We thank P. Jeremy Wang (University of Pennsylvania) for helpful edits and discussions. We also thank Sigrid Eckardt for language editing. K. Z. was supported by National Key R&#38;D Program of China (2016YFA0500902, 2018YFC1003500) and National Natural Science Foundation of China (31771653). L. Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province.&#160;J. N. was supported by Zhejiang Medical Science and Technology Project (2019KY499).&#160;M. L. was supported by grants of National Natural Science Foundation of China (31771588) and the 1000 Youth Talent Plan.

1. Protein preparation
<ol>
	<li>MEIOB and MOV10 expression constructs
	<ol>
		<li>Generate cDNA expression constructs encoding mouse MEIOB-A, C, and E (Figure 1A) and MOV10.
		<ol>
			<li>Set up the polymerase chain reaction (PCR) reactions for each fragment. Mix 1 &#956;L of mouse cDNA (from C57BL/6 mouse testis), 1 &#956;L of dNTP, 2 &#956;L of 10 &#956;M forward primer, 2 &#956;L of 10 &#956;M reverse primer, 1 &#956;L of DNA polymerase, 25 &#956;L of 2x PCR buffer, and 18 &#956;L of double distilled H2O (ddH2O) in a final volume of 50 &#956;L. 
			NOTE: The primers for the amplification of Meiob and Mov10 gene fragments are listed in Table 1.</li>
			<li>Perform PCR reactions using the following programs: 95 &#176;C for 5 min, 35 cycles of heating at 95 &#176;C for 15 s, annealing at 64 &#176;C for 15 s, extension at 72 &#176;C for 20 s (2 min for extending full length MOV10), and final extension at 68 &#176;C for 7 min. 
			NOTE: Use primer pair MEIOB-E (F1) and MEIOB-E-mut (R1) and MEIOB-E-mut (F2) and MEIOB-E (R2) to amplify two segments that contain the mutant sequence within an overlapping sequence at the 3&#39;&#160;and 5&#39;&#160;ends, respectively, to generate a mutant PCR template.</li>
		</ol>
		</li>
		<li>Analyze the amplified PCR DNA by gel electrophoresis, cut the band of required size from the gel quickly under a UV lamp, and place into a centrifuge tube. 
		NOTE: The expected product sizes visible on the agarose gel for MEIOB-A is 536 bp, MEIOB-C is 296 bp, MEIOB-E are 312 bp and 229 bp, and MOV10 is 3015 bp.</li>
		<li>Purify the PCR DNA with a gel extraction kit following the manufacturer&#39;s protocol.
		<ol>
			<li>Add an equal volume of dissolving buffer into the centrifuge tube from step 1.1.2 and melt gel in a 50-55 &#176;C water bath for 5-10 min, ensuring that the gel pieces melt completely. Centrifuge briefly to collect any droplets from the wall of the tube. 
			NOTE: The mass/volume concentration of the gel and the dissolving buffer is 1 mg/&#956;L.</li>
			<li>Place the adsorption column in the collection tube, transfer the solution containing the dissolved gel fragment to the adsorption column, and centrifuge at 12,000 x g for 2 min.</li>
			<li>Discard the filtrate at the bottom of the collection tubes. Add 600 &#956;L of the wash buffer to the column, centrifuge at 12,000 x g for 1 min, and discard the filtrate.</li>
			<li>Repeat step 1.1.3.3 once.</li>
			<li>Place the column back into the collection tube, and centrifuge at 12,000 x g for 2 min to remove all the remaining wash buffer.</li>
			<li>Place the adsorption column in a 1.5 mL sterilized centrifuge tube, add 50 &#956;L of ddH2O to the center of the adsorption column and centrifuge at 12,000 x g for 1 min. Measure the DNA concentration of the eluate using spectrophotometer.</li>
		</ol>
		</li>
		<li>Restriction digestion of plasmids
		<ol>
			<li>Digest pGEX-4T-1 vector with BamHI and NotI. To do so, mix 4 &#956;g of pGEX-4T-1 vector, 5 &#956;L of 10x digest buffer, 1 &#956;L of BamHI, and 1 &#956;L of NotI and ddH2O to a final reaction volume of 50 &#956;L. Incubate at 37 &#176;C for 2 h.</li>
			<li>Digest pRK5 vector with BamHI and XhoI by mixing 4 &#956;g of pRK5 vector, 5 &#956;L of 10x digest buffer, 1 &#956;L of BamHI, and 1 &#956;L of XhoI and ddH2O to a final reaction volume of 50 &#956;L. Incubate at 37 &#176;C for 2 h.</li>
		</ol>
		</li>
		<li>Analyze the vector DNA by gel electrophoresis, cut the desired size band from the gel quickly with a scalpel under a UV lamp and place it into a centrifuge tube.</li>
		<li>Purify the vector DNA with a gel extraction kit as 1.1.3 following the manufacturer&#39;s instruction. 
