Name: rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing
Uploaded: 2016-09-27T12:30:00
Description: Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

This study was supported by the Student Research Grant (13-14) of University of Ulsan College of Medicine, Seoul, Korea and a grant (2014-464) from the Asan Institute for Life Sciences, Seoul, Korea.

1. Cell Line
<ol>
	<li>Culture 106 telomerase-immortalized human corneal epithelial cells (HCECs) in a 6-well plate (density: 1039.9 cell/mm2) in a 37 &#176;C incubator with a 5% CO2 atmosphere using bronchial epithelium growth medium (BEGM) until they reach 95% confluence.</li>
</ol>
2. Western Blot Analysis after Creating Epithelial Scratch Wounds
<ol>
	<li>Streak a sterile 200 &#181;l pipette tip across the surface of a well of confluent cultured HCECs four ...

<ol>
	<li>Dua, H. S., Gomes, J. A., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+wound+healing.">Corneal epithelial wound healing.</a> Br. J. Ophthalmol. 78 (5), 401-408 (1994).</li><li>Estil, S., Primo, E. J., Wilson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+shed+human+corneal+cells.">Apoptosis in shed human corneal cells.</a> Invest. Ophthalmol. Vis. Sci. 41 (11), 3360-3364 (2000).</li><li>Guay, J., 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=Regulation+of+actin+filament+dynamics+by+p38+map+kinase-mediated+phosphorylation+of+heat+shock+protein+27.">Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.</a> J. cell. Sci. 110, 357-368 (1997).</li><li>Park, J. W., 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=Differential+expression+of+heat+shock+protein+mRNAs+under+in+vivo+glutathione+depletion+in+the+mouse+retina.">Differential expression of heat shock protein mRNAs under in vivo glutathione depletion in the mouse retina.</a> Neurosci. Lett. 413 (3), 260-264 (2007).</li><li>Rane, M. J., 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=Heat+shock+protein+27+controls+apoptosis+by+regulating+Akt+activation.">Heat shock protein 27 controls apoptosis by regulating Akt activation.</a> J. Biol. Chem. 278 (30), 27828-27835 (2003).</li><li>Shin, K. D., 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=Blocking+tumor+cell+migration+and+invasion+with+biphenyl+isoxazole+derivative+KRIBB3,+a+synthetic+molecule+that+inhibits+Hsp27+phosphorylation.">Blocking tumor cell migration and invasion with biphenyl isoxazole derivative KRIBB3, a synthetic molecule that inhibits Hsp27 phosphorylation.</a> J. Biol. Chem. 280 (50), 41439-41448 (2005).</li><li>Jain, S., 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=Expression+of+phosphorylated+heat+shock+protein+27+during+corneal+epithelial+wound+healing.">Expression of phosphorylated heat shock protein 27 during corneal epithelial wound healing.</a> Cornea. 31 (7), 820-827 (2012).</li><li>Alekseev, O. M., Richardson, R. T., Alekseev, O., O'Rand, M. G. <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+gene+expression+profiles+in+HeLa+cells+in+response+to+overexpression+or+siRNA-mediated+depletion+of+NASP.">Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP.</a> Reprod. Biol. Endocrinol. 7, 45 (2009).</li><li>Park, H. Y., Kim, J. H., Lee, K. M., Park, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+prostaglandin+analogues+on+tear+proteomics+and+expression+of+cytokines+and+matrix+metalloproteinases+in+the+conjunctiva+and+corea.">Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and corea.</a> Exp. Eye. Res. 94 (1), 13-21 (2012).</li><li>Voegeli, T. S., Currie, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+knocks+down+Hsp27+and+increases+angiotensin+II-induced+phosphorylated+NF-kappaB+p65+levels+in+aortic+smooth+muscle+cells.">siRNA knocks down Hsp27 and increases angiotensin II-induced phosphorylated NF-kappaB p65 levels in aortic smooth muscle cells.</a> Inflamm. Res. 58 (6), 336-343 (2009).</li><li>Shi, B., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenite+pre-conditioning+reduces+UVB-induced+apoptosis+in+corneal+epithelial+cells+through+the+anti-apoptotic+activity+of+27+kDa+heat+shock+protein+(HSP27).">Arsenite pre-conditioning reduces UVB-induced apoptosis in corneal epithelial cells through the anti-apoptotic activity of 27 kDa heat shock protein (HSP27).</a> J. Cell. Physiol. 206 (2), 301-308 (2006).</li><li>Shen, E. P., 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=Comparison+of+corneal+epitheliotrophic+capacity+among+different+human+blood-derived+preparations.">Comparison of corneal epitheliotrophic capacity among different human blood-derived preparations.</a> Cornea. 30 (2), 208-214 (2011).</li><li>Song, I. S., 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=Heat+shock+protein+27+phosphorylation+is+involved+in+epithelial+cell+apoptosis+as+well+as+epithelial+migration+during+corneal+epithelial+wound+healing.">Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.</a> Exp Eye Res. 118 (1), 36-41 (2014).</li></ol>

