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.).

1. Reagent Setup
<ol>
	<li>Reagents used for Cell Synchronization
	<ol>
		<li>To generate Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 2.5 mM thymidine, dissolve 30.28 mg of thymidine per 1 mL of tissue culture grade water to prepare 125 mM thymidine. Filtrate the solution with a 0.22 &#956;m syringe filter unit. Add 1 mL of freshly prepared 125 mM thymidine per 50 mL of DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS).</li>
		<li>To generate DMEM containing 0.1 &#956;g/mL of nocodazole, dissolve nocodazole in dimethyl sulfoxide (DMSO) to prepare 5 &#956;g/&#956;L nocodazole. Add 1 &#181;L of 5 &#181;g/ &#181;L nocodazole per 50 mL of DMEM supplemented with 10% (vol/vol) FBS.</li>
		<li>To generate DMEM containing 0.002% (vol/vol) of DMSO, add 1 &#181;L of DMSO per 50 mL of DMEM supplemented with 10% (vol/vol) FBS.</li>
	</ol>
	</li>
	<li>Reagents used for IP-RdRP assay
	<ol>
		<li>Prepare lysis buffer A: 150 mM NaCl, 20 mM Tris-HCl (pH 7.4) and 0.5% (vol/vol) Nonidet P-40 (NP-40). Store the reagent at 4 &#176;C.</li>
		<li>Prepare 5x acetate buffer: 50 mM 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)-KOH (pH 7.8), 500 mM potassium acetate and 20 mM MgCl2. Store the reagent at room temperature.</li>
		<li>Prepare Wash buffer 1: 1x acetate buffer, 10% (vol/vol) glycerol, 0.1% (vol/vol) Triton X-100 and 0.06x protease inhibitor cocktail, ethylenediamenetetraacetic acid (EDTA)-free. Prepare Wash buffer 1 on the day of use or the day before use. Store the reagent at 4 &#176;C.</li>
		<li>Prepare AGC solution: 1x acetate buffer, 10% (vol/vol) glycerol and 0.02% (wt/vol) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS). Store the reagent at 4 &#176;C.</li>
		<li>To generate AGC solution containing 2 mM CaCl2, add 100 &#181;L of 1 M CaCl2 per 50 mL of AGC solution. Store the reagent at 4 &#176;C.</li>
		<li>To generate AGC solution containing 3 mM ethylene glycol bis (b-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), add 375 &#181;L of 0.4 M EGTA (pH 7.5) per 50 mL of AGC solution. Store the reagent at 4 &#176;C.</li>
		<li>Prepare Wash buffer 2: 1x acetate buffer and 0.02% (wt/vol) CHAPS. Store the reagent at 4 &#176;C.</li>
		<li>Prepare HMD solution: 0.2 M HEPES-KOH (pH 7.8), 40 mM MgCl2 and 2 mM dithiothreitol (DTT). Store the reagent at -20 &#176;C. 
		NOTE: HMD solution should be renewed at least in three months.</li>
		<li>Prepare 2x Proteinase K buffer: 20 mM Tris-HCl (pH 7.6), 20 mM EDTA and 1% (wt/vol) sodium dodecyl sulfate (SDS). Store the reagent at -20 &#176;C.</li>
		<li>Prepare 2x loading buffer: 95% (vol/vol) formamide, 20 mM EDTA and 1% (wt/vol) Orange G. Store the reagent at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
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 &#176;C.
<ol>
	<li>Synchronization of HeLa cells in mitosis
	<ol>
		<li>Plate 1 x 106 of HeLa cells with 10 mL of DMEM supplemented with 10% (vol/vol) FBS in a 10 cm culture dish.</li>
		<li>Two days after plating, replace the medium with 10 mL of DMEM containing 2.5 mM thymidine and 10% (vol/vol) FBS. Culture the cells for 24 h.</li>
		<li>Wash the cells three times with 10 mL of phosphate buffered saline (PBS) (-), and culture the cells with 10 mL of DMEM containing 10% (vol/vol) FBS for 6 h.</li>
		<li>Replace the medium with 10 mL of DMEM containing 0.1 &#181;g/mL of nocodazole and 10% (vol/vol) FBS, and culture the cells for 14 h.</li>
		<li>Shake the culture dish gently, and retrieve the supernatant containing the detached mitotic cells. Count the cell number for the IP-RdRP assay.</li>
		<li>Centrifuge the sample at 490 x g for 5 min at 4 &#176;C, and discard the supernatant. 
		NOTE: The cell pellet can be stored at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of unmanipulated HeLa cells
	<ol>
		<li>Plate 1 x 106 of HeLa cells with 10 mL of DMEM supplemented with 10% (vol/vol) FBS in a 10 cm culture dish.</li>
