参见作者原研究论文

本实验方案简略版
Sep 2021

本文章节


 

Immunoprecipation Assay to Quantify the Amount of tRNAs associated with Their Interacting Proteins in Tissue and Cell Culture
免疫沉淀法定量组织和细胞培养中与其相互作用蛋白相关的tRNAs的数量   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Transfer RNAs (tRNAs) are highly abundant species and, along their biosynthetic and functional path, they establish interactions with a plethora of proteins. The high number of nucleobase modifications in tRNAs renders conventional RNA quantification approaches unsuitable to study protein-tRNA interactions and their associated functional roles in the cell. We present an immunoprecipitation-based approach to quantify tRNA bound to its interacting protein partner(s). The tRNA-protein complexes are immunoprecipitated from cells or tissues and tRNAs are identified by northern blot and quantified by tRNA-specific fluorescent labeling. The tRNA interacting protein is quantified by an automated western blot and the tRNA amount is presented per unit of the interacting protein. We tested the approach to quantify tRNAGly associated with mutant glycyl-tRNA-synthetase implicated in Charcot-Marie-Tooth disease. This simple and versatile protocol can be easily adapted to any other tRNA binding proteins.


Graphic abstract:



Figure 1. Schematic of the tRNA-Immunoprecipitation approach.


Keywords: tRNA (tRNA), In-vivo crosslinking (体内交联), tRNA-immunoprecipitation (tRNA-免疫沉淀), tRNA-interacting proteins (tRNA相互作用蛋白), Aminoacyl-tRNA-synthetase (氨酰-tRNA-合成酶)

Background

tRNAs are ubiquitous molecules, representing 4–10% of all cellular RNAs. tRNAs undergo complex biogenesis during which they interact with different protein entities, including tRNA-splicing proteins, tRNA-base modifying enzymes, tRNA-charging enzymes, 3’-end modification and repair enzymes, and various nucleases, thereby generating active tRNA fragments or completely degrading tRNAs (Betat and Mörl, 2015; Kirchner and Ignatova, 2015; Fernández-Millán et al., 2016; Barciszewska et al., 2016; Schmidt and Matera, 2020; Tosar and Cayota, 2020). tRNAs are crucial components of the translation machinery and are charged at the 3’ ends with their cognate amino acid by an aminoacyl-tRNA-synthetase. Mutations in tRNAs or genes encoding tRNA-interacting partners are linked to complex human pathologies, with intricate heterogeneity at both the cell and tissue levels that modulates the disease (Abbott et al, 2014). Thus, a method for quantitative detection of tRNA-binding-protein interactions that can be widely used to study disease-related alterations of tRNA interactome in living cells and tissues is an urgent need.


Traditional methods to detect RNA-protein interactions include RNA immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP), both of which use antibodies to immunoprecipitate RNA-protein complexes, followed by identification of RNAs by sequencing. Unlike mRNA sequencing, despite recent advances (Zheng et al., 2015; Behrens et al., 2021) tRNA sequencing still has limited quantitative resolution towards many tRNA isoacceptors, likely because of their complex modification pattern (Orioli, 2017; Kimura et al., 2020; Warren et al., 2021). Combining immunoprecipitation (IP) of the RNA-protein complexes with tRNA-tailored detection (Figure 1), we have developed a new twist of the classic IP method that is suitable for quantifying tRNA-protein interactions in living cells. In a recent study, we have used this approach to quantify alterations in tRNA binding to mutated glycyl-tRNA-synthetase (GlyRS), which has been implicated in Charcot-Marie-Tooth (CMT) disease (Zuko et al., 2021). In a CMT-mouse model GarsC201R/+, we observed a stronger association of tRNAsGly with the mutant GlyRS, thus depleting the glycyl-tRNAGly pool and causing ribosome stalling at Gly codons (Zuko et al., 2021). The tRNA-IP methodology identifies and quantifies tRNAs bound to GlyRS in native conditions in tissues. This protocol is easily adapted to other aminoacyl-tRNA-synthetases or any tRNA-binding proteins, to quantify interactions in native conditions, in both cell culture and tissue.

Materials and Reagents

  1. Pipette tips (filtered) (Sarstedt)

  2. Syringe 1 mL with 26 G needles (BD Plastipak)

  3. Cell Scraper, DNase/RNase free (Techno Plastic Products)

  4. RNase-free 1.5 mL microtubes (Carl Roth, catalog number: CNT2.1)

  5. RNase-free 1.5 mL amber microtubes (Carl Roth, catalog number: ENH0.2)

  6. 15 cm cell culture dishes (Sarstedt, catalog number: 83.3903)

  7. 3.5 cm cell culture dishes (Sarstedt, catalog number: 82.1135.500)

  8. GarsC201R/+ mice (C3H.C-GarsC201R/H) (Achilli et al., 2009)

  9. 293 (HEK293) cell line 293 (HEK293) (ATCC CRL-1573TM)

  10. Gibco Dulbecco's modified Eagle medium (DMEM) (Pan Biotech, catalog number: P04-03500)

  11. Fetal bovine serum (FBS) (Pan Biotech, catalog number: P30-3302)

  12. L-glutamine (Serva, catalog numbers: 22942)

  13. PBS (GibcoTM, catalog number: 70011044)

  14. 0.25% Trypsin-EDTA (Pan Biotech, catalog number: P10-019500)

  15. Triton® X-100 (Sigma, catalog number: T9284)

  16. Tris Base (Sigma, catalog number: T6066)

  17. cOmplete TM, EDTA-free protease inhibitor cocktail (Roche, catalog number: 11873580001)

  18. Urea (Carl Roth, catalog number: 57-13-6)

  19. NaCl (Carl Roth, catalog number: HN00.2)

  20. Acid-phenol:chloroform, pH 4.5 (Invitrogen, catalog number:AM9720)

  21. 3 M sodium acetate, pH 5.2 (Carl Roth, catalog number: 6773.2)

  22. Glycogen (Invitrogen, catalog number: AM9510)

  23. Absolute ethanol (Carl Roth, catalog number: 9065.4)

  24. Isopropanol (Carl Roth, catalog number: 7343.2)

  25. 40% polyacrylamide (Carl Roth,catalog number: A516.1 )

  26. RNase inhibitor SUPERase-In (Invitrogen, catalog number: AM2694)

  27. NP-40 (Sigma, catalog number: 6507)

  28. DTT (Carl Roth, catalog number: 6908.2)

  29. SDS (Carl Roth, catalog number: CN30.3)

  30. Sodium deoxycholate detergent (SDC) (Thermo, catalog number: 89904)

  31. EDTA (AppliChem, catalog number: A5097,1000)

  32. BSA (Carl Roth, catalog number: T844.3)

  33. Trisodium citrate (Carl Roth, catalog number: 3580.1)

  34. Hybond-N membrane (GE Healthcare Life Sciences, RPN2250B)

  35. T4 DNA ligase (NEB, catalog number: M0202)

  36. DMSO (Sigma, catalog number: D8418)

  37. Salmon sperm DNA (Invitrogen, catalog number: 15632011)

  38. Anti-GARS antibodies (Abcam, catalog number: ab42905; Proteintech, catalog number: 15831-AP)

  39. Pierce protein G magnetic beads (Thermo, catalog number: 88847)

  40. RevertAid H Minus reverse transcriptase (Thermo, catalog number: K1631)

  41. 10 mM DNTPs (Thermo, catalog number: R0181)

  42. T7 RNA polymerase (Thermo, catalog number: EP0113)

  43. NTPs (Thermo, catalog number: R0481)

  44. Crush and soak buffer (see Recipes)

  45. 2× RNA loading formamide dye (see Recipes)

  46. 20× saline sodium citrate buffer (SSC buffer; see Recipes)

  47. Cell lysis buffer (see Recipes)

  48. Tissue lysis buffer (see Recipes)

  49. Wash buffer for cell culture (see Recipes)

  50. Wash buffer for tissue (see Recipes)

  51. 1× SDS buffer (see Recipes)

  52. Hybridization buffer (see Recipes)

    Note: Except for the anti-GARS antibodies, all other reagents can be purchased from any other supplier.

Equipment

  1. Standard molecular biology equipment

  2. UV crosslinker (Analytik Jena, catalog number: CL-1000)

  3. CellCrusher tissue pulverizer (Kisker, catalog number: 538004)

  4. Eppendorf tube rotator (Eppendorf, catalog number: R5010)

  5. Spectrophotometer UV-vis (DeNovix DS-C)

  6. ChemiDocTM MP Imaging System multiplex fluorescence, chemiluminescence (Bio-Rad)

  7. Magnetic bead separator (Thermo Fisher, catalog number: CS15000)

  8. Jess automated western blots system (Protein Simple)

Software

  1. Fiji ImageJ (ImageJ, https://imagej.net/software/fiji/)

  2. Compass software for simple western (Protein Simple, a biotechne brand, https://www.proteinsimple.com/software_compass_simplewestern.html)

Procedure

Notes:

  1. The starting material can be any tissue of interest, or mammalian cell culture endogenously expressing, stably transfected, or ectopically expressing the tRNA-interacting protein of interest. It is recommended to test and optimize the protocol with easily accessible material, e.g., cell culture, before performing experiments in tissue.

