10 mM Tris/HCl, pH 7.5

    1 mM Na2-EDTA

  6. 3× RNA-editing reaction mix

    60 mM HEPES/KOH, pH 7.5

    30 mM MgCl2

    90 mM KCl

    1.5 mM DTT

    1.5 mM ATP

    0.3 mM UTP

  7. 2× Denaturing PAGE gel-loading buffer

    1× TBE buffer, pH 8.3

    8 M Urea

    0.01% (w/v) Bromophenol Blue (C19H10Br4O5S)

    0.01% (w/v) Xylene cyanol FF, Na+-salt (C25H27N2NaO6S2)

  8. 6× Native PAGE gel-loading buffer

    10 mM Tris/HCl, pH 7.6

    0.03% (w/v) Bromophenol Blue, Na+-salt (C19H10Br4O5S)

    0.03% (w/v) Xylene cyanol FF, Na+-salt (C25H27N2NaO6S2)

    60 mM Na2EDTA

    60% (v/v) Glycerol

Acknowledgments

This work was funded by the German Research Foundation (DFG-GO 516/8-1) and the Dr. Illing-Foundation for Molecular Chemistry. We thank Cristian Del Campo, Paul Reißig, Robert Knieß and Andreas Völker for experimental input. Del Campo et al. (2020) is the original paper from which this protocol was derived.

Competing interests

The authors have no competing interests to declare.

References

  1. Blum, B., Bakalara, N. and Simpson, L. (1990). A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell 60(2): 189-198.
  2. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  3. Brun, R. and Schönenberger, M. (1979). Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop 36(3): 289-292.
  4. Böhm, C., Katari, V.S., Brecht, M. and Göringer, H.U. (2012). Trypanosoma brucei 20 S editosomes have one RNA substrate-binding site and execute RNA unwinding activity. J Biol Chem 287(31): 26268-26277.
  5. Del Campo, C., Leeder, W.M., Reißig, P. and Göringer, H.U. (2020). Analyzing editosome function in high- throughput. Nucleic Acids Res 48(17): e99.
  6. Gerace, E. and Moazed, D. (2015). Affinity purification of protein complexes using TAP tags. Methods Enzymol 559: 37-52.
  7. Giddings, M. C., Brumley Jr., R. L., Haker, M. and Smith, L. M. (1993). An adaptive, object oriented strategy for base calling in DNA sequence analysis. Nucleic Acids Res 21(19): 4530-4540.
  8. Giddings, M. C., Severin. J., Westphall, M., Wu, J. and Smith, L.M. (1998). A software system for data analysis in automated DNA sequencing. Genome Res 8(6): 644-665.
  9. Göringer, H. U. (2012a). RNA editing in African trypanosomes: A U-ser's G-U-ide. In: A. Bindereif (Ed.). RNA Metabolism in Trypanosomes. Nucleic Acids Mol Biol 28: 149-165.
  10. Göringer, H. U. (2012b). ‘Gestalt,’ composition and function of the Trypanosoma brucei editosome. Annu Rev Microbiol 66: 65-82.
  11. Golas, M. M., Böhm, C., Sander, B., Effenberger, K., Brecht, M., Stark, H. and Göringer, H. U. (2009). Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo. EMBO J 28(6): 766-778.
  12. Hermann, T., Schmid, B., Heumann, H. and Göringer, H. U. (1997). A three-dimensional working model for a guide RNA from Trypanosoma brucei. Nucl Acids Res 25(12): 2311-2318.
  13. Igo, Jr. R. P., Palazzo, S. S., Burgess, M. L., Panigrahi, A. K. and Stuart, K. (2000). Uridylate addition and RNA ligation contribute to the specificity of kinetoplastid insertion RNA editing. Mol Cell Biol 20(22): 8447-8457.
  14. Igo. Jr. R. P., Lawson, S. D. and Stuart, K. (2002). RNA sequence and base pairing effects on insertion editing in Trypanosoma brucei. Mol Cell Biol 22(5): 1567-1576.
  15. Johansson, G., Isaksson, R. and Harang V. (2003). Migration time and peak area artifacts caused by systemic effects in voltage controlled capillary electrophoresis.J Chromatogr A 1004(1-2): 91-98.
  16. Kable, M. L., Seiwert, S. D., Heidmann, S. and Stuart, K. (1996). RNA editing: a mechanism for gRNA-specified uridylate insertion into precursor mRNA. Science 273(5279): 1189-1195.
  17. Seiwert, S. D. and Stuart, K. (1994). RNA editing: Transfer of genetic information from gRNA to precursor mRNA in vitro. Science 266(5182): 114-117.
  18. Stuart, K., Kable, M. L., Allen, T. E. and Lawson, S. (1998). Investigating the mechanism and machinery of RNA editing.Methods 15(1): 3-14.