		<li>Two days after plating, replace the medium with 10 mL of DMEM containing 10% (vol/vol) FBS. Culture the cells for 30 h.</li>
		<li>Replace the medium with 10 mL of DMEM containing 0.002% (vol/vol) DMSO and 10% (vol/vol) FBS, and culture the cells for 14 h.</li>
		<li>Collect the cells by trypsinization using 0.7 mL of trypsin (2.5 g/L) containing 1 mM EDTA. Count the cell number for the IP-RdRP assay.</li>
		<li>Centrifuge the sample at 490 x g for 5 min at 4 &#176;C, and discard the supernatant. 
		NOTE: The cell pellet can be stored at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. IP-RdRP Assay
<ol>
	<li>Protein A-agarose bead preparation
	<ol>
		<li>Transfer 1 mL (bed volume 500 &#181;L) of affinity resin to a 1.5 mL microcentrifuge tube. Centrifuge at 800 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 800 &#181;L of Lysis buffer A to the beads, and mix well by inverting the tube several times. Centrifuge at 800 x g for 3 min at 4 &#176;C, and discard the supernatant. Repeat this step twice (total three washes).</li>
		<li>Add 800 &#181;L of Lysis buffer A to the beads, mix well by inverting the tube several times. Rotate the tube overnight (or for at least 4 h) at 4 &#176;C on a rotary shaker.</li>
		<li>Centrifuge the tube at 800 x g for 3 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 500 &#181;L of Lysis buffer A to the beads, and mix well by inverting the tube several times. Store the beads at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of cell lysate
	<ol>
		<li>(Optional) If the cells are frozen in step 2, place and thaw the frozen cells on ice until the cells become loose.</li>
		<li>Wash the cells once with 10 mL of PBS (-). Centrifuge the sample at 1,450 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Lyse the cells with ice-cold Lysis buffer A (1 mL of Lysis buffer A per 1 x 107 of the cells) by gentle pipetting.</li>
		<li>Sonicate the sample in a 1.5 mL tube with following conditions; set amplification of the sonicator at 25%, and sonicate for 10 s.</li>
		<li>Centrifuge the sample at 20,400 x g for 15 min at 4 &#176;C.</li>
		<li>Collect the supernatant. Transfer 1 mL each of the supernatant into 1.5 mL microcentrifuge tubes. 
		CAUTION: Perform all procedures under RNase-free conditions.</li>
	</ol>
	</li>
	<li>Immunoprecipitation
	<ol>
		<li>Pre-clear the lysate from step 3.2.6 by adding 40 &#181;L (bead volume 20 &#181;L) of prewashed protein A-agarose beads per 1 mL of the lysate. Mix well by inverting the tube several times and rotate the sample for 30 min at 4 &#176;C.</li>
		<li>Centrifuge the sample at 13,000 x g for 1 min at 4 &#176;C, and recover the supernatant.</li>
		<li>Add 40 &#181;L (bed volume 20 &#181;L) of prewashed protein A-agarose beads and 10 &#181;g of anti-human TERT antibody (clone: 10E9-2)8 into the pre-cleared lysate. Mix well by inverting the tube several times and rotate the sample at 4 &#176;C over night.</li>
		<li>Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Wash the beads with Wash buffer 1: add 1 mL of Wash buffer 1 to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant. Repeat this step three additional times.</li>
		<li>Wash the beads once with AGC solution containing 2 mM CaCl2: add 1 mL of AGC solution containing 2 mM CaCl2 to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Treat the beads with Micrococcal nuclease (MNase): prepare the MNase reaction mixture described in Table 1. Resuspend the beads in 60 &#181;L of the MNase reaction mixture by gentle pipetting. Shake the sample gently (20 to 25 rpm) on a turntable of a shaker set in a Peltier-type incubator for 15 min at 25 &#176;C. 
		NOTE: It is very important to use an undulating shaker and strictly follow the speed limit in this step. Other type of shakers, such as rotating mixers, are not recommended.</li>
		<li>Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant.</li>