		NOTE: The length of linearized plasmids: pGEX-4T-1, 4.4 kb; pRK5, 4.7 kb.</li>
		<li>Set up a standard recombinant ligation reaction by combining 0.03 pmol of linearized vector, 0.06 pmol of cDNA fragment, 2 &#956;L of ligase, and 4 &#956;L of 5x ligase buffer and ddH2O in a final reaction volume of 10 &#956;L. 
		NOTE: Clone MEIOB-A, C, and E into a pGEX-4T-1 vector and MOV10 into a pRK5 vector.</li>
		<li>Incubate the mixture at 37 &#176;C for 30 min, and then cool the reaction immediately for 5 min on ice. Transform MEIOB recombinant plasmids into BL21 bacteria and MOV10 recombinant plasmids into DH5&#945;&#160;bacteria. 
		NOTE: Verify all recombinant constructs by Sanger sequencing.</li>
		<li>Prepare glycerol stocks of bacterial cultures containing verified recombinant plasmids by adding an equal volume of 50% glycerol to liquid cultures, and store at -80 &#176;C. 
		NOTE: For each subsequent experiment, streak out bacteria from glycerol stocks onto a fresh agar plate and use a single colony for the expansion as described in step 1.2.</li>
	</ol>
	</li>
	<li>MEIOB protein extracts from bacteria
	<ol>
		<li>Pick one colony of each BL21 strain transfected with the empty or recombinant pGEX-4T-1 plasmid verified by sequencing and inoculate in 3 mL LB containing 100 &#181;g/mL ampicillin. Grow overnight at 37 &#176;C with shaking at 220 rpm.</li>
		<li>Inoculate 300 mL LB containing 100 &#181;g/mL ampicillin from 3 mL overnight culture (from step 1.2.1). Grow the cultures with shaking at 37 &#176;C for 2 h till OD600 reaches 0.5-1.0.</li>
		<li>Induce the protein expression by adding isopropyl beta-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM. Incubate the cultures for an additional 3 h with shaking at 180 rpm and 18 &#176;C.</li>
		<li>Centrifuge the bacterial culture at 3,500 x g and 4 &#176;C for 20 min.</li>
		<li>Resuspend the pellet in 20 mL of ice-cold Dulbecco&#39;s phosphate buffered saline (DPBS) buffer. Sonicate the bacterial suspension on ice for 25 cycles in short 10 s bursts (output power 20%) followed by 2-3 s resting on ice.</li>
		<li>Centrifuge the lysate at 12,000 x g and 4 &#176;C for 15 min. Transfer all the supernatant to a fresh tube.</li>
		<li>Pre-wash beads.
		<ol>
			<li>Add 300 &#956;L of glutathione-sepharose beads to a fresh 15&#160;mL tube and wash the beads with 10 mL of ice-cold PBS buffer.</li>
			<li>Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads and discard the wash solution.</li>
		</ol>
		</li>
		<li>Add the lysate to the washed beads and incubate at 4 &#176;C for 2 h. Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads. Rinse the beads in 10 mL of ice-cold PBS 8x.</li>
		<li>Elute the beads with 1 mL of the elution buffer (10 mM glutathione in 50 mM Tris-HCl at pH 8.0) 6x, incubating at 4 &#176;C for 10 min prior to each elution step.&#160;Centrifuge at 750 x g and 4 &#176;C for 1 min to pellet the beads.&#160;Collect and pool the 6 fractions.</li>
		<li>Transfer the eluted proteins into a centrifugal filter and concentrate by centrifugation at 7,500 x g to obtain a final volume of 100-200 &#956;L.</li>
	</ol>
	</li>
	<li>MOV10 protein extracts from HEK293T cells
	<ol>
		<li>Transiently express the MOV10 proteins in cultured HEK293T cells.
