Immunoprecipitation of Tri-methylated Capped RNA

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Proceedings of the National Academy of Sciences of the United States of America
Jun 2017



Cellular quiescence (also known as G0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27Kip1. Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm by the Exportin-1 (XPO1) protein. We used an antibody against (TMG)-caps (which does not cross-react with the (m7G)-caps that most pri-miRNAs or mRNAs contain [Luhrmann et al., 1982]) to perform RNA immunoprecipitations from total RNA extracts of proliferating or quiescent HFFs. The novelty of this assay is the specific isolation of pri-miRNAs as well as other non-coding RNAs containing a TMG-cap modification.

Keywords: m2,2,7G-cap RNA (m2,2,7G帽RNA), TMG-cap RNA (TMG帽RNA), Tri-methylated RNA (三甲基化RNA), RNA immunoprecipitation (RNA免疫沉淀), Pri-miRNA (Pri-miRNA)


Cellular quiescence, a type of reversible growth arrest, is an important cellular state involved in wound healing, organ regeneration, and preventing neoplasia (Coller, 2011; Valcourt et al., 2012). Small non-coding RNAs such as miRNAs have been found involved in the regulation of cellular quiescence. miRNAs are small non-coding RNAs ~22-nucleotides long that regulate the expression of protein-coding genes by base-pairing with the 3’ untranslated region (3’UTR) of messenger RNAs (mRNAs) (Esteller, 2011). The canonical miRNA biogenesis pathway is based on a stepwise processing machinery (Ha and Kim, 2014; Kim et al., 2016). miRNAs are transcribed to produce a primary miRNA (pri-miRNA) with an imperfect loop structure that is recognized by the enzyme Drosha and its binding partner DGCR8 in the nucleus. Cleavage of the pri-miRNA generates a precursor miRNA (pre-miRNA) that is recognized and transported to the cytoplasm by the Exportin-5 (XPO5) protein. The pre-miRNA is cleaved by the enzyme Dicer (mature miRNA) and loaded into the RNA-induced silencing complex (RISC). On the other hand, precursors of small nuclear RNAs (snRNAs) involved in mRNA processing such as U1, U2, U4, and U5 have a (m7G)-cap, which is recognized by cap-binding complex (CBC) and the phosphorylated adaptor for RNA export (PHAX) in the nucleus to enable their export to the cytoplasm by XPO1 (Ohno et al., 2000). These snRNAs are then recognized by Sec1/Munc18 (Sm) proteins (by binding to Sm binding site sequences) in the cytoplasm and TGS1 is recruited to hypermethylate the (m7G)-cap into a (m2,2,7G, TMG)-cap. This modification is recognized by Snuportin-1 in association with Importin-β and other factors to import the snRNAs back into the nucleus (Palacios et al., 1997; Kiss, 2004). Interestingly, XPO1 also has high affinity for the (TMG)-capped small nucleolar RNA (snoRNA) U3 in the nucleus and transports it from Cajal bodies to the nucleoli (Boulon et al., 2004). A previous study showed that TGS1 enhances Rev-dependent HIV-1 RNA expression by (TMG)-capping viral mRNAs in the nucleus, thereby increasing recognition by XPO1 for transport to the cytoplasm (Yedavalli and Jeang, 2010). These findings suggest that TMG-capping of RNAs gives plasticity to different types of RNA molecules in order to regulate their processing and cellular localization. Our recent findings demonstrated the existence of a group of pri-miRNAs modified with a 2,2,7-trimethylguanosine (TMG)-cap by TGS1 protein and transported to the cytoplasm by XPO1 during quiescence. Previous publications have shown the ability to pull-down (TMG)-cap RNAs, such as snRNAs and snoRNAs, with specific antibodies against (TMG)-cap RNAs (Luhrmann et al., 1982). Our previous publications demonstrated for the first time the pull-down of (TMG)-cap pri-miRNAs in human cells (Martinez et al., 2017). Understanding which RNAs could be modified with a TMG-cap will provide new important insights into RNA biogenesis in normal or disease-related conditions.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-129 )
  2. 15 ml conical centrifuge tubes (DNase-/RNase-free) (Corning, catalog number: 430052 )
  3. GilsonTM EXPERTTM University Fit pipette filter tips (Gilson, catalog numbers: F1731031 , F1733031 , F1735031 , F1737031 )
  4. Gel-loading pipet tips (Fisher Scientific, catalog number: 02-707-139 )
  5. Large-orifice pipet tips (Fisher Scientific, catalog number: 02-707-134 )
  6. Sterile polystyrene disposable serological pipettes (Greiner Bio One International, catalog number: 710180 )
  7. Serological pipettes
    2 ml serological pipettes (Fisher Scientific, catalog number: 13-678-11C )
    5 ml serological pipettes (Fisher Scientific, catalog number: 13-678-11D )
    10 ml serological pipettes (Fisher Scientific, catalog number: 13-678-11E )
    25 ml serological pipettes (Fisher Scientific, catalog number: 13-678-11 )
  8. 150 cm2 vented tissue culture treated flasks (Corning, Falcon®, catalog number: 355001 )
  9. 100 mm TC-treated cell culture dish (Corning, Falcon®, catalog number: 353033 )
  10. Cell scrapers (Fisher Scientific, catalog number: 08-100-242 )
  11. HFF cells (obtained from the Yale Skin Disease Research Center) (Alternative source of HFF cells from ATCC: Hs27 (ATCC, catalog number: CRL-1634 )
  12. DMEM (Sigma-Aldrich, catalog number: D7777 )
  13. Minimum essential medium (MEM) non-essential amino acids, 100x (Thermo Fisher Scientific, catalog number: 11140076 )
  14. 0.05% trypsin-EDTA with phenol red (Thermo Fisher Scientific, catalog number: 25300054 )
  15. 10x PBS (Sigma-Aldrich, catalog number: P5493 )
  16. Nuclease-free water (not DEPC-Treated) (Thermo Fisher Scientific, catalog number: AM9937 )
  17. TRIzolTM Reagent (Thermo Fisher Scientific, catalog number: 15596026 )
  18. RNase AWAYTM Surface Decontaminant (Thermo Fisher Scientific, catalog number: 7002 )
  19. Chloroform (Sigma-Aldrich, catalog number: C2432 )
  20. Ethanol, molecular biology grade (Fisher Scientific, catalog number: BP2818-500 )
  21. Isopropanol, molecular biology grade (Fisher Scientific, catalog number: BP26184 )
  22. GlycoBlueTM Coprecipitant (15 mg/ml) (Thermo Fisher Scientific, catalog number: AM9516 )
  23. TURBO DNA-freeTM Kit (Thermo Fisher Scientific, catalog number: AM1907 )
  24. Anti-m3G-cap, rabbit polyclonal, antiserum (Synaptic Systems)*
    Note: *Synaptic Systems discontinued the production of the Anti-m3G-cap, rabbit polyclonal, antiserum. Creative Diagnostics has a rabbit anti-TMG antibody (Anti-m3G-cap polyclonal antibody, Creative Diagnostics, catalog number: DPAB29202 ) that would be similar to the one we previously used but the experimental conditions have to be re-evaluated.
  25. Protein G Sepharose® 4 Fast Flow Beads (GE Healthcare, catalog number: 17061801 )
  26. Normal rabbit serum (control, EMD Millipore, catalog number: NS01L-1ML )
  27. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-3 )
  28. NP-40 (Thermo Fisher Scientific, catalog number: 28324 )
  29. Tris base (Fisher Scientific, catalog number: BP152-5 )
  30. Hydrochloric acid (HCl) (VWR, catalog number: BDH7204-1 )
  31. RNasinTM Plus RNase inhibitor (Promega, catalog number: N2611 )
  32. NaOAc (AMRESCO, catalog number: 0602 )
  33. Ethylenediaminetetraacetate acid (EDTA), pH 8 (Thermo Fisher Scientific, catalog number: AM9260G )
  34. Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0105 )
  35. Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: AM9822 )
  36. Phenol/Chloroform/Isoamyl Alcohol; 125:24:1 mixture, pH 4.5 (Thermo Fisher Scientific, catalog number: AM9720 )
  37. Agarose LE (Denville Scientific, catalog number: CA3510-8 )
  38. iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, catalog number: 1708891 )
  39. Sso AdvancedTM Universal SYBR® Green Supermix (Bio-Rad Laboratories, catalog number: 1725274 )
  40. PARISTM Kit (Thermo Fisher Scientific, catalog number: AM1921 )
  41. Boric acid (Fisher Scientific, catalog number: A73-1 )
  42. NET-2 Buffer (see Recipes)
  43. G-50 Buffer (see Recipes)
  44. 1x TBE (see Recipes)