  2. For quantitative assessment, it is important to perform the experiment in multiple independent biological replicates, i.e., at least ≥4 to enable statistical assessment.

  3. All steps should be performed in an RNase-free environment.


  1. Sample preparation

    a. In-vivo UV crosslinking to stabilize transient tRNA-protein interactions in cell culture (here HEK293T cells, hereafter named only HEK)

    1. Culture HEK cells in a 15 cm cell culture dish in DMEM supplemented with 10% FBS and 2.5 mM L-glutamine in 5% CO2 at 37°C.

      Note: For one experiment, approximately 20 million cells are required. However, for scarcely available cell culture material, as little as 6 million cells can be used.

    2. To prepare the cells for UV-crosslinking, aspirate the medium and gently add 5 mL of cold 1× PBS.

      Note: The PBS volume should be adjusted in each experiment. It should minimal, but suffient to cover the specimen or the surface of the plate in adherent cell culture.

    3. Place the cell culture plate on ice and illuminate with the UV light source (254 nm) of a crosslinker at 150 mJ/cm2 radiation.

      Note: In parallel, cells treated the same way without UV crosslinking (i.e., omitting Step 3) could be used as a control.

    4. Keep the cell culture dish on ice, gently aspirate the PBS solution, and add 800 µL of pre-cooled cell lysis buffer. Harvest the cells using a cell scraper and transfer them into a pre-cooled 1.5 mL Eppendorf tube.

    5. Using a 1 mL syringe with a 26-gauge needle, pass the lysate through eight times, to further shear open the cells and facilitate lysis. To obtain a clear lysate, centrifuge at 16,000 × g and 4°C for 10 min. This is the starting material for the IP (Step C-a).


    b. In-vivo UV Crosslinking to stabilize transient tRNA-protein interactions in tissue samples

    Note: To choose the most appropriate tissue for the experiment, one can refer to the Human Protein Atlas. In our experiment, we use brain tissue from 3- to 6-week-old CMT model mice (GarsC201R/+; Achilli et al., 2009) and compare it to wild-type littermates (i.e., mice expressing WT GlyRS (C57Bl/6J). GarsC201R/+ mice carry an ENU-induced dominant point mutation that causes a cysteine to arginine substitution at residue 201 of the GlyRS protein. Heterozygous stock colonies of GarsC201R mice were maintained in the C57Bl/6J background. One hemisphere of the mouse brain tissue was enough to obtain a sufficient amount of tRNA and GlyRS for the IP. For scarcely expressed proteins of interest, organs from several animals (preferably littermates) can be pooled.

    1. Flash freeze the freshly dissected brain tissue sample in liquid nitrogen and pulverize it in a pre-cooled CellCrusher tissue pulverizer. Transfer the pulverized tissue into a 3.5 cm cell culture dish placed on ice.

      Note: Use homogenous tissue powder, as clumps and bigger tissue chunks would limit the crosslinking effectiveness.

    2. Place the dish on an ice bath under the UV light source (254 nm) inside the UV crosslinker and apply 400 mJ/cm2 radiation.

      Note: Dependent upon tissue availability, a non-crosslinked control should also be used.

    3. Add 500 µL of cold tissue lysis buffer and mechanically shear by pipetting up and down, using pre-cooled wide-bore pipette tips (or 1,000 µL-pipette tips with the end cut out).

    4. Add an additional 500 µL of cold lysis buffer to the lysed tissue and agitate at 4°C for 1 h. Clear the lysate by centrifugation at 16,000 × g and 4°C for 10 min. This is the starting material for the IP (Step C-b).


  2. Preparation of magnetic beads and antibody coupling

    Note: Select magnetic beads according to the immunoglobulin (Ig) type of the antibody to be used for the IP.


    a. For the HEK cell lysate

    1. Use protein G-coupled Dynabeads®. Use 20 µL of the bead slurry for each antibody coupling reaction.

    2. Place the tube with the beads on the magnetic separator, wash twice with 500 µL of cold 1× PBS, and resuspend them in 50 µL of cell lysis buffer.

    3. Add 2 µg of the antibody to the beads and incubate on a tube rotator for 45 min at room temperature. Leave on ice while preparing the lysates (Step A-a).


    b. For the brain tissue lysate
    1. Use protein G-coupled Dynabeads®. Use 50 µL of the bead slurry for each coupling reaction.

    2. Prepare the beads by washing them twice with 500 µL of cold 1× PBS solution, and resuspend them in 100 µL of tissue lysis buffer.

    3. Add 4 µg of the antibody to the beads and incubate for 45 min on a tube rotator at room temperature. Leave on ice while preparing the lysates (Step A-b).

      Note: For our experiment in both cell culture and mouse tissue, we used a mixture of two different anti-GlyRS antibodies, which we mixed in equal amounts (e.g., 1 or 2 µg each for the HEK sample or mouse brain lysate, respectively). Using a mixture of antibodies from different suppliers enhances the IP reproducibility between various supplier charges.


  3. Immunoprecipitation (IP)

    Note: To determine the efficiency of the antibodies, we suggest to first perform the pulldown with more accessible material (e.g., cell culture), thereby optimizing the amount of the beads with coupled antibody and the IP incubation time. For incubation time, we recommend starting with 1 h, or a few hours, up to overnight incubation. The optimal incubation time will be the one at which the antibodies maximally retain the desired target, with minimal to no non-specific RNA bands detectable on an ethidium bromide stained denaturing polyacrylamide gel.


    a. For the HEK293 cell lysate

    1. Add the cleared supernatant obtained from Step 5A-a (approximately 800–900 µL) to the prepared beads (Step B-a3).

    2. Add 20 U of RNase inhibitor (SUPERase-IN) and rotate at 4°C for 2 h.

    3. Place the tube on a magnetic separator to separate the immunoprecipitated tRNA-GlyRS bound to the antibody-coupled beads. Carefully discard the supernatant.

    4. Wash the beads twice with 500 µL of 1× wash buffer for cell culture, and resuspend them in 500 µL of wash buffer for cell culture.

    5. Withdraw 10 µL of the IP reaction (beads) and keep on ice for protein quantification in Step E.

    6. Perform hot acid-phenol extraction of the remaining IP sample, directly on the beads, to denature the protein (here, GlyRS) and elute the bound tRNAs.

      Note: Briefly, the procedure for hot-acid phenol extraction: add one volume of acid phenol:chloroform (5:1, pH 4.5) pre-heated to 65°C and 0.1 volume of 10% SDS to the reaction, incubate for 5 min, followed by incubation on ice for 5 min. Centrifuge at 21,000 × g for 5 min, remove the aqueous phase and add equal volume of acid phenol:chloroform. Incubate at room temperature, followed by centrifugation at 21,000 × g for 5 min. Collect the aqueous phase and add equal volume of chloroform:isoamyl alcohol (24:1) and 0.1 vol of 3 M NaOAc (pH 5.5). Centrifuge at 21,000 × g for 5 min to collect the final aqueuos phase. Precipiate the RNA in the aqueous phase with isopropanol.

    7. Dissolve the recovered tRNA in 5 µL of sterile nuclease-free water.


    b. For the brain tissue lysate:

    1. Add the cleared supernatant obtained from Step A-b4 to the prepared beads (Step B-b3).

    2. Add 20 U of RNase inhibitor and rotate at 4°C overnight.

    3. Place the tube on a magnetic separator to separate the immunoprecipitated tRNA-GlyRS-coupled beads. Carefully discard the supernatant.

