Name: toeprinting analysis of translation initiation complex formation on mammalian mrnas
Uploaded: 2018-05-10T13:00:00
Description: Watch this Scientific Journal Video about Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs at JoVE.com

This work was funded by a Natural Sciences and Engineering Research Council of Canada-Discovery Grant (RGPIN-2017-05463), the Canada Foundation for Innovation-John R. Evans Leaders Fund (35017), the Campus Alberta Innovates Program and the Alberta Ministry of Economic Development and Trade.

NOTE: RNA is highly susceptible to degradation by ribonucleases (RNases). Take standard precautions to keep the RNA intact. Change gloves frequently. Use filtered pipette tips, nuclease-free plasticware, and nuclease-free chemicals in all steps of the protocol. Use nuclease-free or diethyl pyrocarbonate (DEPC)-treated water for all solutions.
1. Preparation of Solutions
<ol>
	<li>Prepare Toeprinting buffer: 20 mM Tris-HCl (pH 7.6), 100 mM KOAc, 2.5 mM Mg(OAc)2, 5% (w/v) sucrose, 2...

<ol>
	<li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nat Rev Mol Cell Biol. 6 (4), 318-327 (2005).</li><li>Lacerda, R., Menezes, J., Romao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+just+scanning:+the+importance+of+cap-independent+mRNA+translation+initiation+for+cellular+stress+response+and+cancer.">More than just scanning: the importance of cap-independent mRNA translation initiation for cellular stress response and cancer.</a> Cell Mol Life Sci. 74 (9), 1659-1680 (2017).</li><li>Sharma, D. K., Bressler, K., Patel, H., Balasingam, N., Thakor, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Eukaryotic+Initiation+Factors+during+Cellular+Stress+and+Cancer+Progression.">Role of Eukaryotic Initiation Factors during Cellular Stress and Cancer Progression.</a> J Nucleic Acids. 2016, 8235121 (2016).</li><li>Gebauer, F., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+translational+control.">Molecular mechanisms of translational control.</a> Nat Rev Mol Cell Biol. 5 (10), 827-835 (2004).</li><li>Anthony, D. D., Merrick, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+40+S+and+80+S+complexes+with+mRNA+as+measured+by+sucrose+density+gradients+and+primer+extension+inhibition.">Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition.</a> J Biol Chem. 267 (3), 1554-1562 (1992).</li><li>Hartz, D., McPheeters, D. S., Traut, R., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+inhibition+analysis+of+translation+initiation+complexes.">Extension inhibition analysis of translation initiation complexes.</a> Methods Enzymol. 164, 419-425 (1988).</li><li>Shirokikh, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+ribosome-mRNA+complexes+at+different+translation+stages.">Quantitative analysis of ribosome-mRNA complexes at different translation stages.</a> Nucleic Acids Res. 38 (3), 15 (2010).</li><li>Thakor, N., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRES-mediated+translation+of+cellular+messenger+RNA+operates+in+eIF2alpha-+independent+manner+during+stress.">IRES-mediated translation of cellular messenger RNA operates in eIF2alpha- independent manner during stress.</a> Nucleic Acids Res. 40 (2), 541-552 (2012).</li><li>Liwak, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+PDCD4+represses+internal+ribosome+entry+site-mediated+translation+of+antiapoptotic+proteins+and+is+regulated+by+S6+kinase+2.">Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2.</a> Mol Cell Biol. 32 (10), 1818-1829 (2012).</li><li>Thakor, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mRNA+recruits+the+ribosome+via+eIF3-PABP+bridge+to+initiate+internal+translation.">Cellular mRNA recruits the ribosome via eIF3-PABP bridge to initiate internal translation.</a> RNA Biol. , 1-15 (2016).</li><li>Thoma, C., Bergamini, G., Galy, B., Hundsdoerfer, P., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+IRES-mediated+translation+of+the+c-myc+and+BiP+mRNAs+by+the+poly(A)+tail+is+independent+of+intact+eIF4G+and+PABP.">Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP.</a> Mol Cell. 15 (6), 925-935 (2004).</li><li>Baird, S. D., Lewis, S. M., Turcotte, M., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+search+for+structurally+similar+cellular+internal+ribosome+entry+sites.">A search for structurally similar cellular internal ribosome entry sites.</a> Nucleic Acids Res. 35 (14), 4664-4677 (2007).</li><li>Merrick, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+a+single+GTP+is+used+in+the+formation+of+80+S+initiation+complexes.">Evidence that a single GTP is used in the formation of 80 S initiation complexes.</a> J Biol Chem. 254 (10), 3708-3711 (1979).</li><li>Arnaud, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+34-Kilodalton+Isoform+of+Human+Fibroblast+Growth+Factor+2+Is+Cap+Dependently+Synthesized+by+Using+a+Non-AUG+Start+Codon+and+Behaves+as+a+Survival+Factor.">A New 34-Kilodalton Isoform of Human Fibroblast Growth Factor 2 Is Cap Dependently Synthesized by Using a Non-AUG Start Codon and Behaves as a Survival Factor.</a> Mol Cell Biol. 19 (1), 505-514 (1999).</li><li>Dmitriev, S. E., Pisarev, A. V., Rubtsova, M. P., Dunaevsky, Y. E., Shatsky, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+48S+translation+preinitiation+complexes+into+80S+initiation+complexes+as+revealed+by+toeprinting.">Conversion of 48S translation preinitiation complexes into 80S initiation complexes as revealed by toeprinting.</a> FEBS Lett. 533 (1-3), 99-104 (2003).</li><li>Faye, M. D., Graber, T. E., Holcik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+selective+mRNA+translation+in+mammalian+cells+by+polysome+profiling.">Assessment of selective mRNA translation in mammalian cells by polysome profiling.</a> J Vis Exp. (92), e52295 (2014).</li><li>Duncan, C. D. S., Mata, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cycloheximide+on+the+interpretation+of+ribosome+profiling+experiments+in+Schizosaccharomyces+pombe.">Effects of cycloheximide on the interpretation of ribosome profiling experiments in Schizosaccharomyces pombe.</a> Sci Rep. 7 (1), 10331 (2017).</li><li>Beck, H. J., Janssen, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Translation+Initiation+Regulation+Mechanism+in+Escherichia+coli+ptrB+Mediated+by+a+5'-Terminal+AUG.">Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5'-Terminal AUG.</a> J Bacteriol. 199 (14), (2017).</li></ol>