  1. Micropipettes (Gilson, model: Pipetman® L, catalog number: F167370 )
  2. FormaTM Steri-CycleTM CO2 Incubator (Thermo Scientific, model: FormaTM Steri-CycleTM CO2 Incubators, catalog number: 370)
  3. -80 °C freeze
  4. SorvallTM LegendTM Micro 21R Microcentrifuge (Thermo Fisher Scientific, model: SorvallTM LegendTM Micro 21R , catalog number: 75002490)
  5. EppendorfTM ThermomixerTM R (Eppendorf, model: Thermomixer R , catalog number: 05-412-401)
  6. LabquakeTM Tube Shaker/Rotator (Thermo Fisher Scientific, catalog number: C4152110Q )
  7. SorvallTM ST 40R Centrifuge (Thermo Fisher Scientific, model: SorvallTM ST 40R , catalog number: 75004525)
  8. NanoDropTM 2000 Spectrophotometer (Thermo Fisher Scientific, model: NanoDropTM 2000 , catalog number: ND-2000)
  9. T100TM Thermal Cycler (Bio-Rad Laboratories, catalog number: 1861096 )
  10. CFX ConnectTM Real-Time PCR Detection System (Bio-Rad Laboratories, catalog number: 1855200 )
  11. UV transilluminator


  1. Plate HFF cells in serum-free DMEM supplemented with MEM non-essential amino acids at low density (approx. 1 x 106 cells/150 cm2 flask). Incubate HFF cells in humidified atmosphere of 5% CO2 at 37 °C for 48 h (Figure 1).
    1. In order to obtain enough RNA for each experiment, use 4 to 6 150 cm2 flasks.
    2. To plate HFF cells in serum-free DMEM; wash 70-80% confluent HFF cells (100 mm TC-treated cell culture dish) three times each with 10 ml phosphate buffered saline, add 1 ml 0.05% trypsin with phenol red to detach cells, add 10 ml serum-free DMEM media and collect cells in 15 ml conical tube, pellet cells by centrifuging at 300 x g at room temperature, and re-suspend pellet in serum-free DMEM supplemented medium.
    3. It is important to remove trypsin from HFF cells before re-plating since medium doesn’t contain FBS to inactive the trypsin.