    4. Wash twice with 500 µL of 1× wash buffer for tissue, and resuspend the beads in 500 µL of wash buffer for tissue.

    5. Withdraw 10 µL of the IP reaction (beads) and keep on ice for protein quantification in Step E.

    6. Perform hot acid-phenol extraction of the remaining IP to denature the protein (here, GlyRS) and elute bound tRNAs.

    7. Dissolve the recovered tRNA in 5 µL of sterile nuclease-free water.


  4. Identification and quantification of the bound tRNAs in the IP


    a. Detection Method 1: tRNA identification by northern blot

    Note: An in vitro transcribed tRNA of interest is required as a positive control. Here, tRNAGlyGCC was prepared by a standard T7-RNA polymerase run-off transcription reaction, using DNA template as described before (Albers et al., 2021). Two partly overlapping DNA primers were designed to cover the full-length tRNAGlyGCC, and the 5’ end of the forward primer bears the T7 promoter site (5’-TAATACGACTCACTATA-3’; Table 1). Both primers (100 µM each) were dissolved in 20 mM Tris-HCl (pH 7.5), denatured for 2 min at 95°C, and incubated for 3 min at room temperature, for their overlapping parts to anneal. Primer extension was performed for 40 min at 37°C, using 0.4 mM dNTPs and 4 U/μL RevertAid H Minus Reverse Transcriptase. The double-stranded DNA (dsDNA) was purified by phenol/chloroform and ethanol precipitation, and dissolved in DEPC-H2O. In vitro T7 promoter-based transcription of the dsDNA template was performed overnight at 37°C, with 0.6 U/μL T7 RNA polymerase in the presence of 2 mM NTPs, 5 mM GMP, and 1× transcription buffer. The transcribed tRNA was purified by denaturing polyacrylamide gel electrophoresis. tRNA was excised from the gel and eluted in crush and soak buffer rotating at 100 × g and 4°C overnight. Gel pieces were pelleted by centrifugation at 3500 × g for 5 min and tRNA was precipitated with ethanol.


    Table 1. Example of DNA primers for in vitro T7 promoter-driven synthesis of tRNAGlyGCC. The forward primer contains 5’ upstream of the tRNA transcription start site the T7 promoter (underlined).

    Forward 5’-TAATACGACTCACTATAGCATCGGTGGTTCAGTGGTAGAATGCTCGCCTGCCACGCGGGC-3’
    Reverse 5’-TGGTGCATCGGCCGGGAATCGAACCCGGGCCGCCCGCGTGGCAGGCGAGCATTCTA-3’


    1. To prepare the RNA recovered from the IP sample (Step C-a7 or C-b7) for northern blot analysis, mix the sample with RNA loading dye, heat at 95°C for 3 min, and place it on ice.

    2. Load samples on a 10% denaturing polyacrylamide gel and run at 10 W for 30 min. In vitro synthesized tRNA is also loaded on the gel as a positive control (Figures 2A and 3A).

    3. Transfer RNA from the gel onto Hybond-N blotting membrane in pre-cooled 0.5× TAE buffer at 10 V and 4°C overnight.

    4. Immobilize the RNA to the membrane by illumiinating at 365 nm and 999.9 mJ/cm2 dosage.

      Hybridize the membrane with an Atto565-labeled DNA oligo probe (5 µL of 100 µM probe), recognizing the tRNA of interest in hybridization buffer at 28°C overnight.

      Note: If the tRNA-binding protein binds all tRNA isoacceptors of one tRNA family (that are all tRNAs recognizing different codons for a given amino acid, and thus aminoacylated with the same amino acid), we recommend using a mixture of probes to all isoacceptors. Here, we used two probes, including one with degenerate nucleotide sequence that recognizes all three tRNAGly isoacceptors (Table 2). Both probes were labeled with the same Atto-565 fluorophore. However, a mixture of probes labeled with separate fluorophores could have been chosen. Probes were fluorescently labeled at their 5’ ends by the manufacturer.

    5. Wash blots thrice with 6x SSC supplemented with 0.1% SDS.

    6. Wash once with 6× SSC.

    7. Wash once with 2× SSC.

    8. Wash once with 0.2× SSC.

    9. Image the blot on a ChemiDoc TM MP Imaging system.


      Table 2. Sequences of the Atto565-labeled DNA probes used in the northern blot experiment.

      One probe contains degenerate bases, thus recognizing both tRNAGlyCCC and tRNAGlyGCC. The probes are labeled at their 5’ ends with Atto565.

      Probe Sequence
      tRNAGlyTCC 5’-CCCGGGTCAACTGCTTGGAAGGCAGCTAT-3’
      tRNAGlyCCC/GCC 5’-GYCTCCCGCGTGGSAGGCGAG-3’


    b. Detection Method 2: Quantification of the tRNA bound to GlyRS by fluorescent tRNA labeling

    Note: The IP (Step C) yields enough RNA for tRNA detection by northern blot and fluorescent quantification. Since the northern blot is performed only for tRNA identification, we recommend performing it in a single biological replicate, by loading the entire remaining extracted tRNA amount in multiple wells [e.g., 2 wells with equal amount of sample (Figure 2B)] onto the gels, and using them as multiple technical replicates, to average the signal in the fluorescent quantification step.


    1. Label the tRNA from Step C-a7 or C-b7 with fluorescently labeled RNA: DNA hairpin oligonucleotide specific to tRNA with the following sequence: 5’-pCGCACUGCdTdTdTCy3dTdTdGdCdAdGdTdGdCdGdTdGdGdN-3’ or pCGCACUGCdTdTdTAtto647dTdTdGdCdAdGdTdGdCdGdTdGdGdN-3’ (d, denotes deoxyribose or DNA nucleotide, and p is the 5’ monophosphate).

    2. For labeling, prepare the following labeling reaction: 1 µL of 10× T4 ligation buffer (NEB, #M0202), 1.5 µL of DMSO, 0.5 µL of Cy3-labeled 25-mer oligonucleotide (90 µM), 0.5 µL of T4 DNA ligase, and 1.5 µL of DEPC-H2O.

      Note: The fluorescently labeled RNA:DNA hairpin oligonucleotide is designed to basepair to the unique unpaired 3’-NCCA end of the tRNAs, and is used to specifically label tRNAs as described previously (Kirchner et al, 2017).

    3. Combine 5 µL of extracted tRNAs (Step C-a7 or C-b7) with 5 µL of the above labeling mix (Step D-a2 or D-b2) and incubate for 1 h at 25°C in the dark, to protect the fluorophores.

    4. Heat the ligation mixture at 95°C for 3 min and place it on ice immediately.

    5. Run it on a 10% denaturing-polyacrylamide gel at 10 W for 30 min in the dark.

    6. Visualize the tRNA on a ChemiDocTM MP Imaging System in the respective fluorescent channel, and save a good quality image for further quantification of the fluorescent tRNA bands (a representative image is shown in Figures 2B and 3B).


  5. Quantification of the GlyRS protein in the IP.

    1. Use the 10 µL of IP sample reserved in Step C-a5 or C-b5. Use the magnetic rack to separate the beads with the bound antibody and protein-tRNA complexes from the liquid phase.

    2. Add 10 µL of 1× SDS buffer to the beads and incubate at 50°C for 10 min by gentle shaking.

    3. Use the magnetic rack to collect the beads, and transfer the solution to a fresh Eppendorf tube—this eluent consists of the bound tRNA-interacting protein.

    4. Use the Jess automatic western blot system—a capillary-based automated western blot instrument—to quantify the protein (Figures 2C and 3C).

      Note: Jess (Protein Simple) is an automated high-throughput western blotting system, that combines capillary electrophoresis to separate proteins by mass and immunodetection by antibodies in chemiluminescent or fluorescent detection mode. A conventional western blot can also be used instead, though the automated Jess system offers much higher sensitivity. We used purified human wildtype GlyRS protein at varying concentrations to establish a standard curve. Wildtype GlyRS was cloned into pET28 vector and expressed in the Escherichia coli Rosetta strain. The GlyRS sequence was extended by two purification tags, 6xHis and SUMO. The protein was purified to homogeneity using two consecutive chromatography steps, i.e., Ni-NTA-based affinity purification, followed by cleavage of both tags, and purification by size-exclusion chromatography. The detailed purification protocol is described in Zuko et al. (2021).



      Figure 2. Establishing of the experimental conditions and quantification of tRNA bound to GlyRS in HEK cells.

      A. Detection of tRNAGly bound to GlyRS by northern blot using Atto565-labeled probes recognizing all three tRNAGly isoacceptors. In vitro transcribed tRNAGlyGCC was loaded as a positive control and has a size of 74 nt. UV and no UV denote cells treated with UV and with no UV treatment, respectively. B. Quantification of tRNAGly bound to GlyRS with Cy3-labelled fluorescent stem-loop RNA/DNA oligonucleotide. The ligated tRNA product was monitored on a 10% denaturing polyacrylamide gel. Fluorescently labeled extended tRNAs have a size of 98 nt. C. Immunoblot of of the IP analyzed by Jess automated western blot and probed with antibodies recognizing GlyRS. Different dilutions of the IP reactions were analyzed. Protein weight markers are shown on the left in kDa. D. Quantification of tRNA bound to GlyRS. Note that HEK cells were used to optimize the protocol and, hence, performed as a single experiment. UV crosslinking increased the yield of tRNAGly bound to GlyRS.