简介

[摘要]非洲锥虫和其他原生动物生物线粒体内的基因表达依赖于核苷酸特异性的RNA编辑反应。在该过程中,仅将尿苷(U)-核苷酸位点特异性插入序列不足的初级转录物中,并从中缺失,以将其转化为可翻译的mRNA。该反应由0.8 MDa的多蛋白复合物催化,该复合物被称为编辑体。在这里我们描述了一种改进的体外试验,以定量探索Editosome的催化活性。该测定使用合成的,荧光团衍生的寡核糖核苷酸 作为编辑底物,可通过耦合到激光诱导荧光(LIF)检测系统的毛细管电泳(CE)自动分离反应产物。该测定法功能强大,只需要纳克级的材料,并且通过使用多毛细管CE / LIF仪器,可以高度平行的方式进行测定。进一步的改进包括使用硫代磷酸酯修饰的,因此具有RNase耐性的底物RNA,以及用于同时监测U插入和U缺失反应的多重型荧光团标记策略。该测定方法对于研究酶体的机理和酶学是有用的。ħ H但是,它也可以在高通量执行以筛选RNA编辑特异性抑制剂。


图形摘要:


基于荧光的体外U插入/ U缺失RNA编辑(FIDE)分析的特征




[背景]中的RNA编辑反应动质体原生动物如非洲锥虫和利什曼原虫表示一个信使的最显着的转录后修饰(米)的RNA(综述Göringer ,2012A)。该反应的生物体,其中所述单个线粒体内发生是类似于其他真核生物和保持用于和化学渗透系统线粒体翻译装置的部件和电子传输的亚基的遗传信息。然而,不像在其他生物体,这些基因的50%以上为“不完全”编码:小号ubstantial序列信息丢失 –在某些情况下超过50%–除了缺少起始密码子和终止密码子以及存在移码。结果,必须校正,即“编辑”来自这些基因的前体(pre)-mRNA ,以产生功能性mRNA。在生物化学上,编辑反应的特征在于U-核苷酸的插入和/或缺失。在非洲锥虫中,在12种不同的前mRNA中的数百个编辑位点插入了3000多个U残基,并删除了约300 U残基。该过程依赖于小的非编码RNA,称为指导(g )RNA (Blum等,1990; Hermann等,1995)。指导RNA是反式作用的遗传元件。它们通过与预编辑的mRNA反平行碱基配对来启动反应周期,从而形成gRNA / pre-mRNA杂种。杂交RNA具有3螺旋连接拓扑结构,其中包括所谓的“锚” (anchor)–与待编辑序列接壤的螺旋。锚螺旋旁边的未配对gRNA核苷酸指定了U插入事件,pre-mRNA中的非碱基配对的尿嘧啶被删除了。进行加工反应的线粒体机器是高分子蛋白质复合物。类似于核糖体和剪接它先后被评为editosome (综述Göringer ,2012B)。Editosomes充当反应周期所有步骤的催化平台,并以一连串的酶促步骤执行加工反应。这包括核酸内切酶和核酸外切酶,末端尿酸转移酶(TUTase ),RNA连接酶以及RNA伴侣活性。

借助体外RNA编辑测定法已经阐明了编辑反应周期的基础步骤(Seiwert和Stuart,1994; Kable等,1996;Igo等,2000;Igo等,2002)。 。该方案代表化学简化的实验系统,其中底物预编辑的mRNA和反式gRNA均被短RNA寡核苷酸模拟。使用纯化的编辑体制备物,通过插入3个Unt或删除4个U ,以gRNA依赖性的方式编辑pre-mRNA寡核苷酸。重要的是,为了回避限速核酸内预mRNA的裂解,相应的寡核糖核苷酸是“预切割的” ,即,它们被提供为5'-和3'-前mRNA裂解(CL)-fragments 。通常将5'-C1寡核糖核苷酸标记为5'-(32 P),以能够放射性读出体外反应。

在这里,我们描述了一种新的RNA编辑测定法(Del Campo等,2020)。该测试系统是已建立的体外RNA编辑测定法的修订版(Stuart等,1998),但是,它不是使用放射性标记的底物RNA,而是使用了荧光团衍生的RNA寡核苷酸。这允许使用与激光诱导的荧光(LIF)检测系统耦合的自动化,高度平行的毛细管电泳(CE)仪器。它还使多重荧光标记实验能够在一个实验中监控多个RNA。此外,我们设计了新的pre-mRNA和模拟gRNA的寡核苷酸。不同的RNA代表在动质体生物体的线粒体基因组中找不到的合成非天然序列,除了编辑位点外,它们对于U插入和U缺失反应都是相同的。这避免了潜在的序列上下文问题,并能够并排监控一个反应瓶中的两个编辑反应。此外,我们设计了底物RNA的硫代磷酸酯(PS)修饰版本。硫代磷酸酯核苷酸间的连接增加了前mRNA和模拟gRNA的寡核苷酸的RNase稳定性,从而允许使用富含编辑体的线粒体蛋白提取物来代替高度纯化的编辑体。最后,我们通过将材料数量和反应体积调整为多孔板形式来优化测定的工作流程。该测定满足高通量筛选方法的所有信噪比标准(Del Campo等人,2020),并且能够得出催化反应周期每一步的定量数据。因此,该方案应有助于寻找RNA编辑特定的抑制性化合物。所述的一般工作流程˚F基于luorescence我nsertion / d eletion ë谛听(FIDE)-assay总结在图1中。

图1中的工作流˚F基于luorescence我nsertion / d eletion ë谛听= FIDE的测定法。该协议的主要步骤在左侧并用方框标出。右侧显示了质量评估步骤和机器读数。

关键字:RNA编辑, 尿嘧啶插入或删除, 编辑体, 向导RNA, RNA加工, 线粒体基因表达, 非洲锥虫, 寄生性原生虫病

材料和试剂
所有试剂,消耗品和设备均应视为可能的选择。除非另有说明,否则化学品和消耗品应在室温下存储。
A.溶剂和试剂     

MeOH中≥99%的PA (CARL ROTH,目录号:8388)
              的EtOH ROTIPURAN ® ≥99.8%,PA (CARL ROTH,目录号:9065)
              氯仿3 ≥99%,PA (CARL ROTH,目录号:3313)
              罗地® -苯酚(TE-缓冲液平衡,pH值7.5〜8.0)(CARL ROTH,目录号:0038,在4℃存储)
              异戊醇(3-甲基-1-丁醇),pa (Merck,目录号:100979)
甘油≥99.7%,pa (CARL ROTH,目录号:6962,储存于4°C)
Bradford分析试剂–与洗涤剂兼容(ThermoFisher Scientific,目录号:23246S,在4°C下储存)
              苯酚/氯仿/异戊醇(PCI)(小号EE ř ecipes,保存于4℃)