In this present study, we evaluated the potential role of HSP27 in corneal epithelial wounding using in vitro approaches. The critical steps involved siRNA transfection for HSP27 knock-down to observe the function of HSP27 in cells subjected to stress. Notably, a role for HSP27 was revealed by these experiments in epithelial cell migration and apoptosis during corneal epithelial wound healing. Unlike previous studies10 that used rat HSP27-specific siRNA to transfect vascular smooth muscle cells, we us...

Corneal epithelial cells (CECs) are continuously shed into tear film, while they are simultaneously replaced by cells from the limbus and corneal epithelial basal layers.1 Various extrinsic stressors can induce the apoptosis and desquamation of CECs.2 The heat shock proteins (HSPs) are highly conserved and can be divided into two families according to molecular size.3 The largest HSP family includes HSP90, HSP70, and HSP60, and the smaller family includes HSP27.4 The phosphorylation of HSP27 is known to play an important role in cell survival and is required for cell migration because of the role of this protein in actin remodeling.5-7 Therefore, we attempted to test the potential role of HSP27 phosphorylation in CEC migration and apoptosis in an in vitro model of epithelial wound healing.
RNA interference (RNAi) using either small or short interfering RNAs (siRNA) has generated interest in both basic and applied biology, as it potentially allows the expression of any gene of interest to be knocked-down.8 Herein, we used HSP27-specific siRNA to assess the contribution of HSP27 to CEC wound healing and apoptosis. Traditional RNAi methods for gene knock-down in cells use synthetic RNA duplexes, including two unmodified 21-mer oligonucleotides that can be assembled to create siRNAs. The RNAi siRNA that we used in this present study is a simple and highly efficient method to transfect cells, and this reagent works with various immortalized cell lines. In this present study, we demonstrate the methods used for this analysis, including a scratch-induced directional wound assay, western blotting, siRNA transfection assay, immunofluorescence assay, and flow cytometry.

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			<td height="38" style="height:38px;width:212px;">Biological safety cabinet</td>
			<td style="width:247px;">CHC LAB Co.Ltd,&#160; Daejeon, Republic of Korea&#160;</td>
			<td style="width:109px;">CHC-777A2-06</td>
			<td style="width:199px;">Class II, Type A2&#160;</td>
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			<td height="58" style="height:58px;width:212px;">Stealth RNAi&#8482; siRNA</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
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			<td style="width:199px;">RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA</td>
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			<td height="38" style="height:38px;width:212px;">BEGMTM</td>
			<td style="width:247px;">Lonza, Inc., Walkersville, MD</td>
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			<td height="38" style="height:38px;width:212px;">Protease inhibitor&#160;</td>
			<td style="width:247px;">Sigma-Aldrich, Inc., St. Louis, MO</td>
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			<td style="width:199px;">1 uM Pepstatin A, 1 uM Leupetin, 0.1 uM Aprotin</td>
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			<td height="20" style="height:20px;width:212px;">Bradford protein assay&#160;</td>
			<td style="width:247px;">Bio-Rad Laboratories, Hercules, CA</td>
			<td style="width:109px;">#500-0001</td>
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			<td style="width:247px;">Abcam Inc., Cambridge, MA</td>
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			<td style="width:199px;">1:1000 dilution (Total HSP27)</td>
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			<td style="width:247px;">Abcam Inc., Cambridge, MA</td>
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			<td style="width:199px;">1: 1000 dilution HSP27 was phosphorylated at Ser85</td>
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			<td height="42" style="height:42px;width:212px;">Lipofectamine&#174; RNAiMAX reagent&#160;</td>
			<td style="width:247px;">Invitrogen, Carlsbad, CA</td>
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			<td height="57" style="height:57px;width:212px;">Phosphorylated Akt (Ser473)</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
			<td style="width:109px;">No. 4060</td>
			<td style="width:199px;">1: 1000 dilution Akt was phosphorylated at Ser473 (cell survival marker)</td>
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			<td height="38" style="height:38px;width:212px;">Non-phosphorylated Akt&#160;</td>
			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
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			<td style="width:247px;">Cell Signaling Technology, Danvers, MA</td>
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			<td height="57" style="height:57px;width:212px;">GAPDH</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">No. 4063</td>
			<td style="width:199px;">1:1000 loading control&#160; marker (house keeping gene)</td>
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			<td height="43" style="height:43px;width:212px;">Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">NCI1460KR</td>
			<td style="width:199px;">1:10000 dilution</td>
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			<td style="width:247px;">Invitrogen, Carlsbad, CA</td>
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			<td style="width:199px;">reduced serum medium for transfection</td>
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			<td style="width:247px;">Carl Roth GmbH + Co. KG, Karlstruhe, Germany</td>
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			<td height="20" style="height:20px;width:212px;">Tris&#160;</td>
			<td style="width:247px;">Amresco LCC, Inc. Solon, OH</td>
			<td style="width:109px;">No-0497</td>
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			<td height="20" style="height:20px;width:212px;">Sodium Chloride&#160;</td>
			<td style="width:247px;">Amresco LCC, Inc. Solon, OH</td>
			<td style="width:109px;">No-0241</td>
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			<td height="38" style="height:38px;width:212px;">Six well culture plate</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">140675</td>
			<td style="width:199px;">35.00 mm diameter / well</td>
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			<td height="38" style="height:38px;width:212px;">24-well culuture dish</td>
			<td style="width:247px;">Thermo Fisher Scientific, Inc., Waltham, MA</td>
			<td style="width:109px;">142475</td>
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			<td height="20" style="height:20px;width:212px;">Orbital shaker</td>
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			<td height="38" style="height:38px;width:212px;">Bovine serum albumin</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">sc-2323&#160;</td>
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			<td height="23" style="height:23px;width:212px;">BDFACSCantoTM II</td>
			<td style="width:247px;">BD Biosciences, Franklin Lakes, NJ</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">Flow cytometry</td>
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			<td height="38" style="height:38px;width:212px;">X-Ray Film</td>
			<td style="width:247px;">Kodak, Rochester, NY</td>
			<td style="width:109px;"></td>
			<td style="width:199px;">Medical X-Ray Cassette with Green 400 Screen&#160;</td>
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			<td height="38" style="height:38px;width:212px;">western blotting luminol reagent</td>
			<td style="width:247px;">Santa Cruz Biotechnology, Santa Cruz, CA</td>
			<td style="width:109px;">sc-2048&#160;</td>
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			<td height="38" style="height:38px;width:212px;">FITC Annexin V Apoptosis Detection Kit I</td>
			<td style="width:247px;">BD Biosciences, Franklin Lakes, NJ</td>
			<td style="width:109px;">556547</td>
			<td style="width:199px;"></td>
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rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

The expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding compared with unwounded HCECs13. Western blot analysis revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced, whereas the expression of Bax was significantly increased in HSP27-specific siRNA-transfected HCECs (Figure 1A-E). The phosphorylated HSP27 expression was reduced by 30% and 40% in 10 nM and 50 ...

Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

The overall goal of this RNA Transfection assay is to use HSP27 specific small interfering RNA to assess the potential role of HSP27 in corneal epithelial wound healing. This method can help answer key questions regarding the the function of Heat Shock Protein 27 during epithelial wound healing process. The main advantage of this technique is that small interfering RNA is sensitive RNA sucrose created to specifically target a messenger RNA transcript to induce it's degradation.It has been used to identify novel pest space various cellular processes. Though this technique can provide the insight into the corneal epithelial wound healing, it may also apply to another system such as skin and mucosa. Demonstrating the procedure will be Soon-Suk Kang, a technician from my laboratory.To begin this procedure, culture human corneal epithelial cells in a six-well plate with with bronchial epithelium growth medium at 37 degree Celsius and five percent carbon dioxide until they 95 percent confluence. Next, dilute 2.5 micro liters, or 7.5 micro liters of the transfection re-agent with 100 micro liters of reduced serum medium for transfection. Then, dissolve HSP27 specific siRNA and scrambled controlled siRNA in 100 micro liters or reduced serum medium to create 10 nanomolar of HSP27 specific siRNA and 15 nanomolar of scrambled control siRNA respectively.Following that, mix 100 micro liters of siRNA solution with 100 micro liters of diluted transfection reagent and incubate the mixture for 15 minutes at room temperature. Subsequently, add siRNA lipid complexes to the cells. After a four hour incubation, change the medium to the complete bronchial epithelium growth medium, and incubate the cells two days at 37 degrees Celsius before western blot analysis.In this step, extract HSP27 specific and scrambled control siRNA transfected HCECs in a biological safety cabinet using 100 micro liters of ice cold lysis buffer. Then incubate the cells for 30 minutes on ice to induce cell lysis. Next, centrifuge the lysates at 10, 000 times G for 15 minutes.Subsequently, transfer the supernatants to fresh 1.5 milliliter tubes and store them at minus 80 degrees Celsius. After that, determine the protein concentrations of the cell lytates using the Bradford protein assay. Then load the samples with equal amounts of total cell proteins on a 10 percent or 12 percent acrylamide gel.Subject the gel to SDS page, and electrophoretically transfer the separated protein bands to the nitrocellulose filters with 200 milliamps of current for one hour and four degrees Celsius. Next, block the nitrocellulose filter membranes with five percent skimmed milk in TBST for one hour. Then add primary anti-bodies and incubate the membranes overnight at four degrees Celsius on a shaker.The next day, wash the membranes three times with TBST allowing ten minutes for each wash. Then detect the immunolreactive bands using horseradish peroxidase conjugated goat anti-rabbit antibodies in five percent BSA. Subsequently, incubate the membranes in the western blotting luminol reagents for one minute at room temperature.Remove the membrane from the reagent solution. Then, remove excess liquid with an absorbent towel and place them in a plastic sheet protector. Afterward, place the covered membrane in a film cassette with the protein side facing up.Place an x-ray film on top of the membranes and expose it for one minute. In a biological safety cabinet, make a wound by dragging a sterile pipette tip across the confluent cultures of HSP27 specific or scrambled control siRNA transfected HCECs. Immediately after wounding wash the cells twice with 1XPBS and maintain them in bronchial epithelium growth medium at 37 degrees Celsius and five percent carbon dioxide for 24 hours.24 hours after wounding take the HCECs image using an upright microscope at 100x and perform background flattening using the filter