    Figure 1. Main steps diagram of this protocol

  2. Collect HFF cells by scraping flask on ice, pellet at 300 x g for 5 min at 4 °C, and wash two times each with 2 ml of phosphate buffered saline. Cell pellets can be frozen at -80 °C until RNA extraction (cell pellets must be kept on ice until lysed with TRIzolTM) (Figure 1).
    1. To pellet HFF cells, remove old serum-free DMEM from cells and add 3 ml of fresh serum-free DMEM. Use the blade of cell scraper to collect cells in one corner of flask taking care to not splash the cells in flask. Add 1-2% of volume of FBS or BSA to cells on ice to assist in pelleting. Extract RNA from pelleted cells using TRIzolTM Reagent per manufacturer’s instructions.
    2. All steps in this protocol should be performed in an RNase free bench area by using RNase AWAYTM Surface Decontaminant to spray and wipe most surface areas including micropipettes and ice bucket.

    1. Resuspend cell pellets thawed on ice in 1 ml of TRIzolTM Reagent for 5 min to lyse.
    2. Add chloroform (20% of TRIzolTM Reagent volume), mix samples well, and incubate at room temperature for 2-3 min.
    3. Centrifuge samples at 12,000 x g for 15 min at 4 °C to separate red phenol-chloroform (red/lower layer), interphase, and aqueous phase (clear/upper layer).
    4. Collect aqueous layer and precipitate RNA for 10 min at room temperature with isopropanol (0.5 volume of original TRIzolTM Reagent); GlycoBlueTM Coprecipitant (15 μg) can be added at this time to aid RNA precipitation.
    5. Pellet precipitated RNA by centrifugation (12,000 x g for 10 min at 4 °C).
    6. Wash RNA pellets with 75% ethanol (add an equal volume of 75% ethanol to TRIzolTM Reagent and briefly vortex) and re-pellet by centrifuging at 7,500 x g for 5 min at 4 °C.
    7. Air-dry RNA pellets for a few minutes and re-suspend in nuclease-free water.
      Note: Do not completely dry the RNA pellet because it will be difficult to re-suspend. Incubation at 55 °C for 10 min can help solubilize the RNA.
  3. Use DNase from TURBO DNA-freeTM Kit to digest DNA contamination in RNA samples according to manufacturer’s protocol:
    1. Mix RNA with 10x TURBO DNase Buffer (0.1 volume) and 1 μl of TURBO DNase.
    2. Incubate RNA at 37 °C for 20 min and halt the reaction by adding DNase Inactivation Reagent (0.1 volume).
    3. Incubate RNA samples for 5 min with intermittent mixing by flicking tube then centrifuge at 10,000 x g at room temperature for 2 min.
    4. Collect RNA (supernatant) and quantitate with NanoDropTM 2000 Spectrophotometer.
    Note: The average amount of total RNA obtained from each flask is 1.5-2 μg.
  4. Pre-load Protein G Sepharose® 4 Fast beads with rabbit serum control or anti-m3G-cap antibody (Figure 1).
    Note: Use large-orifice pipet tips or clip the tip off of a pipet tip to aid the transfer of Protein G Sepharose® 4 Fast beads.
    1. Wash 4 times the Protein G Sepharose® 4 Fast beads (40 μl slurry/IP sample) each with 1 ml NET-2 Buffer (see Recipes) using short 30 sec spins at 1,000 x g to pellet beads.
      Note: Non-filtered gel-loading pipet tips can be used during ‘wash’ steps to help prevent accidental loss of beads.
    2. Dilute washed Protein G Sepharose beads in 500 μl of NET-2 Buffer and add rabbit anti-m3G-cap antibody (15 μl/IP sample, approximately 150 μg) or rabbit serum control (15 μl/IP sample, approximately 150 μg). To achieve equal loading of beads with antibodies only use one microcentrifuge tube/antibody; aliquot the beads into separate tubes after they are pre-loaded.
    3. Incubate beads/antibody for 1.5 h at room temperature on tube rotator; make sure samples are actually mixing.
    4. Remove excess antibody by washing Protein G Sepharose Beads 5 times each with 1 ml NET-2 Buffer (short 30 sec spins at 1,000 x g); Resuspend beads in NET-2 Buffer (50 μl/sample).
    5. Aliquot antibody-bound Protein G Sepharose Beads into 1.5 microcentrifuge tubes; use enough tubes to have one of each antibody (anti-m3G-cap and control) per sample.
  5. Pre-clear RNA with Protein G Sepharose Beads to reduce non-specific binding:
    1. Add RNasin Plus RNase inhibitor (1 μl) to RNA (10 μg/500 μl diluted with NET-2 buffer) and incubate for 3 to 5 min at 85 °C.
      Note: Plunge RNA into ice immediately after heating to avoid refolding of RNA.
    2. After heating, add an additional 1 μl of RNasin Plus RNase inhibitor to RNA.
      Note: Remember to wipe tubes with RNase AWAYTM Surface Decontaminant before opening to prevent possible contamination and degradation of RNA.
    3. Rotate RNA (10 μg/sample for each antibody) slowly (without vibration) with 40 μl of NET 2-washed Protein G Sepharose 4 Fast Flow beads (without antibody) for 2 h at 4 °C.
    4. Centrifuge RNA at 1,000 x g for 2 min to remove pre-clearing beads.
  6. Rotate pre-cleared RNA slowly (without vibration) with anti-m3G-cap or control pre-loaded Protein G Sepharose® 4 Fast beads for 4 to 16 h at 4 °C. Confirm that samples are mixing (Figure 1).
  7. Collect the beads by centrifuging at 1,000 x g for 2 min and remove supernatant (save 250 μl for RNA extraction).
  8. Wash beads 5 times with 1 ml of NET-2 Buffer using short 30 sec spins at 1,000 x g.
    Note: RNasin Plus RNase inhibitor can be added to NET-2 Buffer (see Recipes) to prevent degradation of RNA.
  9. Resuspend beads in 250 μl of G-50 Buffer (see Recipes) to elute TMG-capped RNA (Figure 1).
  10. Purify RNA by phenol-chloroform-isoamyl alcohol (PCI) extraction (also extract RNA from TMG-capped depleted supernatant)
    1. Add 250 μl of phenol-chloroform-isoamyl alcohol to RNA in G-50 Buffer.
    2. Vortex RNA samples for 20 sec (10 up and 10 angled) and centrifuge samples for 10 min at 12,000 x g at room temperature.
    3. Transfer the aqueous phase with RNA (upper layer) to a clean microcentrifuge tube containing 50 μl of 3 M sodium acetate (NaOAc), pH 5.2.
    4. Add 1 ml of 100% ethanol (precooled to -20 °C) to precipitate RNA; 1 μl of GlycoBlue can be added to help precipitate RNA and visualize pellets.
    5. Incubate RNA at -20 °C for 48 h.
    6. Pellet RNA by centrifuging sample at 12,000 x g for 10 min at 4 °C.
    7. Aspirate ethanol and wash RNA pellet with 500 μl of 75% ethanol (precooled to -20 °C).
    8. Re-pellet purified RNA by short spin for 1 min at 12,000 x g at 4 °C and aspirate 75% ethanol.
    9. Dissolve the dried m3G-capped RNA pellet in nuclease-free water (20 to 30 μl).
    Note: The average amount of pull-down m3G-capped RNA obtained from each experiment (5 flasks) is 1.5-2 μg.
  11. RT-PCR of m3G-capped RNAs by using iScriptTM cDNA Synthesis Kit and 2x SsoAdvancedTM SYBR® Green Supermix PCR according to manufacturer’s protocols:
    Note: Perform RT-PCR using un-processed RNA (input) and RNA extracted from supernatant to determine expression levels of RNA of interest (m3G-capped and uncapped).
    1. Mix 5 μl of m3G-capped RNA with 5x iScript Reaction Mix (0.2 volume) and 1 μl of iScript Reverse Transcriptase.
    2. Process RNA to cDNA in a thermocycler by undergoing the following conditions: 25 °C for 5 min, 46 °C for 20 min, 95 °C for 1 min, and held at 4 °C. The reaction can be stored at 4 °C for short-term storage or -20 °C for long-term storage.
    3. Mix the resulting cDNA (around ¼ of the total cDNA reaction mix) with 2x SsoAdvancedTM SYBR® Green Supermix (0.5 volume) and the desired forward and reverse primers (12.5 μM stock; 0.04 volume).
    4. cDNA then undergoes the following conditions to produce a PCR product: 95 °C for 1.5 min; 40 cycles: 95 °C for 30 sec, 60 °C for 30 sec.
    5. The PCR product is ran on a 1% agarose gel (made with 1x TBE [see Recipes] and ethidium bromide) at 100 V for 2 h, and bands are imaged with a UV transilluminator.