		<li>Wash the beads twice with AGC solution containing 3 mM EGTA: add 1 mL of AGC solution containing 3 mM EGTA to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant. Repeat this step once.</li>
		<li>Wash the beads once with Wash buffer 2: add 1 mL of Wash buffer 2 to the beads, mix well by inverting the tube, and rotate the sample for 5 min at 4 &#176;C. Centrifuge the sample at 3,300 x g for 0.5 min at 4 &#176;C, and remove the supernatant. Keep the beads on ice while setting up RdRP reaction. 
		CAUTION: Perform all procedures under RNase-free conditions.</li>
	</ol>
	</li>
	<li>RdRP reaction
	<ol>
		<li>Prepare a RdRP reaction mixture in a new tube as described in Table 2.</li>
		<li>Add 6 &#181;L of [&#945;-32P]UTP (3,000 Ci/mmol) to the mixture and mix well by pipetting. 
		NOTE: [&#945;-32P]ATP, [&#945;-32P]CTP, or [&#945;-32P]GTP can be used for the reaction instead of [&#945;-32P]UTP.</li>
		<li>Add 1 &#181;L of template RNA (1 &#181;g/&#181;L) to the mixture and mix well by pipetting. 
		NOTE: To establish the RdRP assay, the following RNA template is recommended: 5&#39;-GGGAUCAUGUGGGUCCUAUUACAUUUUAAACCCA-3&#39; 9.</li>
		<li>Resuspend the beads with 20 &#181;L of the RdRP reaction mixture from step 3.4.3 by pipetting.</li>
		<li>Shake the sample gently (20 to 25 rpm) on a turntable of a shaker set in a peltier-type incubator for 2 h at 32 &#176;C.</li>
		<li>Add 5.4 &#181;L of Proteinase K (20 mg/mL) and 45.4 &#181;L of 2x Proteinase K buffer to the reaction mixture. Mix by pipetting and shake (20 to 25 rpm) the sample on a turntable set in a Peltier-type incubator for 30 min at 37 &#176;C.</li>
		<li>Add 109.2 &#181;L of RNase-free water to the sample.</li>
		<li>Add 200 &#181;L of acid phenol-chloroform to the sample. Mix well by vortex, and then centrifuge the sample at 21,100 x g for 5 min at room temperature. Transfer the aqueous phase to a new tube. Repeat this step once.</li>
		<li>Add 20 &#181;L of 3 M sodium acetate (pH = 5.2), 4 &#181;L of precipitation carrier and 250 &#181;L of ethanol to the aqueous phase. Shake the tube well for mixing, then centrifuge the sample at 21,100 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Wash the pellet with 300 &#181;L of cold 70% (vol/vol) ethanol stored at -20 &#176;C. Centrifuge the sample at 21,100 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant and air-dry the pellet. 
		CAUTION: Perform all procedures under RNase-free conditions. 
		NOTE: The pellet from step 3.4.11 can be stored at -20 &#176;C for at least 2 days.</li>
	</ol>
	</li>
	<li>RNase treatment
	<ol>
		<li>Resuspend the pellet from step 3.4.11 in 20 &#181;L of RNase-free water.</li>
		<li>Prepare a RNase reaction mixture in a new tube as described in Table 3.</li>
		<li>Add 180 &#181;L of the RNase reaction mixture to the sample, and incubate the tube for 2 h at 37 &#176;C.</li>
		<li>Add 2.3 &#181;L of 10% (vol/vol) SDS to the sample and incubate for 15 min at 37 &#176;C.</li>
		<li>Add 2 &#181;L of Proteinase K (20 mg/mL) to the sample and incubate for 15 min at 37 &#176;C.</li>
		<li>Add 205 &#181;L of acid phenol-chloroform to the sample. Mix well by vortex, then centrifuge the sample at 21,100 x g for 5 min at room temperature. Transfer the aqueous phase to a new tube.</li>
		<li>Add 20 &#181;L of 3 M sodium acetate (pH 5.2), 4 &#181;L of precipitation carrier and 250 &#181;L of ethanol to the aqueous phase. Shake the tube well for mixing, then centrifuge the sample at 21,100 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Wash the pellet with 300 &#181;L of cold 70% (vol/vol) ethanol stored at -20 &#176;C. Centrifuge the sample at 21,100 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant and air-dry the pellet. 
		NOTE: The pellet from step 3.5.9 can be stored at -20 &#176;C for at least 2 days.</li>
	</ol>
	</li>
	<li>Gel electrophoresis and autoradiography
	<ol>
		<li>Resuspend the sample with 20 &#181;L of RNase-free water.</li>