B.酸和碱     

KOH≥85%,pa (CARL ROTH,目录号:6751)
              HOAC 100%,pa (卡尔·罗斯(CARL ROTH),目录号:3738)
              盐酸≥25%,PA (CARL ROTH,目录号:6331)
              B(OH)3 ≥99.8%,PA (PANREAC APPLICHEM,目录号:131015)


C.缓冲化合物和盐     

三- (羟甲基) -氨基甲烷(TRIS)PUFFERAN ® (CARL ROTH,目录号:4855)             
                            N-2-羟乙基哌嗪-N'-2-乙烷磺酸磺酸(HEPES)PUFFERAN ® ≥99.5%,PA (CARL ROTH,目录号:9105)              
咪唑PUFFERAN ® ≥99%,PA (CARL ROTH,目录号:X998)
                            氯化钾(卡尔·罗斯,目录号:6781)
              氯化镁2· 6H 2 O(CARL ROTH,目录号:2189)
              NaCl (CARL ROTH,货号:3957)


D.还原和螯合剂     

1,4-二硫苏糖醇(DTT)≥99%,pa (CARL ROTH,目录号:6908 ,在4°C下储存)
三(2-羧乙基)膦盐酸盐(TCEP)≥98%(Sigma-Aldrich,目录号:C4706,储存在4°C)
              Na 2-乙二胺四乙酸钠(Na 2 EDTA)· 2H 2 O(CARL ROTH,目录号:8043)
乙二醇双(β-氨基乙基醚)四乙酸(EGTA)≥99%,pa (CARL ROTH,目录号:3054)


E.核糖核苷酸     

5'-三磷酸腺苷(ATP),100 mM ,pH 8 .0 (CARL ROTH,目录号:K045 ,储存在-20°C)             
              尿苷5'-三磷酸(UTP),100 mM ,pH 8 .0 (CARL ROTH,目录号:K046,储存在-20°C)             


F.凝胶电泳耗材     

丙烯酰胺/双丙烯酰胺(19:1)Rotiphorese ®凝胶40(CARL ROTH,目录号:3030,保存于4℃)
              尿素(CARL ROTH,货号:2317)
              过二硫酸铵(CARL ROTH,目录号:9592)
              N,N,N 'N' - Tetramethylethylendiamine (CARL ROTH,目录号:2367)
              溴酚蓝,Na +-盐(默克,目录号:108122)
              二甲苯氰FF,Na +-盐(Sigma-Aldrich,目录号:X4126)
              甲苯胺蓝O(Sigma-Aldrich,目录号:T3260)
              6 ×天然PAGE凝胶加载缓冲液(请参见配方)
              2 ×变性PAGE凝胶加载缓冲液(请参见配方)
              TBE缓冲液,pH 8.3 (请参阅配方)
              凝胶染色液(请参阅配方)
              凝胶脱色溶液(请参见配方)


G.毛细管电泳耗材(见注1)     

              玻璃毛细管(Polymicro Technologies,目录号1068150017)
性能优化的聚合物6(POP-6 TM )(赛默飞世科学,目录号:402837,小号在4℃撕)
              10 × CE / LIF缓冲液(Sigma-Aldrich,目录号:B4930,在4°C下存储)             
                                          ħ 2 ö LiChrosolv ® (默克,目录号:115333)
              高度去离子的甲酰胺(Hi- Di TM Formamide )(ThermoFisher Scientific,目录号:4311320,储存在-20°C)              


H. Editosomes和RNA-编辑反应混合物   

亲和纯化的editosomes在10 mM的HEPES / KOH ,pH为7.5,1 mM的镁(OAC )2 ,75 mM的氯化钠,0.1%(W / V)NP-40,0.1毫TCEP,1个mM的咪唑和10毫EGTA,20 %(v / v)甘油(请参阅注释2,储存在-20°C下)                                                                                                                                                                       
可选地,对于HTS-应用:Editosome富集的锥虫布氏线粒体蛋白提取物在20 mM的HEPES / KOH ,pH为7.5,10 mM的镁(OAC )2 ,30毫摩尔的KCl ,0.5毫摩尔DTT,20%(V / V)甘油(见注2,储存在-20°C)                                                                                                                                                                                                                                                                                                                 

                            3x RNA编辑反应混合物(请参阅食谱,储存在-20°C下)


I.寡核糖核苷酸(见注释3和4)       

标准FIDE分析:


              U插入


5'-FAM-synCl14 ins :FAM- (CH 2 )6 -AAAGGAAAUAUAGU
                            3'-synCl16:pAGGUGAUUCCAUUGAG- (CH 2 )6 -NH 2
              SYN-gRNA INS :CUCAAUGGAAUCACCUAAAACUAUAUUUCCUUU
                            删除


              5'-FAM-synCl17 del :FAM- (CH 2 )6 -AAAGGAAAUAUAGUUUU
              3'-synCl16:pAGGUGAUUCCAUUGAG- (CH 2 )6 -NH 2
                            syn-gRNA del :CUCAAUGGAAUCACCUACUAUAUUUCUCUUU


                            标准FIDE分析–硫代磷酸(PS)修饰(*)RNA:


              U插入


5'-FAM_modCl14英寸:FAM- (CH 2 )6 -AAAGGAAAU * A * U * A * GU             
              3'-modCl 16 :pAG * G * U * GAUUCCAUUGAG-(CH 2 )6 -NH 2             
                            modgRNA ins :C * U * C * AAUGGAAUCACCUAAAACUAUAUUUCC * U * U * U             
              删除


              5'-FAM_modCl17 del :FAM- (CH 2 )6 -AAAGGAAAU * A * U * A * GUUUU             
                            3'-modCl 16 :pAG * G * U * GAUUCCAUUGAG-(CH 2 )6 -NH 2             
              modgRNA del :C * U * C * AAUGGAAUCACCUACUAUAUUUCC * U * U * U             


                                                                      多重(“一锅法”)U插入/ U删除测定:


              U插入


5'-TAMRA_Cl18:TAMRA-(CH 2 )6 -GGAAGUAUGAGACGUAGG             
              3'-Cl13:pAUUGGAGUUAUAG- (CH 2 )6 -NH 2             
              gRNA INS :CUAUAACUCCGAUAAACCUACGUCUCAUACUUCC             
              删除


              5'-FAM_Cl22 :FAM- (CH 2 )6 -GGAAAGGGAAAGUUGUGAUUUU             
              3'-Cl15:pGCGAGUUAUAGAAUA- (CH 2 )6 -NH 2             
              gRNA del :GGUUCUAUAACUCGCUCACAACUUUCCCUUUCC             


              双重荧光检测:


              U插入


5'-TAMRA_Cl18:TAMRA-(CH 2 )6 -GGAAGUAUGAGACGUAGG             
              3'-Cl13_FAM:pAUUGGAGUUAUAG- (CH 2 )6 -FAM             
              gRNA INS :CUAUAACUCCGAUAAACCUACGUCUCAUACUUCC             
                                                        删除


                                          5'-FAM_Cl22 :FAM- (CH 2 )6 -GGAAAGGGAAAGUUGUGAUUUU             
              3'-Cl15_TAMRA :pGCGAGUUAUAGAAUA- (CH 2 )6 -TAMRA             
                            gRNA del :GGUUCUAUAACUCGCUCACAACUUUCCCUUUCC             
           

              RNA大小标准:


3'-FAM_13:AUUGGAGUUAUAG-(CH 2 )6 -FAM             
              5'-FAM_22:FAM-(CH 2 )6 - GGAAAGGGAAAGUUGUGAUUUU
              5'-FAM_37:FAM-(CH 2 )6 - GGAAAGGGAAAGUUGUGAUUUUGCGAGUUAUAGAAUA
              5'-FAM_44:FAM-(CH 2 )6 -CUAGUACUCUCAUCAACAUAAGUCUCAUACUUCCCGACAUGCA-CG
              反应瓶:


              薄壁聚丙烯反应管0.2 ml(VWR peqlab ,目录号:732- 3215)                                         
              薄壁聚丙烯反应管0.5 ml(VWR peqlab ,目录号732-3207 )                                         
                            1.5毫升聚丙烯反应管(Sarstedt ,目录号:72.706)


设备



分光光度计(Thermo Scientific,型号:NanoDrop 2000C)
              热循环仪(Biometra ,T3)
              基于CE / LIF的DNA测序仪(Applied Biosystems,型号:ABI PRISM 310遗传分析仪)
              台式离心机(Eppendorf,型号:5417 R)
              SpeedVac真空浓缩器(Savant Instruments,型号:SVC-100)
              干式加热系统(授权,型号:QBD2)
              涡旋混合器(科学仪器,型号:Vortex Genie 2)
              直流高压电源(Biometra ,型号:P30)
                            凝胶电泳槽(BIO-RAD,型号:迷你PROTEAN ®四小区)
              微量移液器(Gilson,型号:Pipetman P10,P20,P200,P1000)
              凝胶文件系统(INTAS,型号:Gel iX20 Imager)
           

软件



R(用于统计计算的R项目,https://www.r-project.org/)
              程序员类型的文本编辑器(例如Vim,https://www.vim.org或Atom,https://atom.io)
           

程序



gRNA / pre-mRNA底物形成
使用两个0.5 ml的薄壁聚丙烯反应管分别组装三聚体U插入和U缺失gRNA / pre-mRNA底物RNA。最终的gRNA / pre-mRNA浓度为10 nM 。


将3'-pre-mRNA Cl-片段,荧光标记的5'-pre-mRNA Cl-片段和相应的gRNA寡核糖核苷酸在TE缓冲液(pH 7.5 )中分别以30nM的最终浓度混合。您需要10 µl gRNA / pre-mRNA底物才能进行30 µl编辑反应。
涡旋混合(5 s),并通过短暂离心(10 s快速旋转)收集试管中的内容物。
在75°C下热解寡核糖核苷酸2分钟,然后通过以0.08°C / s的速度冷却至27°C来退火3个RNA。
注意:必须在单独的分析中验证gRNA / pre-mRNA杂合物的形成(请参见注释5)。三聚体RNA杂交的产率应≥90%。                         