Data analysis

The recovery of (TMG)-capped RNAs was measured by RT-PCR amplification of well-known hypermethylated RNAs such as small nuclear RNA (snRNA) U7 (Figure 2) or small nucleolar RNA (snoRNA) U3 (Martinez et al., 2017).

Figure 2. RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs). RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.


This protocol could be modified to determine the location of TMG-capped RNA by separation of nuclear and cytoplasmic RNA fractions from fresh cultured cells (PARISTM Kit).


Note: All reagents should be made with nuclease-free water and autoclaved.

  1. NET-2 Buffer
    150 mM NaCl
    0.05% NP-40
    50 mM Tris-HCl, pH 7.4
  2. G-50 buffer
    20 mM Tris, pH 7.5
    300 mM NaOAc
    2 mM EDTA, pH 8
    0.25% SDS
  3. 1x TBE
    216 g Tris base
    110 g boric acid
    80 ml 0.5 M EDTA, pH 8
    1.5 L water
    (Bring solution to pH 8.3)


We thank Dr. Joan Steitz’s laboratory for sharing part of this protocol. I.M., J.A.B., and K.E.H. were supported in part by a WVU Foundation Fund (2V882). K.E.H. was supported in part by funding from the Ladies Auxiliary to the VFW of the United States (CK 003229). M.X. is supported by NIH grant R00 CA190886. J.A.S. is an Investigator of the Howard Hughes Medical Institute. This protocol was modified from previous work (Yu et al., 1998). We do not have any conflict of interest or competing interests.


