Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.
Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.
Human telomerase reverse transcriptase (TERT) is well known as the catalytic subunit of telomerase, and it elongates telomere using telomerase RNA component (TERC), the specific RNA template1. Although TERT polymerizes telomeric DNA as a component of telomerase, the structural and phylogenetic analyses indicate that TERT is closely related with viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase, and shares domains with these polymerases2,3,4. RdRP is the enzyme that generates complementary RNA strand to a template RNA. The enzyme is encoded not only in viruses but also in model organisms, such as plant, yeast and worm, and double-stranded RNA synthesis by RdRP contributes to transcriptional and post-transcriptional gene silencing in these organisms5,6. Although human RdRP had been missing for a long time, we found RdRP activity in human TERT in 20097.
We first confirmed RdRP activity of TERT with recombinant protein7, then established a sensitive in vitro assay to detect RdRP activity of endogenous TERT8. Here, we demonstrate the in vitro RdRP assay (IP-RdRP assay) for endogenous TERT. This method starts with immunoprecipitation (IP) of endogenous TERT, and is followed by in vitro RdRP reaction, in which radioactive ribonucleotides are incorporated into nascent RNA strands.
1. Reagent Setup
2. Preparation of HeLa cells
NOTE: Prepare HeLa cells synchronized in mitotic phase (manipulated) or dividing asynchronously (unmanipulated). Culture the cells in the presence of 5% CO2 at 37 °C.
3. IP-RdRP Assay
With the recommended RNA template, radioactive RdRP products are observed between 20 to 30 nucleotides (nt) specifically in HeLa cells in mitotic phase after overnight exposure (Figure 1A). Typically, signal intensity of the RdRP products demonstrates two peaks that are positioned around 25 nt and 30 nt in consonance with the size distribution of the products confirmed by next generation sequencing9. In contrast to mitotic cells, HeLa cells without synchronization demonstrate almost absence of the 20–30 nt radioactive products. Oval signals, that are placed below 20 nt in all samples, are nonspecific drifts.
In case that there is only a ~30 nt product with weak signal in mitotic HeLa cells, it indicates that the RdRP reaction did not go well. For example, if MNase treatment is performed roughly and inappropriately, the signal intensity in mitotic HeLa cells decreases significantly (Figure 1B). RdRP reaction performed with old HMD solution also reduces the products (Figure 1C).
Figure 1: Representative RdRP products of endogenous TERT in mitotic HeLa cells. (A) Three representative results of the IP-RdRP assay in HeLa cells treated with nocodazole (manipulated) or DMSO (unmanipulated). The chemically synthesized 34 nt RNA was used as a template. (B) IP-RdRP assay in mitotic HeLa cells performed with inappropriate MNase treatment. (C) IP-RdRP assay in mitotic HeLa cells performed with either fresh or old HMD solution. Please click here to view a larger version of this figure.
Reagents | Volume |
Micrococcal Nuclease, 20 U/µL | 1 µL |
Micrococcal Nuclease Buffer, 10x | 8 µL |
RNase-free water | 51 µL |
Table 1: MNase reaction mix component.
Reagents | Volume |
CHAPS, 0.07% (wt/vol) | 6 µL |
HMD solution | 2 µL |
rATP, 80 mM | 0.5 µL |
rGTP, 8 mM | 1 µL |
rUTP, 0.4 mM | 1 µL |
rCTP, 8 mM | 1 µL |
RNase inhibitor, 40 U/µL | 0.5 µL |
RNase-free water | 1 µL |
Table 2: RdRP reaction mix component.
Reagents | Volume |
RNase ONE Ribonuclease, 10 U/µL | 0.2 µL |
Reaction Buffer, 10x | 20 µL |
RNase-free water | 159.8 µL |
Table 3: Ribonuclease reaction mix component.
The IP-RdRP assay is a sensitive method to detect RdRP activity of human TERT. TERT protein is highly expressed in mitotic HeLa cells, in which TERT forms the RdRP complex8,9,10. This suggests that mitotic HeLa cells are an optimal material to detect RdRP activity. In the protocol described above, mitotic and non-synchronized HeLa cells are included as a positive and a negative example, respectively. As shown in Figure 1A, RdRP assay products from the recommended RNA template in mitotic HeLa cells show broad radioactive signals between 20 to 30 nt. In addition to HeLa cells, we have performed the assay with different types of cell lines, and found that the signal pattern can be changed in different cell types9: some show a strong signal only around 30 nt, some show a similar pattern with HeLa cells. We have also performed the assay with RNA templates other than the 34 nt template8, short and long (~300 nt) RNAs with various sequences, and successfully obtained RdRP products from those templates although there may be some preference. For the first trial, however, we recommend using HeLa cells in mitotic phase and the 34 nt RNA template. RdRPs can generate double-stranded RNA both in a primer-dependent and in a primer-independent manner11. We have reported that TERT preserves this property as human RdRP7,9; TERT synthesizes dsRNA from an RNA template with 3'-foldback structure through a back-priming mechanism7. For the 34 nt RNA template, TERT synthesizes complementary strands without using primers9. To specifically detect RdRP products synthesized in a primer-independent manner, i.e. de novo synthesized RNA products, [α-32P]NTP can be replaced with [γ-32P]NTP in the RdRP reaction9.