RNA编辑反应和样品净化
将等体积的3 ×编辑反应混合物添加到步骤1c中的退火gRNA / pre-mRNA底物中。
分装20微升这种预混物到0.2毫升薄壁聚丙烯反应管中。
用ddH 2 O调节体积,以使最终体积(包括编辑小体的体积(稍后添加))为30 µl。
涡旋混合(5 s),并通过短暂离心(10 s快速旋转)收集试管中的内容物。
添加Editosomes,并通过轻轻上下吹打进行混合(请参见注释6)。
在27°C下孵育30分钟至3小时(请参见注释6)。
将样品转移到1.5 ml反应管中,并加入17 µl ddH 2 O,3 µl 125 mM Na 2 EDTA和50 µl PCI终止反应。剧烈混合。
在4°C下以> 15,000 × g旋转5分钟。
将H 2 O相转移到新的1.5 ml反应管中。加入5 µl 3 M NaOAc / HOAc (pH 4.8 )和121 µl 100%(v / v)冷(-20°C)乙醇。混合并立即在> 15,000 × g的溶液中于4°C旋转30分钟以沉淀RNA。
吸出上清液,然后加入0.75 ml 70%(v / v)的冷(-20°C)EtOH 。
涡旋洗涤RNA颗粒,并在> 15,000 × g的温度下于4° C旋转5分钟。
重复步骤2j-2k。
抽吸除去任何液体,并在真空中干燥RNA沉淀2分钟。
将RNA颗粒溶于15 µl Hi- Di TM甲酰胺中。通过短暂离心(10 s快速旋转)收集试管中的内容物。
通过在95°C下孵育2分钟使RNA样品变性。在冰上快速冷却。


CE / LIF分离
将3 µl样品转移到0.5 ml反应管中(请参见注释13)。
在4个大小标准的RNA寡核苷酸中各添加4 fmol 。
加入Hi- Di TM甲酰胺至终体积为20 µl。混合并旋转。
切掉盖子,将试管放入CE / LIF仪器的自动进样器中。
运行毛细管电泳
上的CE / LIF-ABI PRISM ® 310 DNA-测序用50厘米的玻璃毛细管使用以下设置:模块SEQ POP6(1毫升); 激光功率:7毫瓦; 注入电压:2.5 kV; 运行电压:12.2 kV; 样品注入时间:10 s(见注7);运行时间:25分钟。                                                                                               

完成CE / LIF运行后,检查原始电泳图。由于只能定量分析非饱和CE / LIF迹线,因此可能有必要以更高的稀释度重新运行样品。


数据分析



CE / LIF分离的输出是未处理的电泳图,它作为二进制数据文件检索。为了提取电泳图数据点并进行下游处理,我们使用编程语言R。R是用于统计计算和图形的免费软件,它可以在UNIX,macOS和Windows操作系统上编译并运行。


打开R。
              可以直接在R-console中键入命令。通过按ENTER / RETURN,将执行该命令。然而,对于更复杂的程序,更方便所有键入命令到一个文本编辑器或R的内置编辑器(见注8),其次是复制的代码到R-控制台。                                                                   

              OS X:文件新建文档           

                            Windows:文件新脚本                                       



安装Bioconductor和R软件包“ pracma ”和“ sangerseqR ”。
              Bioconductor提供了软件包“ sangerseqR ”,该软件包用于从二进制测序文件中提取相对荧光单位(RFU)中的荧光信号强度(FI)。所述包“ pracma ”提供了函数“ peakfind ”。基本的R- package集合中不包含“ pracma ” 。                                                                                                                           



                            if (!requireNamespace (“ BiocManager ”,静默= TRUE))


                                  install.packages(“ BiocManager”)


                                          BiocManager :: install(version =“ 3.11”)



              install.packages(“实用”)


              install.packages(“ sangerseqR”)



为您的FIDE实验创建一个文件夹作为工作目录。
              将CE / LIF二进制数据文件移动到此文件夹。
              将实验文件夹设置为当前R会话的工作目录。活动会话期间生成的所有文件将保存在该位置。                           


              setwd (“ / folder1 / folder2 /…/ myExperimentFolder ”)



              上载下游分析所需的所有软件包。


              图书馆(“实践”)


              库(“ sangerseqR ”)



二进制CE / LIF-文件使用功能“上传到R-工作区read.abif从” “ sangerseqR内Bioconductor的提供”包。它们被转换为abif类对象,从该对象中提取CE / LIF原始数据(即,相对荧光单位(RFU))。                           


              数据文件< -read.abif (“ myAbiFile ”)



              ceData < -data.frame (datafile @data$DATA.1 ,datafile @data$DATA.2 ,


              datafile @ data $ DATA.3,datafile @ data $ DATA.4)



                                                        分析和处理CE / LIF原始数据。
可视化电泳图。


              plot(ceData [,1],类型=“ l”,col =“蓝色”,xlab = “ EM”,ylab =“ FI / RFU”,main =“原始数据”)                                                                   

              行(ceData [,2],col =“绿色”)


              lines(ceData [,3],col =“ orange”)


              线(ceData [,4],COL = “暗红色”)


              图例(“ topleft ”,c(“ 1”,“ 2”,“ 3”,“ 4”),fill = c(“ blue”,“ green”,“ orange”,“ darkred ”))           

           

选择适当的轨迹并将通道号传递到R环境。


              trace < -traceNumberForAnalysis (注意:参数是一个真实的整数)



执行基线校正(请参见注释9)。


                            dataBg < -ceData [,trace]-中位数(ceData [500:2000,trace])             



识别峰(请参见注释10)。


              峰值< -data.frame (findpeaks (dataBg ,阈值= 50,minpeakheight = 100,零=“-”))           

              colnames (peaks)<-c(“ height”,“ max”,“ start”,“ end”)



积分峰面积。


              面积<-c()


              for(i in 1:nrow (peaks)){


                                 开始<-峰[i,3]


                                 停止<-峰[i,4] -1


                   sum < -sum (dataBg [ start:stop ])