Toeprinting is a powerful technique to directly measure the ability of an RNA of interest to support the formation of translation initiation complexes under highly controlled circumstances. This protocol describes a simplified technique for toeprinting mammalian RNAs. Rabbit reticulocyte lysate (RRL) is used as a convenient source of ribosomes, eIFs, initiator tRNA, and IRES trans-acting factors (ITAFs). The experimenter provides their RNA of choice, and can also supplement the toeprinting reaction with specific...

As translation consumes most cellular energy, it makes sense that translation is tightly regulated1. Conversely, dysregulation of translation-and the consequent alterations in protein output-is often observed in stress-response and disease states, such as cancer1,2. A major advantage of translational control is the speed with which cells can alter their protein output in order to respond to various stimuli3. Translation regulation thus represents an important mechanism that can influence cell survival and death1,2,3. Of the steps of translation, initiation is the most highly regulated and complex3. Briefly, most eukaryotic mRNAs contain a 5&#39; m7G cap that is almost always essential for their translation. Cap-dependent initiation requires eukaryotic initiation factors eIF4E, eIF4A, and eIF4G (the cap-recognition complex) to interact with the 5&#39; end of the mRNA. The 43S preinitiation ribosome complex, which contains eIF2-bound initiator tRNA and eIF3, is recruited to the 5&#39; end of the mRNA via an interaction of eIF4G with eIF3. The preinitiation complex is thought to scan mRNA, aided by eIF4A (an RNA helicase) until the start codon (AUG) is located. The 48S initiation complex is subsequently formed and tRNA is delivered into the P-site of the ribosome. Finally, the 60S and 40S ribosome subunits are united to form the 80S initiation complex, followed by translation elongation1,3,4. In contrast, internal ribosome entry sites (IRESes) bypass the requirement for a 5&#39; cap by recruiting the 40S ribosomal subunit directly to the initiation codon3. Physiological stress conditions attenuate global mRNA translation due to modifications of key general eukaryotic initiation factors (eIFs). However, non-canonical translation initiation mechanisms allow for selective translation of certain mRNAs which often encode stress-response proteins, and dysregulation of non-canonical translation initiation is implicated in disease states like cancer1,2. Discreet RNA regulatory elements, such as cellular IRESes, allow for the translation of such mRNAs2,3.
One particularly interesting aspect of translational control is to understand mechanisms of canonical versus non-canonical translation of a given mRNA. Toeprinting is a technique that allows the detailed mechanistic study of translation initiation of specific RNAs in vitro. The overall goal of toeprinting is to assess the ability of an RNA of interest to nucleate the formation of a translation initiation complex with the ribosome under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are required for efficient translation initiation. For instance, ribosome recruitment might be hindered in the absence of a 5&#39; cap but stimulated by the presence of an IRES.
The principle of the technique is to in vitro transcribe an RNA of interest, incubate it in the presence of cellular extracts containing translation components (or the purified components) to allow initiation complexes to form, and to reverse transcribe the RNA with a specific primer. Stable RNA-ribosome complexes will cause reverse transcription to stall at the 3&#39; edge of the ribosome-the so-called &#39;toeprint&#39;5,6,7.
In this protocol, the ribosomal subunits, eIFs, tRNAs, and IRES trans-acting factors (ITAFs) are conveniently contributed by rabbit reticulocyte lysate (RRL). Another advantage of this protocol is the use of a fluorescently-labeled primer and fluorescence gel-based imager, rather than a radiolabeled primer. This eliminates extra steps, including radiolabeling the primer, as well as drying the gel and exposing it to an intensifying screen. The fluorescent bands are recorded in real time, as the gel runs, allowing for greater resolution. Uncapped X-linked inhibitor of apoptosis protein (XIAP) IRES RNA is used as an example here, although capped mRNAs can also be analyzed by this technique8.