		<ol>
			<li>Prepare MOV10-pRK5 plasmid at a concentration &#62;500 ng/&#956;L.</li>
			<li>Seed HEK293T cells in 15 cm dishes. When the cell density reaches ~70%-90%, replace the cell culture medium with fresh Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.</li>
			<li>For one transfection, dilute 60 &#956;g of MOV10-pRK5 plasmid DNA in 3 mL of the reduced serum medium, then add 120 &#956;L of the transfection enhancer reagent, and mix well.</li>
			<li>In a separate tube dilute 90 &#956;L of the transfection reagent with 3 mL of reduced serum medium (without penicillin-streptomycin) and mix well.</li>
			<li>Add the diluted DNA to each tube of diluted transfection reagent. Incubate at room temperature for 15 min.</li>
			<li>Add the transfection mixture to the cell culture, and culture cells for ~36-48 h.</li>
		</ol>
		</li>
		<li>After 36-48 h, collect cells from each plate in a 50 mL tube. Centrifuge at 500 x g for 5 min at 4 &#176;C. Wash each pellet with 10 mL of ice-cold PBS, and collect cells by centrifugation at 500 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the pellet in 3 mL of cell lysis buffer containing complete ehylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail. Incubate for 30 min on ice. Centrifuge the lysate at 20,000 x g and 4 &#176;C for 20 min.</li>
		<li>Add 100 &#956;L of anti-FLAG magnetic beads per dish of cells in a 1.5 mL tube.</li>
		<li>Wash the magnetic beads 2x with K150 buffer (50 mM HEPES at pH 7.5, 150 mM KoAc, 1 mM DTT, 0.1% NP-40).
		<ol>
			<li>Resuspend the magnetic beads in 1 mL of ice-cold K150 buffer.</li>
			<li>Incubate the magnetic beads for 2 min at 4 &#176;C with gentle rotation and pellet the magnetic beads with the help of a magnet. Remove and discard the supernatant.</li>
		</ol>
		</li>
		<li>Add the magnetic beads to the cell lysate supernatant from step 1.3.3 and incubate at 4 &#176;C for 2 h.</li>
		<li>Wash the protein bound magnetic beads 3x with K150 buffer, then 2x with K150 containing 250 mM NaCl, then 3x with K150 buffer as 1.3.5.</li>
		<li>Resuspend the beads in 300 &#956;L of FLAG elution buffer (100 mM NaCl, 20 mM Tris-HCl at pH 7.5, 5 mM MgCl2, 10% glycerol), add FLAG peptide to a final concentration of 0.5 &#956;g/&#956;L, and incubate with beads on a rotator at 4 &#176;C for 1 h, then pellet the magnetic beads with the help of a magnet.</li>
		<li>Collect the supernatant which contains the eluted MOV10 proteins, determine the concentration, and store at -80 &#176;C for future use.</li>
	</ol>
	</li>
</ol>
2. Nucleic acid preparation
<ol>
	<li>Purchase DNA and RNA oligonucleotides (oligos) with or without biotin labels from a suitable source. Dilute each oligo in RNase-free ddH2O to 20 &#956;M and keep it at -80 &#176;C for the future use. 