                   面积<-舍入(c(area,sum ),0)


              }


              结果< -data.frame (峰,面积)



在R控制台中查看结果。


结果



将结果写入文件。


              myOutName <-“ myNameForResults ”


              write.table (data.frame (NUM = rownames (结果),结果),文件=粘贴(myOutName “.TXT”,月= “”),追加= F,九月= “\ t”的,癸=”。 “,row.names = F,col.names = T,引用= F)                                          


可视化CE配置文件中的峰(图2)。


              情节(peaks [peaks ,, 2],peaks [,1],type =“ h”,col =“ red”,  xlim = c(1000,1700 ),

              ylim = c(0,max(dataBg )+200),xlab =“ EM”,ylab =“ FI / RFU”)


              文本(峰[2],峰值[1] 150,rownames (峰),CEX = 1)


              lines(dataBg,type =“ l”,col =“ black”)



将图保存到文件。


              pdf(file = paste(myOutName ,“ .pdf”,sep =“”)))


              情节(peaks [peaks ,, 2],peaks [,1],type =“ h”,col =“ red”,  xlim = c(1000,1700 ),

              ylim = c(0,max(dataBg )+200),xlab =“ EM”,ylab =“ FI / RFU”)


              文本(峰[2],峰值[1] 150,rownames (峰),CEX = 1)


              lines(dataBg,type =“ l”,col =“ black”)


              dev.off ()





图2 。基于R的数据分析输出。A.处理后数据的所有峰的表格表示,指定高度,最大(最大),起始,终止和峰面积。B.所述的剧情电泳(FI = F(EM))。FI =荧光强度;RFU =相对荧光单位;EM =电泳迁移(请参见注释11)。



峰分配。
在分配的不同峰的电泳用荧光团修饰的尺寸标准的帮助下执行的寡核糖核苷酸(见材料和试剂,分项I.寡核糖核苷酸)。我们建议使用四个FAM衍生的RNA寡核苷酸,其核苷酸长度分别为13 nt ,22 nt ,37 nt和44 nt。可以将少量的四个RNA (每个4 fmol )掺入样品中,并用于推断每个RNA物种的nt长度。如图3A所示,标记寡核糖核苷酸的电泳迁移在宽的分离范围内是线性的。图3B显示了“掺入” FIDE U缺失检测的典型示例,图3C显示了U插入和U缺失检测的代表性FIDE结果,其中每个RNA的同一性和nt长度-species已指定。



计算编辑活动。
              为了比较不同的样品,存在几种定义和计算RNA编辑活性的可能性(请参见注释12):                                       

完全编辑的RNA产物的峰面积与所有指定峰的总和之比。
所有连接的RNA产物在所有指定峰上的峰面积比。
与所有指定峰相比,所有已加工RNA物种的峰面积比。
所有处理过的RNA种类的峰面积比与RNA输入峰的面积之比。




图3.代表性的FIDE结果。一。上图:CE / LIF分离10个大小标准的寡核糖核苷酸(从13 nt至44 nt)的电泳图。下图:标记RNA寡核苷酸相对于其电泳迁移率(EM)的分子量(MW)的图。数据点的线性拟合得出R平方(r 2 )为0.995。B.代表电泳用FIDE U形缺失分析(绿色迹线)的“掺入入”标记的RNA寡核苷酸13个核苷酸,22个核苷酸,37个核苷酸和44个核苷酸的长度(黑色迹线)。C. FIDE U插入(左)和U删除(右)测定的典型电泳图。各个峰的分配方式为:RNA输入,反应中间体,部分编辑的RNA,完全编辑的RNA产物和副产物。数字指定了不同RNA种类的nt长度。FI =荧光强度。RFU =相对荧光单位。



对标准FIDE协议的修改



A.高通量国际棋联     

对于高通量筛选(HTS)应用,可以缩小FIDE的比例,以使该测定法适合多孔板。两种编辑反应的下限均约为5-10 fmol ,每个编辑体和荧光团修饰的pre-mRNA / gRNA杂交体的反应量均≤5µl。由于样品中蛋白质和RNA的含量很少,可以通过直接添加Hi- Di TM甲酰胺来终止反应。这可以方便地消除CE / LIF分离之前的苯酚萃取和EtOH沉淀步骤。此外,FIDE只能使用部分纯化的线粒体提取物作为编辑体来源进行。由于存在RNase,这就需要使用“硫代磷酸酯” (PS)修饰的材料,因此需要使用“材料和试剂”部分中指定的耐RNase的底物寡核糖核苷酸。


B. “一锅法” U插入和U删除编辑- “矩阵化”                                                             

              FIDE的显着优势是可以同时监视U插入和U删除反应。可以简单地通过对两个底物RNA使用不同的荧光团来完成此操作(请参见材料和试剂,I。寡核糖核苷酸)。但是,由于荧光染料的发射光谱通常会扩散到CE / LIF检测器的多个通道中,因此需要额外的数据处理步骤。此过程称为“矩阵化”(Gidddings等,1993; Gidddings等,1998)。对于这两个荧光标记的寡核糖核苷酸,必须在被分析分开,即,单重CE / LIF-运行(见注13)。在对原始数据进行基线校正之后,每个检测器通道(i -n )中两个荧光团的荧光强度(FI)计算为:FI通道(i )/ ΣFI通道(i- n)。使用补充材料部分中提供的R脚本“ matrix.fun ”,可以方便地从二进制CE / LIF文件创建矩阵。使用以下命令将该函数加载到工作空间中:



源(“ pathToScript / matrix.fun.R ” )