      Figure 3. Quantification of tRNA bound to GlyRS in brain tissue from GarsC201R/+ mice (C201R-IP) and wildtype littermate (WT-IP).

      A. Detection of tRNAGly bound to mutant and wildtype GlyRS by northern blot using Atto565-labelled probes recognizing all three tRNAGly isoacceptors. In vitro transcribed tRNAGlyGCC was loaded as a positive control and has a size of 74 nt. B. Quantification of tRNAGly bound to GlyRS with Cy3-labelled fluorescent stem-loop RNA/DNA oligonucleotide. The ligated tRNA product was loaded on a 10% denaturing polyacrylamide gel. Fluorescently labeled extended tRNAs are with a size of 98 nt. C. Immunoblot of the Ips analyzed by the Jess automated western blot and probed with antibodies recognizing GlyRS. Different dilutions were analyzed. Protein weight markers are shown on the left in kDa. D. Quantification of tRNA bound to GlyRS and normalized to the tRNA/GlyRS ratio of wild-type mice, which is set as 100%. Data are shown as mean ± SEM (n=5 independent biological replicates).

Data analysis

  1. The intensity of the tRNA band was quantified from the gel in Steps D-a6 and D-b6 (Figures 2A and 3A), using ImageJ software. Normalize the intensity of the tRNA band to that of the in vitro transcribed standard tRNA whose precise amount is known.

    Note: Ensure that the samples to be compared are loaded onto the same gel and that the image is taken in grayscale. Use the same area when calculating the intensities and average it from multiple technical replicates. The intensity from a blank lane in the gel should be used to subtract the background signal.

  2. From the Jess electrogram report, quantify the peak area corresponding to the protein of interest using the Compass software for Simple western (ProteinSimple). Determine the concentration using the standard curve with purified protein samples (see the note in Step E).

  3. Divide the tRNA amount by that of the protein.

    Note: If comparing two conditions or the effect of a mutation, normalize to the ratio to that of the wild-type control, whose ratio is set to 1. We used such additional normalization to enable assessment of the increase in the bound tRNA to CMT-mutant GlyRS (Figure 2E and 3E).

Recipes

  1. Cell lysis buffer

    20 mM Tris-HCl, pH 7.4

    15 mM NaCl

    1% NP-40

    0.1% Triton® X-100

    1× Protease Inhibitor

  2. Tissue lysis buffer

    20 mM Tris-HCl, pH 7.4

    15 mM NaCl

    1% NP-40

    0.1% Triton® X-100

    0.5% SDC

    1× Protease inhibitor

  3. Wash buffer for cell culture

    20 mM Tris-HCl, pH 7.4

    100 mM NaCl

    1% NP-40

    0.1% Triton® X-100

  4. Wash buffer for tissue

    20 mM Tris-HCl pH 7.4

    100 mM NaCl

    1% NP-40

    0.1% Triton® X-100

    2% SDC

  5. 1× SDS buffer

    50 mM Tris-HCl pH 6.8

    2% SDS

  6. 20× SSC buffer

    3 M NaCl

    0.3 M Trisodium citrate

  7. Hybridization buffer

    250 mM Na2HPO4, pH 7.2

    1 mM EDTA

    7% SDS

    0.5% BSA

    100 µg/mL salmon sperm DNA

  8. Crush and soak buffer (1–5 mL per sample)

    50 mM KOAc

    200 mM KCl pH 7.0

  9. 2× RNA loading formamide dye

    9.5 mL of formamide

    330 mL of DEPC water

    150 µL of 1% xylene cyanol

    20 µL of 0.5 M EDTA

Note: All buffers are made fresh in DEPC-treated water.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG, IG73/14-2) to Z.I. and the Donders Center for Neuroscience, the Muscular Dystrophy Association (MDA479773), the EU Joint Programme – Neurodegenerative Disease Research (JPND; ZonMW 733051075 (TransNeuro) and ZonMW 733051073 (LocalNMD), the Radala Foundation, AFM-Téléthon, ARSLA, and an ERC consolidator grant (ERC-2017-COG 770244) to E.S. This adpated protocol was orifginally published in our prevous manuscript (Zuko et al., 2021; Doi: 10.1126/science.abb3356).

Competing interests

The authors declare no competing interests.

Ethics

Mouse experiments were performed at the Radboud University (Nijmegen, Netherlands) and approved by the national Dutch ethics committee ‘Centrale Commissie Dierproeven’ (AVD1030020184826/2017-0067).

References

  1. Abbott, J. A., Francklyn, C. S. and Robey-Bond, S. M. (2014). Transfer RNA and human disease. Front Genet 5: 158.
  2. Achilli, F., Bros-Facer, V., Williams, H. P., Banks, G. T., AlQatari, M., Chia, R., Tucci, V., Groves, M., Nickols, C. D. and Seburn, K. L. (2009). An ENU-induced mutation in mouse glycyl-tRNA synthetase (GARS) causes peripheral sensory and motor phenotypes creating a model of Charcot-Marie-Tooth type 2D peripheral neuropathy. Dis Model Mech 2(7-8): 359-373.
  3. Albers, S., Beckert, B., Matthies, M. C., Mandava, C. S., Schuster, R., Seuring, C., Riedner, M., Sanyal, S., Torda, A. E., Wilson, D. N., et al. (2021). Repurposing tRNAs for nonsense suppression. Nat Commun 12: 3850.
  4. Barciszewska, M. Z., Perrigue, P. M. and Barciszewski, J. (2016). tRNA--the golden standard in molecular biology. Mol Biosyst 12(1): 12-17.
  5. Behrens, A., Rodschinka, G. and Nedialkova, D. D. (2021). High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. Mol Cell 81(8): 1802-1815 e1807.
  6. Betat, H. and Morl, M. (2015). The CCA-adding enzyme: A central scrutinizer in tRNA quality control. Bioessays 37(9): 975-982.
  7. Fernández-Millán, P., Schelcher, C., Chihade, J., Masquida, B., Giege, P. and Sauter, C. (2016). Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology. Arch Biochem Biophys 602: 95-105.
  8. Kimura, S., Srisuknimit, V. and Waldor, M. K. (2020). Probing the diversity and regulation of tRNA modifications. Curr Opin Microbiol 57: 41-48.
  9. Kirchner, S. and Ignatova, Z. (2015). Emerging roles of tRNA in adaptive translation, signalling dynamics and disease. Nat Rev Genet 16(2): 98-112.
  10. Kirchner, S., Rauscher, R., Czech, A. and Ignatova, Z. (2017). Microarray-based quantification of cellular tRNAs. protocols.io. dx.doi.org/10.17504/protocols.io.hfcb3iw.
  11. Schmidt, C. A. and Matera, A. G. (2020). tRNA introns: Presence, processing, and purpose. Wiley Interdiscip Rev RNA 11(3): e1583.
  12. Tosar, J. P. and Cayota, A. (2020). Extracellular tRNAs and tRNA-derived fragments. RNA Biol 17(8): 1149-1167.
  13. Warren, J. M., Salinas-Giegé, T., Hummel, G., Coots, N. L., Svendsen, J. M., Brown, K. C., Drouard, L. and Sloan, D. B. (2021). Combining tRNA sequencing methods to characterize plant tRNA expression and post-transcriptional modification. RNA Biol 18: 64-78.
  14. Zheng, G., Qin, Y., Clark, W. C., Dai, Q., Yi, C., He, C., Lambowitz, A. M. and Pan, T. (2015). Efficient and quantitative high-throughput tRNA sequencing. Nat Methods 12(9): 835-837.
  15. Zuko, A., Mallik, M., Thompson, R., Spaulding, E. L., Wienand, A. R., Been, M., Tadenev, A. L. D., van Bakel, N., Sijlmans, C., Santos, L. A., et al. (2021). tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase. Science 373(6559): 1161-1166.