		<li>Add 20 &#181;L of 2x loading buffer.</li>
		<li>Boil the sample for 10 min at 95 &#176;C. Crush the sample on the ice.</li>
		<li>Run the gel. In case the template size is 34 nts (see also step 3.4.3), 7 M Urea-10% polyacrylamide gel is used for denaturing gel electrophoresis.</li>
		<li>Dry the gel with a gel dryer.</li>
		<li>Expose the film to the dried-gel within an X-ray cassette with an intensifying screen at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol><li>Martinez, P., Blasco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomeric+and+extra-telomeric+roles+for+telomerase+and+the+telomere-binding+proteins%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins.</a> Nat Rev Cancer. 11 (3), 161-176 (2011).</li>
<li>Nakamura, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telomerase+catalytic+subunit+homologs+from+fission+yeast+and+human%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Telomerase catalytic subunit homologs from fission yeast and human.</a> Science. 277 (5328), 955-959 (1997).</li>
<li>Gillis, A. J., Schuller, A. P., Skordalakes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+the+Tribolium+castaneum+telomerase+catalytic+subunit+TERT%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of the Tribolium castaneum telomerase catalytic subunit TERT.</a> Nature. 455 (7213), 633-637 (2008).</li>
<li>Salgado, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+an+RNAi+polymerase+links+RNA+silencing+and+transcription%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">The structure of an RNAi polymerase links RNA silencing and transcription.</a> PLoS Biol. 4 (12), e434(2006).</li>
<li>Castel, S. E., Martienssen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+interference+in+the+nucleus%3A+roles+for+small+RNAs+in+transcription%2C+epigenetics+and+beyond%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.</a> Nat Rev Genet. 14 (2), 100-112 (2013).</li>
<li>Sugiyama, T., Cam, H., Verdel, A., Moazed, D., Grewal, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-dependent+RNA+polymerase+is+an+essential+component+of+a+self-enforcing+loop+coupling+heterochromatin+assembly+to+siRNA+production%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production.</a> Proc Natl Acad Sci U S A. 102 (1), 152-157 (2005).</li>
<li>Maida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+RNA-dependent+RNA+polymerase+formed+by+TERT+and+the+RMRP+RNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">An RNA-dependent RNA polymerase formed by TERT and the RMRP RNA.</a> Nature. 461 (7261), 230-235 (2009).</li>
<li>Maida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Involvement+of+telomerase+reverse+transcriptase+in+heterochromatin+maintenance%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Involvement of telomerase reverse transcriptase in heterochromatin maintenance.</a> Mol Cell Biol. 34 (9), 1576-1593 (2014).</li>
<li>Maida, Y., Yasukawa, M., Masutomi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+Novo+RNA+Synthesis+by+RNA-Dependent+RNA+Polymerase+Activity+of+Telomerase+Reverse+Transcriptase%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">De Novo RNA Synthesis by RNA-Dependent RNA Polymerase Activity of Telomerase Reverse Transcriptase.</a> Mol Cell Biol. 36 (8), 1248-1259 (2016).</li>
<li>Okamoto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+tumor+initiating+cells+of+defined+genetic+composition+by+nucleostemin%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Maintenance of tumor initiating cells of defined genetic composition by nucleostemin.</a> Proc Natl Acad Sci U S A. 108 (51), 20388-20393 (2011).</li>
<li>van Dijk, A. A., Makeyev, E. V., Bamford, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initiation+of+viral+RNA-dependent+RNA+polymerization%5BTitle%5D)+AND+%22J+Gen+Virol%22%5BJournal%5D)">Initiation of viral RNA-dependent RNA polymerization.</a> J Gen Virol. 85 (Pt 5), 1077-1093 (2004).</li>
</ol>