	NOTE: The oligo sequences of DNA/RNA substrates used in this study are listed in Table 2.</li>
	<li>Prepare the following mixture for the double-stranded RNA (dsRNA) annealing reaction for MOV10 helicase activity assay: mix 60 mM N-2-hydroxyethylpiperazine-N-ethane-sulphonic acid (HEPES) at pH 7.5, 6 mM KCl, 0.2 mM MgCl2 and RNase-free ddH2O in a final reaction volume of 20 &#956;L.</li>
	<li>Anneal RNA oligos to form RNA duplex by heating a mixture of the biotin-labeled top strand (2 &#956;M, final concentration) and a 1.5-fold of its unlabeled complementary bottom strand in the annealing buffer (step 2.2) at 95 &#176;C for 5 min, and then slowly cool it to room temperature (RT).</li>
</ol>
3. In vitro biochemical assays
<ol>
	<li>EMSA and enzymatic reactions
	<ol>
		<li>For the MEIOB EMSA assay, mix 50 mM Tris HCl at pH 7.5, 2 mM MgCl2, 50 mM NaCl, 10 mM EDTA, 2 mM dithiothreitol (DTT), 0.01% NP-40, 0.8 mM (or other relevant concentrations as shown in Figure 2 and Figure 3) MEIOB protein, and 10 nM biotin-labeled oligonucleotides and ddH2O in a final reaction volume of 20 &#956;L. Incubate at RT for 30 min, and add 5x stop buffer (125 mM EDTA, 50% glycerol) to stop the reaction. &#160;</li>
		<li>Set up MEIOB nuclease activity reactions as described in step 3.1.1 but without the addition of 10 mM EDTA.</li>
		<li>For the MOV10 helicase activity assay, mix 50 mM Tris-HCl at pH 7.5, 20 mM KoAc, 2 mM MgCl2, 0.01% NP-40, 1 mM DTT, 2 U/&#956;L RNase inhibitor, 10 nM biotin-labeled RNA substrate, 2 mM adenosine triphosphate (ATP), 100 nM RNA trap, and 20 ng of MOV10 protein and ddH2O in a final reaction volume of 20 &#956;L. Incubate the reaction mixture at 37 &#176;C for 10 min, 30 min, and 60 min. Add the 5x stop buffer to stop the reaction. 
		NOTE: RNA trap, a biotin-unlabeled oligo with sequence, complementarity to the labeled oligo, which prevents the unwound dsRNA from annealing again.</li>
	</ol>
	</li>
	<li>Polyacrylamide gels
	<ol>
		<li>Wash the gel plates (16 cm x 16 cm) and 1.5 mm combs. Assemble the gel electrophoresis units.</li>
		<li>To prepare a 10% native polyacrylamide gel, mix 14 mL of ddH2O, 1.25 mL of 10x Tris-boric acid-EDTA (TBE), 8.3 mL of 30% acrylamide, 1.25 mL of 50% glycerol, 187.5 &#956;L of 10% freshly prepared ammonium persulfate (APS), and 12.5&#160;&#956;L of tetramethylethylenediamine (TEMED).</li>
		<li>To prepare a 20% native polyacrylamide gel, mix 5.5 mL of ddH2O, 1.25 mL of 10x TBE, 1.25 mL of 50% glycerol, 16.7 mL of 30% acrylamide, 187.5 &#956;L of 10% APS, and 12.5 &#956;L of TEMED.</li>
		<li>Pour the acrylamide solution immediately to the gel and insert the comb. Let the mixture polymerize for approximately 30 min.</li>
	</ol>
	</li>
	<li>Gel running
	<ol>
		<li>Remove the comb, fill the tanks with the electrophoresis running buffer (0.5x TBE).</li>
		<li>Rinse the sample wells with 0.5x TBE buffer, then pre-run the gel at 100 V on ice for 30 min. Replace the running buffer with fresh 0.5x TBE.</li>
		<li>Load 20-25 &#956;L samples into each well.</li>
		<li>Use a 10% native acrylamide gel for the EMSA assay and a 20% native acrylamide gel for the enzymatic assays. Run electrophoresis at 100 V on the ice bath until the bromophenol blue marker has migrated to the bottom quarter of the gel.&#160;</li>
	</ol>
	</li>
	<li>Disassemble the gel plates, trim the gel by removing loading wells and unused lanes. Place the gel in 0.5x TBE buffer.</li>
	<li>Cut the filter paper and the nylon membrane to the size of the gel. Pre-wet the clean filter paper and the nylon membrane.</li>
	<li>Assemble the stack for transfer.
	<ol>
		<li>Place the pre-wet membrane onto the pre-wet filter paper.</li>
		<li>Place the gel on the membrane.</li>
		<li>Cover the gel with another layer of pre-wet filter paper.</li>
		<li>Remove all air bubbles by rolling a clean pipette from center to edge.</li>
	</ol>
	</li>
	<li>Transfer the samples from the gel to the membrane in a semi-dry electrophoretic apparatus at 90 mA for 20 min.</li>
	<li>Stop the transfer, and then dry the membrane on a new filter paper for 1 min.</li>
	<li>Crosslink the samples by irradiating the membrane at 120 mJ/cm2 for 45-60 s in a UV-light crosslinker equipped with 254 nm bulbs (auto crosslink function). Air dry the membrane at RT for 30 min.</li>
	<li>Chemiluminescence detection
	<ol>
		<li>Protocol 1: Use a standard volume of commercial chemiluminescent nucleic acid detection kit.