简介



[摘要]转移 RNA (tRNA) 是一种高度丰富的物种,在其生物合成和功能路径上,它们与大量蛋白质建立了相互作用。 tRNA 中的大量核碱基修饰使传统的 RNA 定量方法不适合研究蛋白质-tRNA 相互作用及其在细胞中的相关功能作用。我们提出了一种基于免疫沉淀的方法来量化与其相互作用的蛋白质伙伴结合的 tRNA。 tRNA-蛋白质复合物从细胞或组织中免疫沉淀,并通过northern印迹鉴定tRNA,并通过tRNA特异性荧光标记进行量化。 tRNA 相互作用蛋白通过自动蛋白质印迹定量,并且 tRNA 量以每单位相互作用蛋白的形式呈现。我们测试了量化与Charcot-Marie-Tooth 病有关的突变甘氨酰-tRNA-合成酶相关的tRNA Gly的方法。这种简单而通用的协议可以很容易地适应任何其他 tRNA 结合蛋白。

图文摘要:


图 1. tRNA-免疫沉淀方法示意图。


[背景] tRNA 是普遍存在的分子,占所有细胞 RNA 的 4-10 % 。 tRNA经历复杂的生物发生过程,在此过程中它们与不同的蛋白质实体相互作用,包括 tRNA 剪接蛋白、tRNA 碱基修饰酶、tRNA 充电酶、3' 端修饰和修复酶以及各种核酸酶,从而产生活性 tRNA 片段或完全降解 tRNA (Betat 和 Mörl,2015;Kirchner 和 Ignatova,2015;Fernández-Millán等,2016;Barciszewska等,2016;Schmidt 和 Matera,2020;Tosar 和 Cayota,2020) 。 tRNA 是翻译机器的重要组成部分,并通过氨酰-tRNA 合成酶在 3' 末端与它们的同源氨基酸一起充电。 tRNA 或编码 tRNA 相互作用伙伴的基因的突变与复杂的人类病理有关,在调节疾病的细胞和组织水平上具有复杂的异质性(Abbott等,2014) 。因此,迫切需要一种定量检测 tRNA 结合蛋白相互作用的方法,该方法可广泛用于研究活细胞和组织中 tRNA 相互作用组的疾病相关改变。
检测 RNA-蛋白质相互作用的传统方法包括RNA 免疫沉淀 (RIP) 和交联和免疫沉淀 (CLIP),这两种方法都使用抗体来免疫沉淀 RNA-蛋白质复合物,然后通过测序鉴定 RNA。与 mRNA 测序不同,尽管最近取得了进展(Zheng等人,2015 年;Behrens等人,2021 年),但 tRNA 测序对许多 tRNA 同工受体的定量分辨率仍然有限,可能是因为它们的修饰模式复杂(Orioli,2017 年;Kimura等人,2021 年)。 ,2020 年;沃伦等人,2021 年) 。将 RNA-蛋白质复合物的免疫沉淀 (IP) 与 tRNA 定制检测相结合(图 1),我们开发了经典 IP 方法的新转折,适用于定量活细胞中的 tRNA-蛋白质相互作用。在最近的一项研究中,我们使用这种方法来量化 tRNA 与突变的甘氨酰-tRNA-合成酶 (GlyRS) 结合的变化,这与 Charcot-Marie-Tooth (CMT) 病有关(Zuko et al. , 2021) 。在 CMT 小鼠模型Gars C201R/+中,我们观察到 tRNA Gly与突变 GlyRS 的关联更强,从而耗尽了甘氨酰-tRNA Gly库并导致核糖体在 Gly 密码子处停滞(Zuko等人,2021) 。 tRNA-IP 方法可识别和量化组织中天然条件下与 GlyRS 结合的 tRNA。该协议很容易适应其他氨酰-tRNA 合成酶或任何 tRNA 结合蛋白,以量化细胞培养和组织中天然条件下的相互作用。