必选参数是一个字符向量,其中包含单工CE / LIF文件的名称:



myMatrix的< - matrix.fun (C( “ myFileDye1 ” ,“ myFileDye2 ” ))



该矩阵被存储为制表符分隔的文本,并且可以用于相同的CE / LIF-设备上运行的所有多路复用FIDE的测定法。



多重FIDE分析的数据分析


矩阵化
读取二进制CE / LIF文件以读取R并提取原始数据,如:数据分析1-7中所述。
如果已在另一个R环境中创建矩阵,则将矩阵文件上载到工作区。


myMatrix < -data.matrix (read.delim (“ pathAndNameOfMatrixFile ” ,header = FALSE))



执行基线校正。


对于(i in 1:ncol (ceData )){


  ceData [,i ] < -ceData [,i ]-中位数(ceData [500:2000,i])


}



负值的矩阵化和校正。
对于(i in 1:nrow(ceData )){


  ceData [ i ,] = data.matrix (ceData )[ i ,]%*%t(solve(myMatrix ))


}


ceData < -replace (ceData ,ceData <0,0)



              峰检测和定量
在将迹线编号传递给R之后,按照单重FIDE / CE测定法(请参阅数据分析8,d- i )中的描述,将U删除和U插入测定与相应的迹线分开进行分析。



trace < -traceNumberForAnalysis分别用于U缺失或U插入测定的染料通道号。(注意:该参数是一个真实的整数)。                                                                     



dataBg < -ceData [,trace]



              按照以下内容继续操作:数据分析,8,d- i 。



C.双荧光FIDE     

通过使用5'-荧光团衍生的5'-mRNA寡核苷酸和同时使用3'-荧光团标记的3'-mRNA寡核苷酸,可以在一个实验中监测两个mRNA前片段的反应途径。可以对U插入和U缺失反应进行同样的操作,相应的寡核糖核苷酸列在“材料和试剂”部分。在这种情况下,不同的RNA-寡核苷酸的序列衍生自布鲁氏锥虫的线粒体ATP合酶(A6)的编码mRNA的前mRNA 。类似地,可以通过使用5'-荧光团修饰的gRNA-寡核糖核苷酸来追踪gRNA的反应途径。



笔记



对于CE / LIF仪器,我们仅使用化学等级最高的试剂。举例来说,我们使用LiChrosolv ® -H 2 O代表清洁的目的和用于稀释CE / LIF缓冲区。高度去离子的甲酰胺(Hi- Di TM甲酰胺)用于样品制备。                           
Editosomes不能商业购买。他们需要从昆虫阶段的非洲纯化锥虫(锥虫布氏在无菌细胞培养(培养)布伦和Schönenberger ,1979)。将复合物或者通过串联亲和纯化(TAP)使用转基因纯化T.布氏细胞有条件地表达的TAP标记的变体线editosomal蛋白(戈拉斯等人,2009;杰拉切和Moazed ,2015)或从线粒体去污剂提取物野生型锥虫(Böhmet al。,2012)。典型地,5升数中期的T.布氏细胞培养物(5×10 10 -8×10 10个细胞)产率约50微克TAP纯化的editosomes 。相同数量的细胞可产生约0.5 mg的具有编辑活性的线粒体蛋白质提取物。通过考马斯亮蓝G-250结合(Bradford测定法[Bradford,1976])测定蛋白质浓度。                                                                                                               
使用2'-O-(叔丁基)二甲基甲硅烷基(TBDMS)保护的亚磷酰胺,在固孔玻璃(CPG)-珠上通过固相化学合成法合成RNA-寡核苷酸。200 nmol的合成规模通常可产生≥200 µg的纯寡核糖核苷酸。RNA寡核苷酸应该是高效液相色谱法(HPLC)通过反相或阴离子-纯化任一交换HPLC和通过质谱证实。我们还建议仔细检查8 M含脲的聚丙烯酰胺凝胶中合成RNA的纯度。荧光团修饰被引入不同寡核糖核苷酸的5'-或3'-末端。这可以共存或后合完成。应避免使用内部荧光团标记策略来维持RNA分子的碱基配对能力。我们还建议在荧光基团和RNA之间引入接头序列,例如六亚甲基接头,以最大程度地减少与核碱基的空间冲突。许多荧光团可以用作取代基。具体选择取决于上的激光诱导荧光(LIF) -检测系统的波长规范。实例包括5-羧基四甲基-罗丹明(TAMRA),6-六氯-荧光素(HEX),6-羧基-荧光素(FAM),所述磺基-cyanine染料Cy5和Cy5.5的以及红外荧光染料IRDye800和WellRedD1。可以引入其他修饰,例如硫代磷酸酯骨架修饰,以增加不同RNA的RNase稳定性。通过引入3'-末端的六亚甲基-氨基接头可以抵消3'-末端的核酸外降解。代表3'-pre-mRNA片段的寡核糖核苷酸需要使用T4-多核苷酸激酶(T4-PNK)和ATP引入5'-磷酸基团。                                                                                                                                                                                                                                                                                                                                                                                         
根据在260 nm (A 260 )波长下的紫外线吸收率测量值计算出RNA浓度。对于不同的RNA的摩尔消光系数(ε)可以从最近的邻居来导出使用计算OligoAnalyzer ® :工具在www.idtna.com/scitools。可以借助NanoDrop型紫外分光光度计仪器进行测量。寡核糖核苷酸浓度≥0.2µg / µl和≤2.5µg / µl恰好在仪器的动态线性范围内,可产生可靠的测量结果,而无需使用更敏感的基于报告荧光团的方法。寡核糖核苷酸被存储在TE缓冲液(pH为7.5 )(见ř ecipes)在-20℃下以100μM的浓度。                                         
在单独的设置中,有必要确认和定量三聚体gRNA / pre-mRNA杂交RNA的形成/产量。为此,将0.4 nmol的荧光标记的5'-pre-mRNA Cl-寡核糖核苷酸,3'-pre-mRNA Cl-寡核糖核苷酸和相应的gRNA结合起来。如上所述,将样品热展开并退火(步骤1c)。加入含有2 mM MgCl 2的6 ×天然PAGE凝胶上样缓冲液,并将样品分离为18%(w / v)聚丙烯酰胺凝胶(凝胶尺寸:7.2 cm × 8.3 cm × 0.1 cm)。在4°C和8 W(恒定)的TBE (pH 8.3 ),2 mM MgCl 2中运行凝胶,直到溴酚蓝标记物到达凝胶底部为止。作为对照加载所有3个寡核糖核苷酸独立地和任选地所述RNA寡核苷酸的所有双分子组合。染色/脱色凝胶(配方2和3),并通过光密度测定法确定gRNA / pre-mRNA杂合体形成的程度。在大多数染色材料(≥90%)的应出现在频带与最低电泳迁移率,其表示三聚gRNA /前体mRNA杂交。单体寡核糖核苷酸应忽略不计。典型结果如图4所示。                                                                                                                                                                                                                                                           
所需的量editosomes和温育时间将取决于具体的编辑活动的的(EA)(EA /蛋白质量)editosome制备。在我们的手中,在FIDE U缺失检测中,通常在60分钟内,将50 ng TAP提纯的编辑小体产生完全编辑的产品的≥20%。                                                       
延长注射时间可能会导致峰值异常/伪影(Johansson等人,2003)。
使用代码/程序员类型的文本编辑器,并注意R区分大小写。
用于背景校正数据区域适于将CE / LIF-ABI PRISM ® 310 DNA-测序用50厘米的玻璃毛细管,并且可以被调整用于其它CE / LIF的仪器。             
“阈值”和“最小峰值”的设置将影响峰值检测的灵敏度。
可以组合使用这些命令以批量处理多个文件。根据作者的要求提供了脚本。             
对于方法I-III,如果处理的底物少于50%,则比率与编辑活性线性相关。方法IV显示较高的动态范围,但与编辑活动没有线性关系。方法III和IV不能说明连接活性,即缺乏连接酶活性的编体制备被认为是有活性的。                                                                                                               
它的本质是,CE / LIF-的峰值电泳低于饱和。这可能需要以更高的稀释度重新运行样品。