  1. Boulon, S., Verheggen, C., Jady, B. E., Girard, C., Pescia, C., Paul, C., Ospina, J. K., Kiss, T., Matera, A. G., Bordonne, R. and Bertrand, E. (2004). PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli. Mol Cell 16(5): 777-787.
  2. Coller, H. A. (2011). Cell biology. The essence of quiescence. Science 334(6059): 1074-1075.
  3. Esteller, M. (2011). Non-coding RNAs in human disease. Nat Rev Genet 12(12): 861-874.
  4. Ha, M. and Kim, V. N. (2014). Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15(8): 509-524.
  5. Kim, Y. K., Kim, B. and Kim, V. N. (2016). Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis. Proc Natl Acad Sci U S A 113(13): E1881-1889.
  6. Kiss, T. (2004). Biogenesis of small nuclear RNPs. J Cell Sci 117(Pt 25): 5949-5951.
  7. Luhrmann, R., Appel, B., Bringmann, P., Rinke, J., Reuter, R., Rothe, S. and Bald, R. (1982). Isolation and characterization of rabbit anti-m3 2,2,7G antibodies. Nucleic Acids Res 10(22): 7103-7113.
  8. Martinez, I., Hayes, K. E., Barr, J. A., Harold, A. D., Xie, M., Bukhari, S. I. A., Vasudevan, S., Steitz, J. A. and DiMaio, D. (2017). An Exportin-1-dependent microRNA biogenesis pathway during human cell quiescence. Proc Natl Acad Sci U S A 114(25): E4961-E4970.
  9. Ohno, M., Segref, A., Bachi, A., Wilm, M. and Mattaj, I. W. (2000). PHAX, a mediator of U snRNA nuclear export whose activity is regulated by phosphorylation. Cell 101(2): 187-198.
  10. Palacios, I., Hetzer, M., Adam, S. A. and Mattaj, I. W. (1997). Nuclear import of U snRNPs requires importin β. EMBO J 16(22): 6783-6792.
  11. Valcourt, J. R., Lemons, J. M., Haley, E. M., Kojima, M., Demuren, O. O. and Coller, H. A. (2012). Staying alive: metabolic adaptations to quiescence. Cell Cycle 11(9): 1680-1696.
  12. Yedavalli, V. S. and Jeang, K. T. (2010). Trimethylguanosine capping selectively promotes expression of Rev-dependent HIV-1 RNAs. Proc Natl Acad Sci U S A 107(33): 14787-14792.
  13. Yu, Y. T., Shu, M. D. and Steitz, J. A. (1998). Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing. EMBO J 17(19): 5783-5795.


蜂窝静止(因此已知为G <子> 0 骤停)是由降低的DNA复制,增加自噬表征,并且增加的细胞周期蛋白依赖性激酶p27蛋白上标kip1 表达。静止对伤口愈合,器官再生和瘤形成是必不可少的。先前的发现表明微小RNA(miRNA)在调节细胞静止过程中起重要作用。我们最近的出版物静止期间以实例阐述在原代人替代miRNA生物途径包皮成纤维(HFF)细胞的存在。实际上,我们已经发现了一组与由trimethylguanosine合酶1(TGS1)蛋白的2,2,7- trimethylguanosine(TMG)带肩改性PRI-的miRNA(其成熟miRNA发现静止期期间诱导的)的并运输到细胞质通过Exportin-1(XPO1)蛋白质。我们用来抗体针对(TMG)兴趣盖(不与第(m交叉反应 7 G)兴趣帽了大部分PRI-的miRNA或mRNA的含有[鲁曼等人的,1982]),以从增殖或静态HFFS的总RNA提取RNA进行免疫沉淀。该测定的新颖性是PRI-miRNA的以及含有一个TMG-帽修改以外的非编码RNA的特异性分离。

【背景】蜂窝静止,类型可逆生长停滞的,是在伤口愈合,器官再生,和预防瘤形成涉及一种重要的细胞状态(科勒,2011;瓦尔古等人 2012)。已发现小的非编码RNA如miRNA参与细胞静止的调节。 miRNA是小的非编码RNA〜22个核苷酸长做调节的蛋白质编码基因通过碱基配对与所述3“非翻译区信使RNA的(3'UTR)(的mRNA)(Esteller,2011)的表达。规范的miRNA生物发生途径基于逐步加工机制(Ha和Kim,2014,Kim等人,2016)。 miRNA被转录产生初级miRNA(pri-miRNA),其具有由Drosha及其结合伴侣DGCR8在细胞核中识别的不完全环结构。 pri-miRNA的切割产生被输出蛋白-5(XPO5)蛋白识别并转运至细胞质的前体miRNA(pre-miRNA)。 pre-miRNA被Dicer(成熟miRNA)切割并加载到RNA诱导的沉默复合物(RISC)中。另一方面,与U1,U2,U4和U5等mRNA加工有关的小核RNA(snRNA)的前体具有(m 7 G)帽,其被帽子识别结合复合物(CBC)和磷酸化的适配器用于核输出中的RNA输出(PHAX)以使其能够通过XPO1输出到细胞质(Ohno等,2000)。然后这些snRNAs通过SEC1 / Munc18(SM)蛋白识别(通过结合至Sm结合位点序列)在细胞质和TGS1被招募到hypermethylate(M 7 G)带肩成(米2,2,7-G,TMG) - 帽。这种修改是通过Snuportin-1与输入蛋白-β和其它因素协认识导入snRNAs回核(Palacios的等人,1997;吻,2004)。有趣的是,XPO1因此具有用于(TMG)高亲和力在细胞核中加帽小核仁RNA(的snoRNA)U3和从卡哈尔体到核仁传输它(Boulon 等人,2004年)。先前的研究表明确实TGS1增强版本依赖性HIV-1 RNA的表达由(TMG)在细胞核-capping病毒mRNA,由此通过XPO1运输增加识别到细胞质(Yedavalli和Jeang,2010)。这些研究结果表明TMG封顶的RNA为不同类型的RNA分子提供可塑性,以调节其加工和细胞定位。我们最近的发现实例阐述的基团与2,2,7- trimethylguanosine(TMG)改性PRI-的miRNA通过帽TGS1蛋白的存在和静止期间由XPO1运至细胞质。以前的出版物已经显示下拉(TMG)的RNA带肩的能力:如snRNA的和snoRNAs,用针对(TMG)的RNA帽特异性抗体(鲁曼等人,1982年。)。我们之前的出版物展示了人类细胞中(TMG) - 帽pri-miRNA的下拉(Martinez等人,2017)。了解哪些RNA可以用TMG帽进行修饰,将为正常或疾病相关条件下的RNA生物合成提供新的见解。