关键字:tRNA, 体内交联, tRNA-免疫沉淀, tRNA相互作用蛋白, 氨酰-tRNA-合成酶


材料和试剂
1. 移液器吸头(过滤)(Sarstedt) 2. 注射器 1 mL,带 26 G 针头(BD Plastipak) 3. 细胞刮刀,不含 DNase/RNase(Techno Plastic Products) 4. 不含 RNase 的 1.5 mL 微管(Carl Roth,目录号: CNT2.1) 5. 不含 RNase 的 1.5 mL 琥珀色微管(Carl Roth,目录号:ENH0.2) 6. 15 cm细胞培养皿( Sarstedt,目录号: 83.3903 ) 7. 3.5 cm 细胞培养皿( Sarstedt,目录号:82.1135.500) 8. C201R /+小鼠 (C3H.C-GarsC201R/H ) (阿奇利等人,2009) 9. 293 (HEK293) 细胞系293 (HEK293) (ATCC CRL-1573 TM ) 10. Gibco Dulbecco 改良的 Eagle 培养基(DMEM) (Pan Biotech,目录号: P04-03500 ) 11. 胎牛血清(FBS)(Pan Biotech,目录号: P30-3302 ) 12. L-谷氨酰胺(Serva,目录号: 22942 ) 13. PBS( Gibco TM ,目录号:70011044) 14. 0.25% 胰蛋白酶-EDTA(Pan Biotech,目录号: P10-019500 ) 15. Triton ® X-100(Sigma,目录号:T9284) 16. Tris Base(Sigma,目录号:T6066) 17. 无EDTA蛋白酶抑制剂混合物 (罗氏,目录号:11873580001) 18. 尿素(Carl Roth,目录号: 57-13-6 ) 19. NaCl(Carl Roth,目录号:HN00.2) 20. 酸 - 苯酚:氯仿,pH 4.5(Invitrogen,目录号:AM9720) 21. 3 M乙酸钠,pH 5.2(Carl Roth,目录号:6773.2) 22. 糖原(Invitrogen,目录号:AM9510) 23. 无水乙醇(Carl Roth,目录号: 9065.4 ) 24. 异丙醇(Carl Roth,目录号:7343.2) 25. 40% 聚丙烯酰胺(Carl Roth,目录号: A516.1 ) 26. RNase抑制剂SUPERase-In (Invitrogen,目录号:AM2694) 27. NP-40(Sigma,目录号:6507) 28. DTT (Carl Roth,目录号:6908.2) 29. SDS(Carl Roth,目录号: CN30.3) 30. 脱氧胆酸钠洗涤剂(SDC)( Thermo,目录号:89904) 31. EDTA(AppliChem,目录号:A5097,1000) 32. BSA(Carl Roth,目录号: T844.3) 33. 柠檬酸三钠(Carl Roth,目录号:3580.1) 34. Hybond-N 膜(GE Healthcare Life Sciences, RPN2250B ) 35. T4 DNA连接酶(NEB,目录号:M0202) 36. DMSO(Sigma,目录号:D8418) 37. 鲑鱼精子DNA(Invitrogen,目录号:15632011) 38. 抗 GARS 抗体( Abcam,目录号:ab42905; Proteintech,目录号:15831-AP) 39. Pierce 蛋白 G 磁珠(Thermo,目录号:88847) 40. RevertAid H Minus 逆转录酶(Thermo,目录号:K1631) 41. 10 mM DNTP(Thermo,目录号:R0181) 42. T7 RNA聚合酶(Thermo,目录号:EP0113) 43. NTP (Thermo,目录号:R0481) 44. 粉碎和浸泡缓冲液(见食谱) 45. 2 × RNA 负载甲酰胺染料(见配方) 46. 20 ×柠檬酸钠盐缓冲液(SSC 缓冲液;见配方) 47. 细胞裂解缓冲液(参见配方) 48. 组织裂解缓冲液(见配方) 49. 用于细胞培养的洗涤缓冲液(参见配方) 50. 组织洗涤缓冲液(参见食谱) 51. 1 × SDS 缓冲液(参见配方) 52. 杂交缓冲液(见配方) 注:除抗 GARS 抗体外,所有其他试剂均可从任何其他供应商处购买。
设备
1. 标准分子生物学设备 2. UV交联剂(Analytik Jena,目录号:CL-1000) 3. CellCrusher 组织粉碎机(Kisker,目录号:538004) 4. Eppendorf管旋转器(Eppendorf,目录号:R5010) 5. 紫外-可见分光光度计 (DeNovix DS-C) 6. ChemiDoc TM MP 成像系统 多重荧光、化学发光(Bio-Rad) 7. 磁珠分离器(Thermo Fisher,目录号:CS15000) 8. Jess 自动化蛋白质印迹系统(Protein Simple)
软件 
1. 斐济 ImageJ(ImageJ, https: //imagej.net/software/fiji/ ) 2. 用于简单西方的指南针软件(Protein Simple,一个生物技术品牌, https://www.proteinsimple.com/software_compass_simplewestern.html )
程序
笔记: a. 起始材料可以是任何感兴趣的组织,或内源性表达、稳定转染或异位表达感兴趣的tRNA相互作用蛋白的哺乳动物细胞培养物。建议在组织中进行实验之前,使用易于获取的材料(例如细胞培养)测试和优化协议。 b. 对于定量评估,重要的是在多个独立的生物复制中进行实验,即至少≥4 以进行统计评估。 c. 所有步骤都应在无 RNase 的环境中执行。
A. 样品制备
一个。体内紫外交联以稳定细胞培养中的瞬时 tRNA-蛋白质相互作用(此处为 HEK293T 细胞,以下仅命名为 HEK) 1. 在 37°C 下,在 15 cm细胞培养皿中的 DMEM 中培养 HEK 细胞,并在 5% CO 2中添加 10% FBS 和2.5 mM L-谷氨酰胺。 注意:对于一项实验,大约需要 2000 万个细胞。然而,对于几乎没有可用的细胞培养材料,可以使用少至 600 万个细胞。 2. 要准备细胞进行 UV 交联,请吸出培养基并轻轻加入 5 mL 的冷 1 × PBS。 注意:PBS 体积应在每个实验中进行调整。它应该最少,但足以覆盖贴壁细胞培养中的标本或板表面。 3. 将细胞培养板放在冰上,用交联剂的紫外光源(254 nm)以 150 mJ/cm 2辐射照射。 注意:平行地,以相同方式处理但没有 UV 交联(即省略步骤 3)的细胞可用作对照。 4. 将细胞培养皿放在冰上,轻轻吸出 PBS 溶液,加入 800 μL 的预冷细胞裂解缓冲液。使用细胞刮刀收获细胞并将其转移到预冷却的 1.5 mL Eppendorf 管中。 5. 使用带有 26 号针头的 1 mL 注射器,将裂解液通过 8 次,以进一步剪切打开细胞并促进裂解。要获得澄清的裂解物,请在 16,000 × g和 4°C 下离心 10 分钟。这是 IP 的起始材料(步骤 Ca)。
湾。体内紫外交联以稳定组织样本中的瞬时 tRNA-蛋白质相互作用 注意:要选择最适合实验的组织,可以参考人类蛋白质图谱。在我们的实验中,我们使用来自 3 至 6 周龄 CMT 模型小鼠的脑组织( Gars C201R /+ ; Achilli 等人,2009)并将其与野生型同窝仔鼠(即表达 WT GlyRS 的小鼠)进行比较。 C57Bl/6J ). Gars C201R /+ 小鼠携带 ENU 诱导的显性点突变,导致 GlyRS 蛋白的残基 201 处半胱氨酸取代为精氨酸。 GarsC201R 小鼠的杂合原种菌落维持在 C57Bl/6J 背景中。小鼠脑组织的一个半球足以获得足够量的 tRNA 和 GlyRS 用于 IP 。对于几乎不表达的感兴趣的蛋白质,可以汇集来自几种动物(最好是同窝仔)的器官。