command in image analysis software. Using the select measurements command, define the area of interest with the same sized polygonal shape that can cover the initial wound perpendicularly from end to end. Then, determine three different area of interest in the wounded area of each sample.Automatically count the cell number in each field using the count size measure menu option. Western blot analysis shown here, revealed that the expression of phosphorylated HSP27 and phosphorylated Akt were both significantly reduced. Where as the expression of backs was significantly increased in HSP27 specific siRNA transfected HCECs.The phosphorylated HSP27 expression was reduced by 30 percent and 40 percent in 10 nanomolar and 15 nanomolar of HSP27-specific siRNA transfected cells respectively, compared with control siRNA transfected cells. Moreover, the nonphosphorylated HSP27 expression was reduced by 20 percent and 30 percent in ten nanomolar and 15 nanomolar of HSP27 specific siRNA transfected cells respectively. The scratch induced directional wound assay indicated that 24 hours after wounding, HSP27-specific siRNA transfected cells at 10 and 15 nanomolar exibited reduced migration.In addition HSP27-specific siRNA transfected HCEC's underwent more apoptotic and necrotic cell death compared with the scrambled control siRNA transfected cells shown by flow cytometry. Once mastered RNA transfection technique can be done in 24 hours if it is performed properly. Following these procedures an episode like lock mouth can be generated in order to answer additional question like the initial function of Heat Shock Protein 27 in cornea epithelial wound healing.After watching this video, you should have a good understanding of how to use Heat Shock Protein 27 specific small interfering RNA to assess the potential role of Heat Shock Protein 27 in cornea epithelial wound healing. Don't forget that working with small interfering RNA and reagents can be hazardous and precursors such as a biological safety cabinet and protective mask should always be used while performing these procedures.

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Research

JoVE Journal

Genetics

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.

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 ...

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding RNA&#39;s family. Although their biological functions remain largely unknown, the number of these lncRNAs has steadily increased and it is now estimated that humans may have more than 10,000 such transcripts. Some of these are known to be involved in important regulatory pathways of gene expression which take place at the transcriptional level, but also at different steps of RNA co- and post-transcriptional maturation. In the latter cases, RNAs that are targeted by the lncRNA have to be identified. That&#39;s the reason why it is useful to develop a method enabling the identification of RNAs associated directly or indirectly with a lncRNA of interest.
This protocol, which was inspired by previously published protocols allowing the isolation of a lncRNA together with its associated chromatin sequences, was adapted to permit the isolation of associated RNAs. We determined that two steps are critical for the efficiency of this protocol. The first is the design of specific anti-sense DNA oligonucleotide probes able to hybridize to the lncRNA of interest. To this end, the lncRNA secondary structure was predicted by bioinformatics and anti-sense oligonucleotide probes were designed with a strong affinity for regions that display a low probability of internal base pairing. The second crucial step of the procedure relies on the fixative conditions of the tissue or cultured cells that have to preserve the network between all molecular partners. Coupled with high throughput RNA sequencing, this RNA pull-down protocol can provide the whole RNA interactome of a lncRNA of interest.

This RNA pull-down method allows identifying the RNA targets of a long non-coding RNA (lncRNA). Based on the hybridization of home-made, designed anti-sense DNA oligonucleotide probes specific to this lncRNA in an appropriately fixed tissue or cell line, it efficiently allows the capture of all RNA targets of the lncRNA.

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...