Unlike western analysis, which measures the final output of the translation process in cell lysates, toeprinting is an in vitro approach to measure translation initiation complex formation on an RNA. This reductionist approach allows for the highly detailed study of substrates or factors that regulate translation initiation (e.g., capped or un-capped mRNA, IRES structure, presence or absence of poly-A tail, provision of specific protein factors, etc.). Hence, toeprinting can be used to study different modes of translation8 or the effects of mRNA structures, such as IRESes, on protein synthesis9,10.

<table border="0" cellpadding="0" cellspacing="0" style="width:1045px;" width="1043">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:405px;">DEPC (Diethyl pyrocarbonate)</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:191px;">D5758-100ML</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TRIS base, Ultrapure</td>
			<td>JT Baker</td>
			<td>4109-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KOAc (Potassium acetate)</td>
			<td>Bio Basic</td>
			<td>PB0438</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mg(OAc)2 (Magnesium acetate tetrahydrate)</td>
			<td>Bio Basic</td>
			<td>MB0326</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose, molecular biology grade</td>
			<td>Calbiochem</td>
			<td>573113-1KG</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spermidine</td>
			<td>Sigma</td>
			<td>85558</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GMP-PNP (Guanosine 5&#8242;-[&#946;,&#947;-imido]triphosphate trisodium salt hydrate) 0.1 M solution</td>
			<td>Sigma</td>
			<td>G0635</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ATP (Adenosine 5&#8242;-triphosphate) disodium salt, 100 mM solution</td>
			<td>Sigma</td>
			<td>A6559</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">19:1 Acrylamide:bis-acrylamide, 40%</td>
			<td>Bio Basic</td>
			<td>A0006</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Urea</td>
			<td>Bio Basic</td>
			<td>UB0148</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">500mL bottle top filtration units, 0.2 &#181;m</td>
			<td>Sarstedt</td>
			<td>83.1823.101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Formamide</td>
			<td>Sigma</td>
			<td>F9037-100ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">EDTA (disodium salt, dihydrate)</td>
			<td>Bio Basic</td>
			<td>EB0185</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SDS</td>
			<td>Bio Basic</td>
			<td>SB0485</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bromophenol blue</td>
			<td>Bio Basic</td>
			<td>BDB0001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylene cyanol FF</td>
			<td>Bio Basic</td>
			<td>XB0005</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEGAshortscript T7 transcription kit</td>
			<td>Ambion</td>
			<td>AM1354</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">mMESSAGE mMACHINE T7 transcription kit</td>
			<td>Ambion</td>
			<td>AM1344</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Acid Phenol:Chloroform (5:1)</td>
			<td>Ambion</td>
			<td>AM9722</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:405px;">25:24:1 Phenol:Chloroform:Isoamyl Alcohol</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:191px;">15593-049</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rabbit Reticulocyte Lysate (RRL). Should NOT be nuclease-treated.</td>
			<td>Green Hectares, USA</td>
			<td></td>
			<td>Contact Green Hectares, ask for 1:1 RRL:water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RiboLock RNase Inhibitor (40 U/&#181;L)</td>
			<td>Thermo Fisher</td>
			<td>E00382</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">100 mM dNTPs</td>
			<td>Invitrogen</td>
			<td>56172, 56173, 56174, 56175</td>
			<td>Mix equal parts for a stock of 25 mM each.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AMV-RT, 10 U/&#181;L</td>
			<td>Promega</td>
			<td>M5101</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sequenase Version 2.0 DNA Sequencing Kit</td>
			<td>Thermo Fisher</td>
			<td>707701KT</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Model 4200 IR2 DNA analyzer</td>
			<td>LI-COR</td>
			<td></td>
			<td>Product has been discontinued</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APS (Ammonium Persulfate)</td>
			<td>Bio Basic</td>
			<td>AB0072</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TEMED</td>
			<td>Bio Basic</td>
			<td>TB0508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Phusion High Fidelity Polymerase</td>
			<td style="width:159px;">New England Biolabs</td>
			<td style="width:191px;">M0530</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Turbo Dnase</td>
			<td>Thermo Fisher</td>
			<td>AM2238</td>
			<td></td>
		</tr>
	</tbody>
</table>