关键字:m2,2,7G帽RNA, TMG帽RNA, 三甲基化RNA, RNA免疫沉淀, Pri-miRNA


  1. 1.5ml微量离心管(Fisher Scientific,目录号:05-408-129)。

  2. 15 ml锥形离心管(不含DNase / RNase)(Corning,目录号:430052)。
  3. 吉尔森 TM EXPERT TM 大学适合吸移管过滤嘴(吉尔森,目录号:F1731031,F1733031,F1735031,F1737031)
  4. 凝胶加样移液枪头(Fisher Scientific,目录号:02-707-139)。
  5. 大口径移液枪头(Fisher Scientific,目录号:02-707-134)。

  6. 无菌聚苯乙烯一次性血清移液器(Greiner Bio One International,目录号:710180)
  7. 血清移液器
    2毫升血清移液器(Fisher Scientific,目录号:13-678-11C)。
    5毫升血清移液器(Fisher Scientific,目录号:13-678-11D)。
    10毫升血清移液器(Fisher Scientific,目录号:13-678-11E)。
    25毫升血清学移液器(Fisher Scientific,目录号:13-678-11)。
  8. 150厘米 2 排出组织培养瓶治疗中(Corning,隼®,目录号:355001)
  9. 100毫米TC处理的细胞培养皿(Corning,Falcon ,产品目录号:353033)
  10. 细胞刮刀(Fisher Scientific,目录号:08-100-242)。
  11. HFF细胞(来自ATCC HFF细胞的替代来源:HS27(ATCC,目录号:CRL-1634))(从耶鲁皮肤病研究中心获得)
  12. DMEM(Sigma-Aldrich,目录号:D7777)
  13. 最小基本培养基(MEM)非必需氨基酸,100x(Thermo Fisher Scientific,目录号:11140076)。
  14. 0.05%胰蛋白酶-EDTA与酚红(Thermo Fisher Scientific,目录号:25300054)。
  15. 10x PBS(Sigma-Aldrich,目录号:P5493)
  16. 无核酸酶的水(未经DEPC处理)(Thermo Fisher Scientific,目录号:AM9937)。
  17. TRIzol TM试剂(Thermo Fisher Scientific,目录号:15596026)。
  18. RNase AWAY TM表面去污剂(Thermo Fisher Scientific,目录号:7002)
  19. 氯仿(Sigma-Aldrich,目录号:C2432)。
  20. 乙醇,分子生物学级(Fisher Scientific,目录号:BP2818-500)。
  21. 异丙醇,分子生物学级(Fisher Scientific,目录号:BP26184)。
  22. GlycoBlue TM 共沉淀(15毫克/毫升)(赛默飞世尔科技,产品目录号:AM9516)
  23. TURBO DNA-free TM试剂盒(Thermo Fisher Scientific,目录号:AM1907)
  24. 抗m3G帽,兔多克隆,抗血清(突触系统)*
  25. 蛋白G Sepharose 4 Fast Flow Beads(GE Healthcare,产品目录号:17061801)
  26. 正常兔血清(对照,EMD Millipore,目录号:NS01L-1ML)。
  27. 氯化钠(NaCl)(Fisher Scientific,目录号:S671-3)。
  28. NP-40(Thermo Fisher Scientific,目录号:28324)。
  29. Tris碱(Fisher Scientific,目录号:BP152-5)。
  30. 盐酸(HCl)(VWR,目录号:BDH7204-1)。
  31. RNasin TM Plus RNase抑制剂(Promega,目录编号:N2611)
  32. NaOAc(AMRESCO,目录号:0602)
  33. 乙二胺四乙酸(EDTA),pH8(Thermo Fisher Scientific,目录号:AM9260G)。
  34. 乙二胺四乙酸(EDTA)(AMRESCO,目录号:0105)
  35. 十二烷基硫酸钠(SDS)(Thermo Fisher Scientific,目录号:AM9822)
  36. 苯酚/氯仿/异戊醇; 125:24:1混合物,pH4.5(Thermo Fisher Scientific,目录号:AM9720)。
  37. 琼脂糖LE(Denville Scientific,目录号:CA3510-8)。
  38. iScript TM cDNA合成试剂盒(Bio-Rad Laboratories,目录号:1708891)。
  39. Sso Advanced TM Universal SYBR Green Supermix(Bio-Rad Laboratories,目录号:1725274)
  40. PARIS TM Kit(Thermo Fisher Scientific,目录号:AM1921)
  41. 硼酸(Fisher Scientific,目录号:A73-1)。
  42. NET-2缓冲区(见食谱)
  43. G-50缓冲液(见食谱)
  44. 1倍TBE(见食谱)


  1. 微量移液管(Gilson,型号:Pipetman L,目录号:F167370)
  2. FORMA TM 免缝周期 TM CO <子> 2 培养箱(Thermo Scientific的,型号:FORMA TM 免缝周期 TM培养箱,目录号:370)
  3. -80°C冷冻
  4. SORVALL TM 图例 TM 微21R微量(赛默飞世尔科技,型号:SORVALL TM 图例 TM 微21R目录号码:75002490)
  5. 的Eppendorf TM Thermomixer中 TM R(的Eppendorf,型号:Thermomixer中R,目录号:05-412-401)
  6. Labquake TM Tube Shaker / Rotator(Thermo Fisher Scientific,产品目录号:C4152110Q)。
  7. SORVALL TM ST 40R离心机(赛默飞世尔科技,型号:SORVALL TM ST 40R,目录号:75004525)
  8. 纳米滴 TM 2000分光光度计(Thermo Fisher Scientific公司,型号:纳米滴 TM 2000,目录号:ND-2000)
  9. T100 TM热循环仪(Bio-Rad Laboratories,目录号:1861096)。
  10. CFX Connect TM实时PCR检测系统(Bio-Rad Laboratories,目录号:1855200)
  11. 紫外透照器