		<ol>
			<li>Add 20 mL of blocking buffer to the membrane and incubate for 15-30 min with gentle shaking on a rotator at 20-25 rpm.</li>
			<li>Prepare conjugate/blocking buffer solution by adding 66.7 &#956;L stabilized streptavidin-horseradish peroxidase conjugate to 20 mL of blocking buffer.</li>
			<li>Gently remove the blocking buffer and replace it with conjugate/blocking buffer. Incubate for 15 min on a rotator at 20-25 rpm.</li>
			<li>Wash the membrane 4x with shaking at 40-45 rpm for 5 min each.</li>
			<li>Add 30 mL of substrate equilibration buffer to the membrane. Incubate the membrane for 5 min with shaking at 20-25 rpm.</li>
			<li>Prepare substrate working solution by adding 6 mL of luminol/enhancer solution to 6&#160;mL of stable peroxide solution. Avoid light.&#160;</li>
			<li>Cover the entire surface of the membrane with substrate working solution and incubate for 5 min.</li>
			<li>Scan the membrane in a chemiluminescent imaging system for 1-3 s.</li>
		</ol>
		</li>
		<li>Protocol 2: Use 2x diluted commercial chemiluminescent nucleic acid detection kit and follow the steps 3.10.1.1-3.10.1.8.</li>
		<li>Protocol 3: Use self-made reagents6.
		<ol>
			<li>Prepare blocking buffer: mix 0.1 M Tris-HCl at pH 7.5, 0.1 M NaCl, 2 mM MgCl2, and 3% bovine serum albumin Fraction V. AP 7.5 buffer: mix 0.1 M Tris-HCl at pH 7.5, 0.1 M NaCl, and 2 mM MgCl2. AP 9.5 buffer: mix 0.1 M Tris-HCl at pH 9.5, 0.1 M NaCl, and 50 mM MgCl2. TE buffer: mix 10 mM Tris-HCl at pH 8.0, 1 mM EDTA at pH 8.0.</li>
			<li>Soak the membrane in blocking buffer at 30 &#176;C for 1 h.</li>
			<li>Add 8.5 &#956;L of streptavidin alkaline phosphatase to 10 mL of AP 7.5 Buffer. Shake the membrane gently in this solution at RT for 10 min. Then, wash the membrane 2x in 15 mL of AP 7.5 buffer for 10 min.</li>
			<li>Wash the membrane one more time in 20 mL of AP 9.5 buffer for 10 min. Add 20 mL of TE buffer to stop the reaction.</li>
			<li>Add 7.5 mL of development solution onto the membrane, and scan the membrane in a chemiluminescent imaging system for 1-3 s.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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H., 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=MOV10+Is+a+5'+to+3'+RNA+helicase+contributing+to+UPF1+mRNA+target+degradation+by+translocation+along+3'+UTRs.">MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.</a> Molecular Cell. 54 (4), (2014).</li><li>Talwar, T., 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=The+DEAD-box+protein+DDX43+(HAGE)+is+a+dual+RNA-DNA+helicase+and+has+a+K-homology+domain+required+for+full+nucleic+acid+unwinding+activity.">The DEAD-box protein DDX43 (HAGE) is a dual RNA-DNA helicase and has a K-homology domain required for full nucleic acid unwinding activity.</a> The Journal of Biological Chemistry. 292 (25), 10429-10443 (2017).</li><li>Nagy, N. M., Konya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+fast+and+slow+consecutive+processes+by+heterogeneous+isotope+exchange+using+P-32+radiotracer.">Study of fast and slow consecutive processes by heterogeneous isotope exchange using P-32 radiotracer.</a> Journal of Radioanalytical And Nuclear Chemistry. 318 (3), 2349-2353 (2018).</li><li>Wilson, D. L., Beharry, A. A., Srivastava, A., O'Connor, T. R., Kool, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Probes+for+ALKBH2+Allow+the+Measurement+of+DNA+Alkylation+Repair+and+Drug+Resistance+Responses.">Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses.</a> Angewandte Chemie. 57 (39), 12896-12900 (2018).</li><li>Wilchek, M., Bayer, E. A., Livnah, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+biorecognition:+the+(strept)avidin-biotin+system+as+a+model+for+protein-protein+and+protein-ligand+interaction.">Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.</a> Immunology Letters. 103 (1), 27-32 (2006).