MNase treatment of TERT immune complexes on beads is a critical step to achieve desired results. If the MNase treatment is performed too long or with intensive shaking, the RdRP products would be reduced remarkably. To avoid such a trouble, we strongly recommend to strictly follow the protocol carefully. HMD solution is another critical factor for success. If you find that the signals are very weak, replace the HMD solution to a newly prepared one.
Throughout the protocol, one should take great care to avoid RNase contamination. Ribonuclease treatment should be performed with dedicated equipment. After manipulating the Ribonuclease, one should discard tips and tubes with RNase immediately, eliminate RNase with specialized solution (e.g. RNase Quiet), and change groves.
TERT interacts with not only TERC but also many kinds of endogenous RNAs, and we have reported part of them7. Modification in the IP-RdRP assay, such as the IP-RdRP assay without MNase treatment, will provide a full list of endogenous RNA templates for TERT-associated RdRP activity and bioinformatic guide on their sequence features. We have successfully applied this protocol to tissue lysate. Because TERT is expressed in a variety of normal and tumor tissues, this assay might be useful to investigate non-canonical enzymatic function of TERT in human.
The authors have nothing to disclose.
This work was supported in part by the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT) (15cm0106102h0002, K.M.) and the Project for Cancer Research and Therapeutic Evolution (P-CREATE) (16cm01061150001, K.M.) from Japan Agency for Medical Research and Development, AMED; the Takeda Science Foundation (Y.M.); Grant of the Princess Takamatsu Cancer Research Fund (13-24520, Y.M.); and JSPS KAKENHI Grant Number JP16K07133 (Y.M.).
Dulbecco’s modified Eagle’s medium (DMEM) | Wako | 044-29765 | |
Fetal bovine serum (FBS) | CORNING | 35-010-CV | |
Penicillin-Streptomycin mixed solution | nacalai tesque | 26253-84 | |
Trypsin-Ethylenediamenetetraacetic acid (EDTA) solution | nacalai tesque | 32777-44 | |
Phosphate buffered saline (PBS(-)) | Wako | 166-23555 | |
Thymidine | nacalai tesque | 07147-61 | |
Nocodazole | Sigma | M1404 | |
Dimethyl sulfoxide (DMSO) | nacalai tesque | 08904-85 | |
Sodium chloride | nacalai tesque | 31333-45 | |
Tris(hydroxymethyl)aminomethane | nacalai tesque | 35434-21 | |
Hydrochloric acid | nacalai tesque | 18321-05 | |
Nonidet P-40 (NP-40) | nacalai tesque | 25223-04 | |
Pierce Protein A Plus Agarose | Thermo scientific | 22812 | |
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) | nacalai tesque | 17514-15 | |
Potassium hydroxide | Wako | 168-21815 | |
Potassium acetate | nacalai tesque | 28405-05 | |
Magnesium chloride hexahydrate (MgCl2•6H2O) | Wako | 135-00165 | |
Glycerol | nacalai tesque | 17045-65 | |
Polyoxyethylene(10) octylphenyl ether (Triton X-100) | Wako | 169-21105 | |
cOmplete, EDTA-free | Roche Applied Science | 11 873 580 001 | |
Calcium chloride dihydrate (CaCl2•2H2O) | nacalai tesque | 06731-05 | |
Micrococcal nuclease (MNase) | Takara Bio | 2910A | |
Ethylene glycol bis (b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) | nacalai tesque | 15214-92 | |
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS) | nacalai tesque | 07957-64 | |
Dithiothreitol (DTT) | nacalai tesque | 14128-91 | |
rCTP, rATP, rUTP, rGTP, 100mM each | Promega | E6000 | |
[a-32P]UTP (3,000 Ci/mmol) | PerkinElmer | NEG507H | Use fresh RI for the assay |
RNase inhibitor | TOYOBO | SIN-201 | |
Proteinase K | Takara | 9033 | |
Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1) | Life Technologies | AM9720 | |
3 M Sodium acetate | NIPPON GENE | 316-90081 | |
Ethanol (99.5) | nacalai tesque | 14713-95 | |
Dr. GenTLE Precipitation Carrier | Takara Bio | 9094 | |
RNase One Ribonuclease | Promega | M4265 | |
Sodium dodecyl sulfate (SDS) | nacalai tesque | 02873-75 | |
Formamide, deionized | nacalai tesque | 16345-65 | |
Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (EDTA) | nacalai tesque | 15130-95 | |
Orange G | nacalai tesque | 25401-22 | |
CO2 incubator | e.g., ASTEC | SCA-325DRS | |
Centrifuge | e.g., TOMY | EX-135 | For step B) |
Micro refrigerated centrifuge | e.g., KUBOTA | 3740 | For step 6.2 |
Sonicator | Qsonica | Q125 | Set a microtip (#4422) on the sonicator, for step 6.4 |
Micro refrigerated centrifuge | e.g., TOMY | MX-305 | For step 6.5 |
Cooled incubator | e.g., Panasonic | MIR-154-PJ | Set a rotary shaker inside |
Rotary shaker | e.g., TAITEC | RT-5 | |
Cooled incubator | e.g., TAITEC | BR-43FL | Set a shaker “MINI WAVE” inside, for step 7.7 |
MINI WAVE | AS ONE | WEV-03 | A shaker for steps 7.7, 8.5 and 8.6 |
Incubator | e.g., TAITEC | HB-80 | Set a shaker “MINI WAVE” inside, for step 8.5 and 8.6 |
Block incubator | e.g., ASTEC | BI-516S |