图4.指导RNA / pre-mRNA底物形成。用于U插入反应(A)和U删除反应(B)的gRNA / pre-mRNA杂合RNA形成的凝胶电泳验证。两种PAA凝胶从左到右显示了荧光团标记的5'-pre-mRNA裂解片段(5'-Cl),3'-pre-mRNA裂解片段(3'-Cl)和相应的gRNA-寡核糖核苷酸紧挨着两个双分子复合物(5'-Cl / gRNA; 3'-Cl / gRNA)和最后的三分子(5'-Cl / 3'-Cl / gRNA)编辑底物(带框的箭头)。三聚体RNA的产率应超过90%。荧光团取代基显示为化学环系统。



菜谱



TBE缓冲液,pH 8.3
                            89 mM Tris- (羟甲基)-氨基甲烷


              89毫米B(OH)3


                            2 mM Na 2 EDTA


苯酚/氯仿/异戊醇(PCI)
              苯酚/ CHCl 3 /异戊醇(25/24/1)[v / v / v]


              凝胶染色液
              H 2 O中的7.5%(v / v)HOAc


0.1%(w / v)甲苯胺蓝O(C 15 H 16 ClN 3 S)


              凝胶脱色液
10%(v / v)甲醇


              1%(v / v)HOAc


              TE缓冲液,pH为7.5
10 mM Tris / HCl ,pH 7.5


              1 mM Na 2 -EDTA


3 × RNA编辑反应混合物
60 mM HEPES / KOH ,pH 7.5


              30毫米MgCl 2


                            90毫米氯化钾


1.5毫米DTT


1.5毫米ATP


              0.3 mM的UTP


2 ×变性PAGE凝胶加载缓冲液
              1 × TBE缓冲液,pH 8.3


尿素800万


0.01%(w / v)溴酚蓝(C 19 H 10 Br 4 O 5 S)


                            0.01%(w / v)二甲苯氰FF,Na +-盐(C 25 H 27 N 2 NaO 6 S 2 )


6 ×天然PAGE凝胶加载缓冲液
              10 mM Tris / HCl ,pH 7.6


0.03%(w / v)溴酚蓝,Na +-盐(C 19 H 10 Br 4 O 5 S)


0.03%(w / v)二甲苯氰FF,Na +-盐(C 25 H 27 N 2 NaO 6 S 2 )


              60 mM Na 2 EDTA


                            60%(v / v)甘油



致谢



这项工作由德国研究基金会(DFG-GO 516 / 8-1)和伊灵博士-分子化学基金会资助。我们感谢克里斯蒂安·坎波,保罗Reißig ,罗伯特Knieß和Andreas沃尔克实验输入。Del Campo等。(2020 )是该协议的原始文件。



利益争夺



作者没有竞争利益要声明。



参考



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引用:Leeder, M., Kruse, E. and Göringer, H. (2021). Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE). Bio-protocol 11(5): e3935. DOI: 10.21769/BioProtoc.3935.
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