1. 将新鲜解剖的脑组织样本快速冷冻在液氮中,并在预冷的 CellCrusher 组织粉碎机中粉碎。将粉碎的组织转移到放置在冰上的 3.5 cm 细胞培养皿中。 注意:使用同质组织粉末,因为团块和较大的组织块会限制交联效果。 2. 将盘子放在 UV 交联剂内的 UV 光源 (254 nm) 下的冰浴上,并施加 400 mJ/cm 2辐射。 注意:根据组织的可用性,还应使用非交联控制。 3. 添加 500 μL 的冷组织裂解缓冲液并通过上下移液进行机械剪切,使用预冷却的大口径移液器吸头(或末端切出的 1,000 μL 移液器吸头)。 4. 加入500 µL 冷裂解缓冲液,并在 4°C 下搅拌 1 小时。通过在 16,000 × g和 4°C下离心 10 分钟来清除裂解物。这是 IP 的起始材料(步骤 Cb)。
B. 磁珠的制备和抗体偶联 注意:根据要用于 IP 的抗体的免疫球蛋白 (Ig) 类型选择磁珠。
一个。对于 HEK 细胞裂解物 1. 使用蛋白 G 偶联 Dynabeads ® 。每次抗体偶联反应使用 20 μL的珠浆。 2. 将带有珠子的管子放在磁分离器上,用 500 μL的冷 1 × PBS 洗涤两次,然后将它们重新悬浮在 50 μL的细胞裂解缓冲液中。 3. 在珠子中加入 2 μg 抗体,并在室温下在管旋转器上孵育 45 分钟。制备裂解液时置于冰上(步骤 Aa)。
湾。用于脑组织裂解物 1. 使用蛋白 G 偶联 Dynabeads ® 。每次耦合反应使用 50 μL的珠浆。 2. 准备珠子,用 500 μL的冷 1 × PBS 溶液洗涤两次,然后将它们重新悬浮在 100 μL的组织裂解缓冲液中。 3. 在珠子中加入 4 μg 抗体,并在室温下在管旋转器上孵育 45 分钟。制备裂解液时置于冰上(步骤 Ab)。 注意:对于我们在细胞培养和小鼠组织中的实验,我们使用了两种不同抗 GlyRS 抗体的混合物,我们以等量混合(例如,对于 HEK 样品或小鼠脑裂解物,分别为 1 或 2 µg) .使用来自不同供应商的抗体混合物可提高不同供应商收费之间的 IP 可重复性。
C. 免疫沉淀 (IP) 注意:为了确定抗体的效率,我们建议首先使用更容易获得的材料(例如细胞培养物)进行下拉,从而优化带有偶联抗体的珠子的数量和 IP 孵育时间。对于孵育时间,我们建议从 1 小时或几个小时开始,直至过夜孵育。最佳孵育时间将是抗体最大限度保留所需靶标的时间,在溴化乙锭染色的变性聚丙烯酰胺凝胶上可检测到最少或没有非特异性 RNA 条带。
一个。对于 HEK293 细胞裂解物 1. 将步骤 5A-a(约 800–900 μL)中获得的清除上清液添加到准备好的珠子(步骤 B-a3)中。 2. 加入 20 U 的 RNase 抑制剂 (SUPERase-IN) 并在 4°C 下旋转 2 小时。 3. 将管子放在磁分离器上,以分离与抗体偶联珠结合的免疫沉淀 tRNA-GlyRS。小心丢弃上清液。 4. ×洗涤缓冲液洗涤珠子两次,用于细胞培养,并将它们重新悬浮在 500 μL 的洗涤缓冲液中进行细胞培养。 5. 在步骤 E 中取出 10 μL 的 IP 反应(珠子)并保持在冰上进行蛋白质定量。 6. 直接在珠子上对剩余的 IP 样品进行热酸-苯酚提取,以使蛋白质(此处为 GlyRS)变性并洗脱结合的 tRNA。 注:简而言之,热酸性苯酚提取的步骤:加入1体积的酸性苯酚:氯仿(5:1,pH 4.5)预热至65°C和0.1体积的10% SDS反应,孵育5分钟,然后在冰上孵育 5 分钟。以 21,000 × g 离心 5 分钟,除去水相并加入等体积的酸性苯酚:氯仿。在室温下孵育,然后以 21,000 × g 离心 5 分钟。收集水相并加入等体积的氯仿:异戊醇 (24:1) 和 0.1 体积的 3 M NaOAc (pH 5.5)。以 21,000 × g 离心 5 分钟以收集最终的水相。用异丙醇沉淀水相中的 RNA。 7. 将回收的 tRNA 溶解在 5 μL 的无菌无核酸酶水中。
湾。对于脑组织裂解物: 1. 将步骤 A-b4 中获得的澄清上清液添加到制备好的珠子中(步骤 B-b3)。 2. 添加 20 U 的 RNase 抑制剂并在 4°C 下旋转过夜。 3. 将管子放在磁分离器上,以分离免疫沉淀的 tRNA-GlyRS 耦合珠。小心丢弃上清液。 4. ×组织洗涤缓冲液洗涤两次,并将珠子重新悬浮在 500 μL 的组织洗涤缓冲液中。 5. 在步骤 E 中取出 10 μL 的 IP 反应(珠子)并保持在冰上进行蛋白质定量。 6. 对剩余的 IP 进行热酸-苯酚提取,以使蛋白质(此处为 GlyRS)变性并洗脱结合的 tRNA。 7. 将回收的 tRNA 溶解在 5 μL 的无菌无核酸酶水中。
D. IP 中结合 tRNA 的鉴定和定量
一个。检测方法一:northern blot tRNA鉴定 注意:需要体外转录的感兴趣的 tRNA 作为阳性对照。在这里,tRNA Gly GCC 是通过标准 T7-RNA 聚合酶径流转录反应制备的,使用如前所述的 DNA 模板 ( Albers et al., 2021) 。设计了两个部分重叠的 DNA 引物以覆盖全长 tRNA Gly GCC,正向引物的 5' 端带有 T7 启动子位点( 5'-TAATACGACTCACTATA-3';表 1)。两种引物(各 100 µM)均溶解在 20 mM Tris-HCl(pH 7.5)中,在 95°C 下变性 2 分钟,在室温下孵育 3 分钟,使其重叠部分退火。引物延伸在 37°C 下进行 40 分钟,使用 0.4 mM dNTPs 和 4 U/μL RevertAid H Minus Reverse Transcriptase。双链 DNA (dsDNA) 通过苯酚/氯仿和乙醇沉淀纯化,溶解在 DEPC-H 2 O 中。 dsDNA 模板的体外 T7 启动子转录在 37°C 下过夜,0.6 U/ μL T7 RNA 聚合酶,存在 2 mM NTP、5 mM GMP 和 1 ×转录缓冲液。通过变性聚丙烯酰胺凝胶电泳纯化转录的tRNA。从凝胶中切下tRNA并在粉碎和浸泡缓冲液中洗脱,在 100 × g和 4°C 下旋转过夜。通过在 3500 下离心沉淀凝胶块 × g 5 分钟,用乙醇沉淀 tRNA。
体外T7 启动子驱动合成 tRNA Gly GCC的 DNA 引物示例。  正向引物在 tRNA 转录起始位点的 5' 上游包含 T7 启动子(下划线)。 向前 5'- TAATACGACTCACTATA GCATCGGTGGTTCAGTGGTAGAATGCTCGCCTGCCACGCGGGC-3' 撤销 5'-TGGTGCATCGGCCGGGAATCGAACCCGGGCCGCCCGCGTGGCAGGCGAGCATTCTA-3'
1. 为了准备从 IP 样品中回收的 RNA(步骤 C-a7 或 C-b7)用于 Northern 印迹分析,将样品与 RNA 上样染料混合,在 95°C 加热 3 分钟,然后将其置于冰上。 2. 将样品加载到 10% 变性聚丙烯酰胺凝胶上,并在 10 W 下运行 30 分钟。体外合成的 tRNA 也加载在凝胶上作为阳性对照(图 2A 和 3A)。 3. 将 RNA 从凝胶转移到 Hybond-N 印迹膜上,使用预冷的 0.5 × TAE 缓冲液在 10 V 和 4°C 下过夜。 4. 通过以 365 nm 和 999.9 mJ/cm 2剂量照射将 RNA 固定在膜上。 将膜与 Atto565 标记的 DNA 寡核苷酸探针(5 µL 的 100 µM 探针)杂交,在 28°C 的杂交缓冲液中识别感兴趣的 tRNA。 注意:如果 tRNA 结合蛋白结合一个 tRNA 家族的所有 tRNA 异受体(所有 tRNA 识别给定氨基酸的不同密码子,因此被相同氨基酸氨酰化),我们建议对所有异受体使用探针混合物.在这里,我们使用了两种探针,其中一种具有可识别所有三种 tRNA Gly同工受体的简并核苷酸序列(表 2)。两种探针都用相同的 Atto-565 荧光团标记。然而,可以选择用单独的荧光团标记的探针混合物。制造商对探针的 5' 端进行荧光标记。 5. 清洗三次,辅以 0.1% SDS。 6. × SSC洗涤一次。 7. × SSC洗涤一次。 8. 用 0.2 × SSC 洗涤一次。 9. 在 ChemiDoc TM上成像印迹 MP成像系统。
表 2. 在 Northern 印迹实验中使用的 Atto565 标记的 DNA 探针的序列。  一种探针包含简并碱基,因此可以识别 tRNA Gly CCC 和 tRNA Gly GCC。探针的 5' 末端用 Atto565 标记。 探测 顺序 tRNA Gly TCC 5'-CCCGGGTCAACTGCTTGGAAGGCAGCTAT-3' tRNA Gly CCC/GCC 5'-GYCTCCCGCGTGGSAGGCGAG-3'