The authors have nothing to disclose.

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&#39;-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, [&#945;-32P]NTP can be replaced with [&#947;-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.

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.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Wako</td><td>044-29765</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>CORNING</td><td>35-010-CV</td><td></td></tr><tr><td>Penicillin-Streptomycin mixed solution</td><td>nacalai tesque</td><td>26253-84</td><td></td></tr><tr><td>Trypsin-Ethylenediamenetetraacetic acid (EDTA) solution</td><td>nacalai tesque</td><td>32777-44</td><td></td></tr><tr><td>Phosphate buffered saline (PBS(-))</td><td>Wako</td><td>166-23555</td><td></td></tr><tr><td>Thymidine</td><td>nacalai tesque</td><td>07147-61</td><td></td></tr><tr><td>Nocodazole</td><td>Sigma</td><td>M1404</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>nacalai tesque</td><td>08904-85</td><td></td></tr><tr><td>Sodium chloride</td><td>nacalai tesque</td><td>31333-45</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>nacalai tesque</td><td>35434-21</td><td></td></tr><tr><td>Hydrochloric acid</td><td>nacalai tesque</td><td>18321-05</td><td></td></tr><tr><td>Nonidet P-40 (NP-40)</td><td>nacalai tesque</td><td>25223-04</td><td></td></tr><tr><td>Pierce Protein A Plus Agarose</td><td>Thermo scientific</td><td>22812</td><td></td></tr><tr><td>2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td><td>nacalai tesque</td><td>17514-15</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Wako</td><td>168-21815</td><td></td></tr><tr><td>Potassium acetate</td><td>nacalai tesque</td><td>28405-05</td><td></td></tr><tr><td>Magnesium chloride hexahydrate (MgCl2&amp;bull;6H2O)</td><td>Wako</td><td>135-00165</td><td></td></tr><tr><td>Glycerol</td><td>nacalai tesque</td><td>17045-65</td><td></td></tr><tr><td>Polyoxyethylene(10) octylphenyl ether (Triton X-100)</td><td>Wako</td><td>169-21105</td><td></td></tr><tr><td>cOmplete, EDTA-free</td><td>Roche Applied Science</td><td>11 873 580 001</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl2&amp;bull;2H2O)</td><td>nacalai tesque</td><td>06731-05</td><td></td></tr><tr><td>Micrococcal nuclease (MNase)</td><td>Takara Bio</td><td>2910A</td><td></td></tr><tr><td>Ethylene glycol bis (b-aminoethylether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>nacalai tesque</td><td>15214-92</td><td></td></tr><tr><td>3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonat (CHAPS)</td><td>nacalai tesque</td><td>07957-64</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>nacalai tesque</td><td>14128-91</td><td></td></tr><tr><td>rCTP, rATP, rUTP, rGTP, 100mM each</td><td>Promega</td><td>E6000</td><td></td></tr><tr><td>[a-&lt;sup&gt;32&lt;/sup&gt;P]UTP (3,000 Ci/mmol)</td><td>PerkinElmer</td><td>NEG507H</td><td>Use fresh RI for the assay</td></tr><tr><td>RNase inhibitor</td><td>TOYOBO</td><td>SIN-201</td><td></td></tr><tr><td>Proteinase K</td><td>Takara</td><td>9033</td><td></td></tr><tr><td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Life Technologies</td><td>AM9720</td><td></td></tr><tr><td>3 M Sodium acetate</td><td>NIPPON GENE</td><td>316-90081</td><td></td></tr><tr><td>Ethanol (99.5)</td><td>nacalai tesque</td><td>14713-95</td><td></td></tr><tr><td>Dr. GenTLE Precipitation Carrier</td><td>Takara Bio</td><td>9094</td><td></td></tr><tr><td>RNase One Ribonuclease</td><td>Promega</td><td>M4265</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>nacalai tesque</td><td>02873-75</td><td></td></tr><tr><td>Formamide, deionized</td><td>nacalai tesque</td><td>16345-65</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate (EDTA)</td><td>nacalai tesque</td><td>15130-95</td><td></td></tr><tr><td>Orange G</td><td>nacalai tesque</td><td>25401-22</td><td></td></tr><tr><td>CO2 incubator</td><td>e.g., ASTEC</td><td>SCA-325DRS</td><td></td></tr><tr><td>Centrifuge</td><td>e.g., TOMY</td><td>EX-135</td><td>For step B)</td></tr><tr><td>Micro refrigerated centrifuge</td><td>e.g., KUBOTA</td><td>3740</td><td>For step 6.2</td></tr><tr><td>Sonicator</td><td>Qsonica</td><td>Q125</td><td>Set a microtip (#4422) on the sonicator, for step 6.4</td></tr><tr><td>Micro refrigerated centrifuge</td><td>e.g., TOMY</td><td>MX-305</td><td>For step 6.5</td></tr><tr><td>Cooled incubator</td><td>e.g., Panasonic</td><td>MIR-154-PJ</td><td>Set a rotary shaker inside</td></tr><tr><td>Rotary shaker</td><td>e.g., TAITEC</td><td>RT-5</td><td></td></tr><tr><td>Cooled incubator&amp;nbsp;</td><td>e.g., TAITEC</td><td>BR-43FL</td><td>Set a shaker &amp;ldquo;MINI WAVE&amp;rdquo; inside, for step 7.7</td></tr><tr><td>MINI WAVE</td><td>AS ONE</td><td>WEV-03</td><td>A shaker for steps 7.7, 8.5 and 8.6</td></tr><tr><td>Incubator</td><td>e.g., TAITEC</td><td>HB-80</td><td>Set a shaker &amp;ldquo;MINI WAVE&amp;rdquo; inside, for step 8.5 and 8.6</td></tr><tr><td>Block incubator</td><td>e.g., ASTEC</td><td>BI-516S</td><td></td></tr></tbody></table>