  1. 板HFF细胞在无血清DMEM补充有以低密度MEM非必需氨基酸(约1×10 6 细胞/150厘米 2 烧瓶)。在5%CO 2的潮湿空气中37°C孵育HFF细胞48小时(图1)。
    1. 为了获得足够的RNA用于每个实验中,使用4至6150厘米 2 烧瓶中。
    2. 在无血清DMEM中培养HFF细胞;洗70-80%汇合的HFF细胞英寸(100毫米TC处理的细胞培养皿)用10ml磷酸盐缓冲盐水各三次,加入1ml 0.05%胰蛋白酶与酚红以分离细胞,加入10毫升的无血清DMEM培养基和将细胞收集在15ml锥形管中,通过在室温下以300xg离心沉淀细胞,并在无血清DMEM补充培养基中重新悬浮沉淀。
    3. 由于培养基不含有FBS以使胰蛋白酶失活,因此在重新接种之前从HFF细胞中去除胰蛋白酶是重要的。
  2. 通过在冰上刮瓶收集HFF细胞,在4℃下300g×5g颗粒沉淀,并用2ml磷酸盐缓冲盐水洗涤。细胞沉淀可以在-80冻结℃,直到RNA提取(细胞沉淀必须保持在冰上直到用TRIzol TM 裂解)(图1)。
    1. 为了沉淀HFF细胞,从细胞中除去无血清的旧DMEM,并加入3ml新鲜的无血清DMEM。使用细胞刮刀的刀片收集烧瓶角落的细胞。在冰上的细胞中加入1-2%体积的FBS或BSA以帮助造粒。
    2. 在这个协议中的所有步骤应在无RNA酶的长椅面积通过使用RNA酶AWAY TM 表面净化剂喷雾和擦拭最表面区域包括微型移液管和冰桶来进行。

    1. 在1ml TRIzol TM试剂中重悬在冰上解冻的细胞沉淀5分钟以裂解。
    2. 加入氯仿(TRIzol TM试剂体积的20%),充分混合样品,并在室温下孵育2-3分钟。
    3. 离心机样品在12000 ×g的在4℃下15分钟以分离红色的苯酚 - 氯仿(红/下层),界面和wässrige相位(清零/上层)。
    4. 用异丙醇(0.5体积的原始TRIzol TM试剂)在室温下收集层和沉淀RNA 10分钟;此时可加入GlycoBlue TM共沉淀剂(15μg)以辅助RNA沉淀。
    5. 沉淀通过离心沉淀RNA(12,000×g,4℃10分钟)。
    6. 洗涤RNA沉淀用75%乙醇(加在等体积的75%乙醇,以TRIzol试剂 TM 试剂,并简要涡流)中并再沉淀通过在7500离心 XG 5分钟在4℃
    7. 空气干燥的RNA沉淀几分钟,重新悬浮在无核酸酶的水中。
  3. 使用来自TURBO DNA-free TM试剂盒的DNA酶根据制造商的方案消化RNA样品中的DNA污染物:
    1. 将RNA与10x TURBO DNase缓冲液(0.1体积)和1μlTURBO DNase混合。
    2. RNA在37°C孵育20分钟,并通过添加脱氧核糖核酸酶灭活试剂(0.1体积)停止反应。
    3. 孵育5分钟间歇搅拌RNA样品通过轻弹管然后以10,000 ×g的离心在室温下搅拌2分钟。
    4. 收集RNA(上清液)并用NanoDrop TM 2000分光光度计定量。
  4. 预负荷蛋白G琼脂糖® 4个快速珠粒与兔血清对照或抗M3G帽抗体(图1)。
    注意:使用大孔移液管尖端或夹前端关闭吸管头,以帮助蛋白质的转移G琼脂糖 ® 4个快珠子。
    1. 洗蛋白G琼脂糖四次® 4个快速珠(40微升浆料/ IP样品)各用1ml NET-2缓冲液(见配方),使用短30秒的转速为1000 XG <去粒珠。
    2. 稀于500微升NET-2缓冲液洗蛋白G琼脂糖珠,并添加兔抗M3G帽抗体(15微升/ IP样品,将大约150微克)或兔血清对照(15微升/ IP样品,将大约150微克)。为了达到与抗体相等的珠粒加载,仅使用一个微量离心管/抗体;
    3. 在管旋转器上在室温下孵育珠/抗体1.5小时;确保样品实际上是混合。
    4. 通过洗涤蛋白G琼脂糖珠除去过量的抗体每5次用1ml NET-2缓冲液(短30秒转速为1.000 ×g的);在NET-2缓冲液中重悬珠(50μl/样品)。
    5. 将抗体结合的蛋白G琼脂糖珠分装到1.5微量离心管中;每个样品使用一种抗体(抗M3G帽和对照)。
  5. 用蛋白G Sepharose珠预清除RNA以减少非特异性结合:
    1. 添加的RNasin加RNase抑制剂(1微升)以RNA(10微克/微升500与NET-2缓冲液中稀释),并在85℃下孵育3至5分钟
    2. 加热后,再加入1μLRNasin Plus RNase抑制剂至RNA。
      注意:请记住AWAY用RNase擦拭管 TM 开口之前的表面去污剂,以防止RNA的可能的污染和降解
    3. 旋转RNA用40微升的NET 2洗涤的蛋白G琼脂糖4快流珠(没有抗体)处理2小时,在4℃(10微克/每个抗体样品)慢慢地(无振动)