湾。检测方法 2:通过荧光 tRNA 标记对与 GlyRS 结合的 tRNA 进行定量 注意:IP(步骤 C)产生足够的 RNA,用于通过 Northern 印迹和荧光定量检测 tRNA。由于 Northern 印迹仅用于 tRNA 鉴定,我们建议在单个生物复制中进行,方法是将整个剩余提取的 tRNA 量加载到多个孔中 [例如,2 个具有等量样品的孔(图 2B)] 到凝胶上,并将它们用作多个技术复制品,以平均荧光定量步骤中的信号。
1. 步骤 C-a7 或 C-b7中的 tRNA:具有以下序列的 tRNA 特异性 DNA 发夹寡核苷酸: 5'-p CGCACUGCdTdTdT Cy3 dTdTdGdCdAdGdTdGdCdGdTdGdGdN -3'或 p CGCACUGCdTdTdT Atto647 dTdTdGdCdGdGdTd -3 '表示脱氧核糖或DNA核苷酸,p是5'单磷酸)。 2. 对于标记,准备以下标记反应:1 µL 10 × T4 连接缓冲液(NEB,#M0202)、1.5 µL DMSO、0.5 µL Cy3 标记的 25 聚体寡核苷酸(90 µM)、0.5 µL T4 DNA 连接酶和 1.5 µL的 DEPC-H 2 O。 注意:荧光标记的 RNA:DNA 发夹寡核苷酸旨在与 tRNA 的独特未配对 3'-NCCA 末端进行碱基配对,并用于专门标记 tRNA,如前所述 (Kirchner 等人,2017 年) 。 3. 5 µL提取的 tRNA(步骤 C-a7 或 C-b7)与 5 µL上述标记混合物(步骤 D-a2 或 D-b2)混合,在 25°C 避光孵育 1 小时,以保护荧光团。 4. 将连接混合物在 95°C 加热 3 分钟,然后立即将其置于冰上。 5. 在 10 W 的 10% 变性聚丙烯酰胺凝胶上在黑暗中运行 30 分钟。 6. 可视化 ChemiDoc TM MP 成像系统上的 tRNA,并保存高质量的图像以进一步量化荧光 tRNA 条带(代表性图像如图 2B 和 3B 所示)。
E. IP 中 GlyRS 蛋白的定量。 1. 使用步骤 C-a5 或 C-b5 中保留的 10 µL IP 样品。使用磁性架将带有结合抗体和蛋白质-tRNA 复合物的珠子从液相中分离出来。 2. 中加入 10 µL 1 × SDS 缓冲液,在 50°C 下轻轻摇动孵育 10 分钟。 3. 使用磁力架收集磁珠,然后将溶液转移到新的 Eppendorf 管中——这种洗脱液由结合的 tRNA 相互作用蛋白组成。 4. 使用 Jess 自动蛋白质印迹系统(一种基于毛细管的自动蛋白质印迹仪器)来量化蛋白质(图 2C 和 3C)。 注意:Jess (Protein Simple) 是一种自动化的高通量蛋白质印迹系统,它结合了毛细管电泳按质量分离蛋白质和通过抗体在化学发光或荧光检测模式下进行免疫检测。 尽管自动化 Jess 系统提供更高的灵敏度,但也可以使用传统的蛋白质印迹法。我们使用不同浓度的纯化人野生型 GlyRS 蛋白来建立标准曲线。野生型 GlyRS 被克隆到 pET28 载体中并在大肠杆菌 Rosetta 菌株中表达。 GlyRS 序列通过两个纯化标签 6xHis 和 SUMO 进行了扩展。使用两个连续的色谱步骤将蛋白质纯化至均质,即基于Ni-NTA的亲和纯化,随后切割两个标签,并通过尺寸排阻色谱纯化。 Zuko 等人描述了详细的纯化方案。 (2021 年) 。
  图 2. 在 HEK 细胞中建立与 GlyRS 结合的 tRNA 的实验条件和定量。 A. 使用 Atto565 标记的探针通过northern 印迹检测与GlyRS 结合的tRNA Gly ,该探针识别所有三种tRNA Gly同工受体。加载体外转录的 tRNA Gly GCC 作为阳性对照,大小为 74 nt。 UV 和无UV 分别表示用UV 和没有UV 处理的细胞。 B.用 Cy3 标记的荧光茎环 RNA/DNA 寡核苷酸对与 GlyRS 结合的 tRNA Gly进行定量。在 10% 变性聚丙烯酰胺凝胶上监测连接的 tRNA 产物。荧光标记的扩展 tRNA 的大小为 98 nt。 C. 通过 Jess 自动蛋白质印迹分析 IP 的免疫印迹并用识别 GlyRS 的抗体进行探测。分析了 IP 反应的不同稀释度。蛋白质重量标记以 kDa 显示在左侧。 D. 与 GlyRS 结合的 tRNA 的定量。请注意,HEK 细胞用于优化协议,因此作为单个实验进行。 UV 交联增加了与GlyRS 结合的 tRNA Gly的产量。
  图 3. Gars C201R/+小鼠 (C201R-IP) 和野生型同窝小鼠 (WT-IP)脑组织中与GlyRS 结合的 tRNA 的定量。 A.使用 Atto565 标记的探针识别所有三种 tRNA Gly同工受体,通过 Northern 印迹检测与突变型和野生型 GlyRS 结合的 tRNA Gly 。加载体外转录的 tRNA Gly GCC 作为阳性对照,大小为 74 nt。 B.用 Cy3 标记的荧光茎环 RNA/DNA 寡核苷酸对与 GlyRS 结合的 tRNA Gly进行定量。将连接的 tRNA 产物加载到 10% 变性聚丙烯酰胺凝胶上。荧光标记的扩展 tRNA 大小为 98 nt。 C. 通过 Jess 自动蛋白质印迹分析 Ips 的免疫印迹并用识别 GlyRS 的抗体进行探测。分析了不同的稀释度。蛋白质重量标记以 kDa 显示在左侧。 D. tRNA 与 GlyRS 结合并标准化为野生型小鼠的 tRNA/GlyRS 比率的量化,该比率设置为 100%。数据显示为平均值±SEM(n = 5 个独立的生物学重复)。  数据分析
1. 使用 ImageJ 软件从步骤 D-a6 和 D-b6(图 2A 和 3A)中的凝胶中量化 tRNA 条带的强度。 将 tRNA 带的强度标准化为精确数量已知的体外转录标准 tRNA 的强度。 注意:确保要比较的样品加载到相同的凝胶上,并且图像是灰度拍摄的。计算强度时使用相同的区域,并从多个技术复制中对其进行平均。凝胶中空白泳道的强度应用于减去背景信号。 2. 从 Jess 电图报告中,使用 Compass 软件 for Simple Western (ProteinSimple) 量化与感兴趣的蛋白质相对应的峰面积。使用纯化蛋白样品的标准曲线确定浓度(参见步骤 E 中的注释)。  3. 将 tRNA 的量除以蛋白质的量。 注意:如果比较两种情况或突变的影响,请标准化为与野生型对照的比率,其比率设置为 1。我们使用这种额外的标准化来评估结合 tRNA 与 CMT 的增加-突变 GlyRS(图 2E 和 3E)。
食谱
1. 细胞裂解缓冲液 20 mM Tris-HCl,pH 7.4 15 毫米氯化钠 1% NP-40 0.1%海卫® X-100 1 ×蛋白酶抑制剂 2. 组织裂解缓冲液 20 mM Tris-HCl,pH 7.4 15 毫米氯化钠 1% NP-40 0.1%海卫® X-100 0.5% SDC 1 ×蛋白酶抑制剂 3. 细胞培养洗涤缓冲液 20 mM Tris-HCl,pH 7.4 100 毫米氯化钠 1% NP-40 0.1%海卫® X-100 4. 组织洗涤缓冲液 20 mM Tris-HCl pH 7.4 100 毫米氯化钠 1% NP-40 0.1%海卫® X-100 2% SDC 5. 1 × SDS 缓冲液 50 mM Tris-HCl pH 6.8 2% SDS 6. 20 × SSC 缓冲器 3 M 氯化钠 0.3 M 柠檬酸三钠 7. 杂交缓冲液 250 mM Na 2 HPO 4 ,pH 7.2 1 毫米乙二胺四乙酸 7% 安全数据表 0.5% BSA 100 µg/mL 鲑鱼精子 DNA 8. 粉碎和浸泡缓冲液(每个样品 1–5 mL) 50 毫米 KOAc 200 毫米氯化钾 pH 7.0 9. 2 × RNA 负载甲酰胺染料 9.5 毫升甲酰胺 330 毫升 DEPC 水 150 µL 1% 二甲苯氰 20 µL 0.5 M EDTA 注意:所有缓冲液都是在 DEPC 处理过的水中新鲜制成的。
致谢
这项工作得到了 Deutsche Forschungsgemeinschaft (DFG, IG73/14-2) to ZI 和 Donders 神经科学中心的支持, 肌肉萎缩症协会 ( MDA479773 )、欧盟联合计划 - 神经退行性疾病研究 (JPND;ZonMW 733051075 (TransNeuro) 和 ZonMW 733051073 (LocalNMD)、Radala 基金会、AFM-Téléthon、ARSLA 和 ERC 整合资助 (ERC-2017) -COG 770244) 到 ES 这个改编的协议最初发表在我们以前的手稿中(Zuko et al. , 2021; Doi: 10.1126/science.abb3356)。
利益争夺
作者声明没有竞争利益。
伦理
小鼠实验在 Radboud 大学(荷兰奈梅亨)进行,并得到荷兰国家伦理委员会“Centrale Commissie Dierproeven”(AVD1030020184826/2017-0067)的批准。
参考
1. Abbott, JA, Francklyn, CS 和 Robey-Bond, SM (2014)。转移RNA与人类疾病。前基因5:158。 2. Achilli, F., Bros-Facer, V., Williams, HP, Banks, GT, AlQatari, M., Chia, R., Tucci, V., Groves, M., Nickols, CD 和Seburn, KL (2009)。 ENU 诱导的小鼠甘氨酰-tRNA 合成酶 (GARS) 突变导致外周感觉和运动表型,创建了 Charcot-Marie-Tooth 型 2D 周围神经病变模型。 Dis Model Mech 2(7-8):359-373。 3. Albers, S., Beckert, B., Matthies, MC, Mandava, CS, Schuster, R., Seuring, C., Riedner, M., Sanyal, S., Torda, AE, Wilson, DN, et al. (2021 年)。重新利用 tRNA 进行无意义的抑制。国家通讯12:3850。 4. Barciszewska, MZ, Perrigue, PM 和 Barciszewski, J. (2016)。 tRNA——分子生物学的黄金标准。 摩尔生物系统12(1):12-17。 5. Behrens, A.、Rodschinka, G. 和 Nedialkova, DD (2021)。通过 mim-tRNAseq 对真核生物中 tRNA 丰度和修饰状态进行高分辨率定量分析。 分子细胞81(8):1802-1815 e1807。 6. Betat, H. 和 Morl, M. (2015)。 CCA 添加酶:tRNA 质量控制中的核心检查器。 生物论文 37(9):975-982 。 7. Fernández-Millán , P.、Schelcher, C.、Chihade, J.、Masquida, B.、Giege, P. 和 Sauter, C. (2016)。转移 RNA:从开创性的晶体学研究到当代 tRNA 生物学。 Arch Biochem Biophys 602:95-105。 8. Kimura, S.、Srisuknimit, V. 和 Waldor, MK (2020)。探索 tRNA 修饰的多样性和调控。 Curr Opin Microbiol 57:41-48。 9. Kirchner, S. 和 Ignatova, Z. (2015)。 tRNA 在适应性翻译、信号动力学和疾病中的新兴作用。 Nat Rev Genet 16(2):98-112。 10. Kirchner, S.、Rauscher, R.、Czech, A. 和 Ignatova, Z. (2017)。基于微阵列的细胞 tRNA 定量。协议.io。 dx.doi.org/10.17504/protocols.io.hfcb3iw。 11. 加利福尼亚州施密特和美国马泰拉 (2020)。 tRNA 内含子:存在、加工和目的。 Wiley Interdiscip Rev RNA 11(3):e1583。 12. Tosar, JP 和 Cayota, A. (2020)。细胞外 tRNA 和 tRNA 衍生片段。 RNA 生物学17(8):1149-1167。 13. Warren, JM, Salinas-Giegé, T., Hummel, G., Coots, NL, Svendsen, JM, Brown, KC, Drouard, L. 和 Sloan, DB (2021)。 结合 tRNA 测序方法来表征植物 tRNA 表达和转录后修饰。 RNA 生物学18:64-78。 14. Zheng, G., Qin, Y., Clark, WC, Dai, Q., Yi, C., He, C., Lambowitz, AM 和 Pan, T. (2015)。高效、定量的高通量 tRNA 测序。 国家方法12(9):835-837。 15. Zuko, A., Mallik, M., Thompson, R., Spaulding, EL, Wienand, AR, Been, M., Tadenev, ALD, van Bakel, N., Sijlmans, C., Santos, LA等。 (2021 年)。 tRNA 过表达可挽救由 tRNA 合成酶突变引起的周围神经病变。 科学373(6559):1161-1166。

登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Das, S., Zuko, A., Thompson, R., Storkebaum, E. and Ignatova, Z. (2022). Immunoprecipation Assay to Quantify the Amount of tRNAs associated with Their Interacting Proteins in Tissue and Cell Culture. Bio-protocol 12(4): e4335. DOI: 10.21769/BioProtoc.4335.
  2. Zuko, A., Mallik, M., Thompson, R., Spaulding, E. L., Wienand, A. R., Been, M., Tadenev, A. L. D., van Bakel, N., Sijlmans, C., Santos, L. A., et al. (2021). tRNA overexpression rescues peripheral neuropathy caused by mutations in tRNA synthetase. Science 373(6559): 1161-1166.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。