semi-quantitative detection of rna-dependent rna polymerase activity of human telomerase reverse transcriptase protein

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.

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&#8211;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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57021/57021fig1.jpg" /> 
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. <a href="https://cloudflare.jove.com/files/ftp_upload/57021/57021fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume&#160;</td>
		</tr>
		<tr>
			<td>Micrococcal Nuclease, 20 U/&#181;L</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>Micrococcal Nuclease Buffer, 10x</td>
			<td>8 &#181;L</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>51 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: MNase reaction mix component.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume&#160;</td>
		</tr>
		<tr>
			<td>CHAPS, 0.07% (wt/vol)&#160;</td>
			<td>6 &#181;L</td>
		</tr>
		<tr>
			<td>HMD solution</td>
			<td>2 &#181;L</td>
		</tr>
		<tr>
			<td>rATP, 80 mM</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>rGTP, 8 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>rUTP, 0.4 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>rCTP, 8 mM</td>
			<td>1 &#181;L</td>
		</tr>
		<tr>
			<td>RNase inhibitor, 40 U/&#181;L</td>
			<td>0.5 &#181;L</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>1 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 2: RdRP reaction mix component.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Volume&#160;</td>
		</tr>
		<tr>
			<td>RNase ONE Ribonuclease, 10 U/&#181;L</td>
			<td>0.2 &#181;L</td>
		</tr>
		<tr>
			<td>Reaction Buffer, 10x</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>159.8 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 3: Ribonuclease reaction mix component.

Watch this Scientific Journal Video about Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein at JoVE.com

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

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 ...

semi-quantitative-detection-rna-dependent-rna-polymerase-activity

Research

JoVE Journal

Genetics

9.8K Views.  National Cancer Center Research Institute. 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.

Video: Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Biochemistry

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase chain reaction (PCR)-based method allows for robust measurements of enzyme activity from most cell lysates. The gel-based TRAP with fluorescently labeled primers limits sample throughput, and the ability to detect differences in samples is restricted to two fold or greater changes in enzyme activity. The droplet digital TRAP, ddTRAP, is a highly sensitive approach that has been modified from the traditional TRAP assay, enabling the user to perform a robust analysis on 96 samples per run and obtain absolute quantification of the DNA (telomerase extension products) input within each PCR. Therefore, the newly developed ddTRAP assay overcomes the limitations of the traditional gel-based TRAP assay and provides a more efficient, accurate, and quantitative approach to measuring telomerase activity within laboratory and clinical settings.

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

We successfully converted the standard telomere repeat amplification protocol (TRAP) assay to be employed in droplet digital polymerase chain reactions. This new assay, called ddTRAP, is more sensitive and quantitative, allowing for better detection and statistical analysis of telomerase activity within various human cells.

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

Cancer Research

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Environment

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

Accurate Telomere Length Assessment in Human Population Studies

Monochrome Multiplex Quantitative PCR Telomere Length Measurement

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. Telomere length (TL) has been associated with various exposures, biological processes, and health outcomes. This article describes the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay protocol routinely conducted in our laboratory for measuring relative mean TL from human DNA. There are several different PCR-based TL measurement methods, but the specific protocol for the MMqPCR method presented in this publication is repeatable, efficient, cost-effective, and suitable&#160;for population-based studies. This detailed protocol outlines all information necessary for investigators to establish this assay in their laboratory. In addition, this protocol provides specific steps to increase the reproducibility of TL measurement by this assay, defined by the intraclass correlation coefficient (ICC) across repeated measurements of the same sample. The ICC is a critical factor in evaluating expected power for a specific study population; as such, reporting cohort-specific ICCs for any TL assay is a necessary step to enhance the overall rigor of population-based studies of TL. Example results utilizing DNA samples extracted from peripheral blood mononuclear cells demonstrate the feasibility of generating highly repeatable TL data using this MMqPCR protocol.

Author Spotlight: Exploring the Impact of Trauma on Cellular Aging

Here we present a protocol for the measurement of relative telomere length (TL) using the monochrome multiplex quantitative polymerase chain reaction (MMqPCR) assay. The MMqPCR assay is a repeatable, efficient, and cost-effective method for measuring TL from human DNA in population-based studies.

Telomeres are ribonucleoprotein structures at the end of all eukaryotic chromosomes that protect DNA from damage and preserve chromosome stability. ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

https://cloudfront.jove.com/CDNSource/teasers/65288.jpg

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Neuroscience

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Biology

Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence

Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

An accurate, short, sophisticated and cheap method is described that assesses telomere length in multiple tissues and species using qRT-PCR. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.

Telomeres  are repeating DNA sequences at the tip ends of the chromosomes that are diverse  in length and in humans can reach a length of 15,000 base ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

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