    4. 1.000克xg离心RNA 2分钟以去除预清除珠粒。
  6. 旋转预澄清RNA缓慢(无振动)用抗M3G帽或控制预加载蛋白质G琼脂糖® 4个快速珠粒对4至16小时,在4℃下确认样本正在混合(图1)。
  7. 通过在1,000 ×g的 2分钟离心收集珠并去除上清液(保存250微升提取RNA)。
  8. 用1ml的NET-2缓冲液洗涤珠子5次,使用1.000gxg的短暂30秒自旋。
    注意:可以将RNasin Plus RNase抑制剂添加到NET-2缓冲液中(请参阅食谱)以防止RNA降解。
  9. 用250μlG-50缓冲液重悬微珠(参见食谱)以洗脱TMG封端的RNA(图1)。
  10. 通过酚 - 氯仿 - 异戊醇(PCI)提取纯化RNA(即从TMG封端的耗尽的上清液中提取RNA)
    1. 在G-50缓冲液中加入250μl酚 - 氯仿 - 异戊醇到RNA中。
    2. 持续20秒涡旋RNA样品(10向上和10成角度的),并离心样品10分钟,在12,000 ×g的在室温下。
    3. 50μl的3M醋酸钠(NaOAc),pH5.2。
    4. 加入1ml 100%乙醇(预冷至-20℃)以沉淀RNA;可以添加1μl的GlycoBlue来帮助沉淀RNA并使颗粒可视化。

    5. 在-20°C孵育RNA 48小时
    6. 通过在4℃下将样品在12,000×gg离心10分钟来沉淀RNA。
    7. 吸取乙醇,用500μl75%乙醇(预冷至-20°C)洗涤RNA沉淀。
    8. 通过在4℃以12,000×gg短时间旋转1分钟重新沉淀纯化的RNA,并抽吸75%乙醇。
    9. 将干燥的m3G-封端的RNA沉淀溶于无核酸酶的水(20至30μl)中。
  11. 通过使用的iScript TM cDNA合成试剂盒和RT-PCR M3G封端的RNA 2X SsoAdvanced TM SYBR ® Green超PCR雅丁制造商的方案:
    1. 混合5微升M3G加盖的RNA与5x iScript反应混合物(0.2体积)和1微升iScript逆转录酶。
    2. 过程RNA成cDNA中通过进行下述条件的热循环:25℃5分钟,46℃20分钟,95℃1分钟,并在4℃下保持该反应可以储存在4°C短期储存或-20°C长期储存。
    3. 混合得到的cDNA(总的cDNA反应混合物的周围1/4)用2x SsoAdvanced TM SYBR ® Green超(0.5体积)和所需的正向和反向引物(12.5 UM股票; 0.04卷)。
    4. PCR产物:95℃1.5分钟; 40个循环:95℃30秒,60℃30秒。
    5. 将PCR产物跑1%琼脂糖凝胶在100V进行2小时(用1x TBE [见配方]和溴化乙锭制造),和条带与一个紫外透射仪成像。




的图2(TMG)的RNA免疫沉淀在静态人类加帽初级miRNA的(PRI-miRNA的)包皮成纤维细胞(HFFS)。 RT-PCR的数据显示在静止期HFFS小学-的miR-34a和小学-MIR-3188的RNA免疫沉淀(以及作为阳性对照的snRNA U7),但不小学-MIR-423使用抗体对( TMG)加帽的RNA。装载有对照抗体(正常兔血清,密理博)或15微升总RNA使用Trizol试剂,其提取,RNA的10微克,其稀释在NET-2缓冲器,预澄清,并用蛋白G琼脂糖4个孵育Fast Flow的珠识别(TMG)帽(抗-m3G帽,兔多克隆,突触系统)的抗体。用NET-2缓冲液漂洗珠子5次,并重新悬浮在G-50缓冲液中。通过苯酚 - 氯仿 - 异戊醇萃取从珠中提取RNA并重新悬浮于20μl无核酸酶的水中。免疫沉淀的三甲基化的加帽的RNA使用的iScript cDNA合成试剂盒(Bio-Rad公司),然后通过RT-PCR,其转化成cDNA,并在凝胶电泳后可见。


该协议可以通过从新鲜培养的细胞(PARIS TM 试剂盒)核和细胞质RNA级分的分离进行修改,以确定矿TMG-加帽的RNA的位置。



  1. NET-2缓冲区
    150 mM NaCl。
    50mM Tris-HCl,pH7.4
  2. G-50缓冲液
    20 mM Tris,pH 7.5。
    300 mM NaOAc。
    2mM EDTA,pH 8
  3. 1x TBE
    80ml 0.5M EDTA,pH8
    (将溶液带到pH 8.3)


我们感谢Dr. Joan Steitz的实验室共享这个协议的一部分。 I.M.,J.A.B.和K.E.H.得到了西弗吉尼亚大学基金会(2V882)的部分支持。 K.E.H.这是由美国VFW辅助女士(CK 003229)支持的。 M.X.由NIH资助R00 CA190886支持。 J.A.S.霍华德休斯医学研究所的调查员。该协议是从以前的工作进行修改(Yu等人,1998)。我们没有任何利益冲突或利益冲突。


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引用:Hayes, K. E., Barr, J. A., Xie, M., Steitz, J. A. and Martinez, I. (2018). Immunoprecipitation of Tri-methylated Capped RNA. Bio-protocol 8(3): e2717. DOI: 10.21769/BioProtoc.2717.