toeprinting analysis of translation initiation complex formation on mammalian mrnas

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

We have previously described the ability of the XIAP IRES to support cap-independent translation initiation in vitro8,10. Toeprinting was the key technique to interrogate the mechanistic details of the XIAP IRES initiation complex. A DNA construct encoding an mRNA containing the XIAP IRES (Figure 1A) was in vitro transcribed and subjected to toeprinting analysis. The...

Watch this Scientific Journal Video about Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs at JoVE.com

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

The overall goal of this toeprinting analysis is to observe the translation initiation complex formation on mammalian mRNAs. This method can help answer key questions in the field of mRNA translation, such as IRES mediated translation initiation. The main advantage of this technique is that one can perform detailed analysis of translation initiation complex formation on a specific mRNA.Assemble the transcription reactions to transcribe RNA from the PCR amplified DNA templates, encoding the XIAP IRES RNA or its mutants. Next, add two units of RNase-free DNase to remove the DNA template to each 20 microliter reaction of RNA. Incubate at 37 degrees Celsius for 30 minutes.Then, dilute the DNase treated RNA with nuclease free water to a final volume of 110 microliters. Next, add 110 microliters of acid phenol to begin RNA purification. Vortex the samples for five seconds and centrifuge at 20, 000 x g for three minutes at room temperature.It's critical to dilute the RNA or CDNA prior to phenol extraction. If the volume is too low, it's gonna be difficult to physically recover the aqueous phase and the final yield will be low. Then, carefully transfer 100 microliters of the RNA-containing aqueous phase to a new microcentrifuge tube.Add 10 microliters of 3 M sodium acetate, pH 5.5, and mix the samples. Then, add three volumes of 100%ethanol to precipitate RNA and vortex the samples for five seconds. Incubate overnight at 20 degrees Celsius.The next day, centrifuge the samples at 20, 000 x g for 30 minutes at four degrees Celsius and discard the supernatant. Then, wash the pellet with 500 microliters of ice cold 70%ethanol and repeat the centrifugation. Carefully aspirate as much supernatant as possible without dislodging the pellet.Then, air dry the pellet for five to 10 minutes. Resuspend the pellet in nuclease-free water to a final concentration of 0.5 micrograms per microliter. To begin the toeprinting assay, add 15 microliters of non-nuclease treated rabbit reticulocyte lysate to each 1.5 milliliter microcentrifuge tube, which represent the wild-type XIAP IRES RNA and its mutants.Add RNase inhibitor, ATP, and GMP-PNP to each tube. Incubate at 30 degrees Celsius for five minutes. After this, add one microliter of the respective RNA to each of the tubes and incubate at 30 degrees Celsius for an additional five minutes.During the incubation period, add DTT and spermidine to the toeprinting buffer. Then, add 22 microliters of toeprinting buffer to each of the tubes and incubate at 30 degrees Celsius for three minutes. Next, add 0.5 microliters of IR dye labeled toeprinting primer and incubate the reaction on ice for 10 minutes.Subsequently, add two microliters of dNTPs, two microliters of magnesium acetate, and one microliter of avian myeoloblastosis virus reverse transcriptase. Bring the final volume to 50 microliters with toeprinting buffer. Then, allow primer extension to occur for 45 minutes at 30 degrees Celsius.It's also critical to add magnesium acetate to the toeprinting reaction, without which, the reverse transcriptase won't function optimally. After this, add 200 microliters of nuclease-free water. Add 250 microliters of 25:24:1 phenol:chloroform:isoamyl alcohol, pH 8.0, to begin the purification of cDNA.After vortexing the samples for five seconds, centrifuge the samples at 20, 000 x g at room temperature for three minutes. Next, carefully transfer the aqueous phase containing the DNA to a new microcentrifuge tube. Then, add three volumes of 100%ethanol to precipitate the DNA and vortex for five seconds.Incubate overnight at 20 degrees Celsius. The next day, centrifuge the tubes at 20, 000 x g for 30 minutes at four degrees Celsius and discard the supernatant. Then, wash the DNA pellet with 500 microliters of ice cold 70%ethanol and centrifuge at 20, 000 x g for 15 minutes at four degrees Celsius.Aspirate as much as supernatant as possible, taking care not to dislodge the pellet. Then, air dry the pellet for five to 10 minutes. Dissolve the DNA pellet in six microliters of nuclease-free water.Then, add three microliters of formamide loading dye. Use a sequencing kit to prepare a dideoxy nucleotide sequencing ladder with the toeprinting primer and the DNA construct encoding the XIAP IRES RNA. Then, mix six microliters of each sequencing reaction with three microliters of the formamide loading dye.After cleaning and assembling the plates, pour the gel, and insert the comb. Following the polymerization of the gel, assemble the sequencing apparatus and fill the reservoirs with 1x TBE. Then, prerun at 1500 volts for 15 minutes until a temperature of 55 degrees Celsius is achieved.Heat all of the samples with formamide dye to 85 degrees Celsius for five minutes. Then, load one microliter of each sample on a 6%polyacrylamide sequencing gel and run the gel at 1500 volts for eight hours. Finally, read the bands in realtime with a fluorescent based imager.This method shows that confirmation of RNA is critical for translation initiation complex formation. The reverse transcription of XIAP mRNA ribosome complex yields typical toeprints 17 to 19 nucleotides downstream of AUG. The 5'PPT mutant shows impaired toeprint formation.However, when transcribed with the 5'cap, initiation complex formation occurs. Similarly, the 3'PPT mutant also shows impaired toeprint formation. However, transcription with the 5'cap leads to initiation complex formation.The double PPT mutant harboring both the 5'PPT and 3'PPT mutations is able to form the initiation complex without the 5'cap. Toeprint formation is strongly impaired for the start codon mutant in the absence of RRL and GMP-PNP and a Poly A tail. The CDNA products are compared with a dideoxy nucleotide sequencing ladder.Human beta globin, or HBB mRNA, shows no toeprint in the absence of the 5'cap. However, the initiation complex is formed on the capped 5'UTR of HBB, yielding toeprints 16 to 18 nucleotides downstream of AUG. Once mastered, this technique can be done in three days, including preparation of in vitro transcription templates and RNA, if performed properly.Following this procedure, other methods like structural probing can be performed in order to answer additional questions like the secondary structure of the wild-type or mutant variants of the IRES. After its development, this technique has paved the way for researchers in the field of mRNA translation to explore IRES mediated translation in vitro. After watching this video, you should have a good understanding of how to perform toeprinting of mammalian mRNA and to analyze the formation of translation initiation complexes.Don't forget that acid-phenol, phenol:chloroform:isoamyl alcohol and acrylamide can be extremely hazardous. Appropriate personal protective equipment should be worn while using these reagents.

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Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

Expanding the Mass Photometry Analysis Concentration Range with Microfluidics

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

Author Spotlight: Characterization of Low-Affinity Protein Interactions in Solution Using MassFluidix Technology

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...