</li><li>Trippier, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+strategies+for+the+biotinylation+of+bioactive+small+molecules.">Synthetic strategies for the biotinylation of bioactive small molecules.</a> ChemMedChem. 8 (2), 190-203 (2013).</li><li>Rodgers, J. T., Patel, P., Hennes, J. L., Bolognia, S. L., Mascotti, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biotin-labeled+nucleic+acids+for+protein+purification+and+agarose-based+chemiluminescent+electromobility+shift+assays.">Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays.</a> Analytical Biochemistry. 277 (2), 254-259 (2000).</li><li>Panda, A. C., Martindale, J. L., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+Pulldown+of+Biotinylated+RNA+for+Detection+of+Protein-RNA+Complexes.">Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.</a> Bio-Protocol. 6 (24), (2016).</li><li>Bednarek, 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=mRNAs+biotinylated+within+the+5'+cap+and+protected+against+decapping:+new+tools+to+capture+RNA+-+protein+complexes.">mRNAs biotinylated within the 5' cap and protected against decapping: new tools to capture RNA - protein complexes.</a> Philosophical Transactions Of the Royal Society B-Biological Sciences. 373 (1762), (2018).</li><li>Souquet, B., 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=MEIOB+Targets+Single-Strand+DNA+and+Is+Necessary+for+Meiotic+Recombination.">MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination.</a> Plos Genetics. 9 (9), (2013).</li><li>Luo, M., 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=MEIOB+exhibits+single-stranded+DNA-binding+and+exonuclease+activities+and+is+essential+for+meiotic+recombination.">MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination.</a> Nature Communications. 4, 2788 (2013).</li><li>Xu, Y., Greenberg, R. A., Schonbrunn, E., Wang, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis-specific+proteins+MEIOB+and+SPATA22+cooperatively+associate+with+the+single-stranded+DNA-binding+replication+protein+A+complex+and+DNA+double-strand+breaks.">Meiosis-specific proteins MEIOB and SPATA22 cooperatively associate with the single-stranded DNA-binding replication protein A complex and DNA double-strand breaks.</a> Biology of Reproduction. 96 (5), 1096-1104 (2017).</li></ol>

No conflicts of interest are declared.

Investigating protein-nucleic acid interactions is critical to our understanding of molecular mechanisms underlying diverse biological processes. For example, MEIOB is a testis-specific protein essential for meiosis and fertility in mammals25,26,27. MEIOB contains an OB domain that binds to single-stranded DNA and exhibits 3&#39;&#160;to 5&#39;&#160;exonuclease activity26, which directly relates to its ph...

Protein-nucleic acid interactions are involved in many essential cellular processes such as DNA repair, replication, transcription, RNA processing, and translation. Protein interactions with specific DNA sequences within the chromatin are required for the tight control of gene expression at the transcriptional level1. Precise posttranscriptional regulation of numerous coding and noncoding RNAs necessitates extensive and complicated interactions between any protein and RNA2. These layers of gene expression regulatory mechanism comprise a cascade of dynamic intermolecular events, which are coordinated by interactions of transcription/epigenetic factors or RNA-binding proteins with their nucleic acid targets, as well as protein-protein interactions. To dissect whether proteins in vivo are directly or indirectly associated with nucleic acids and how such associations occur and culminate, in vitro biochemical assays are conducted to examine the binding affinity or enzymatic activity of proteins of interest on designed substrates of DNA and/or RNA.
Many techniques have been developed to detect and characterize nucleic acid-protein complexes, including the electrophoretic mobility shift assay (EMSA), also termed gel retardation assay or band shift assay3,4,5. EMSA is a versatile and sensitive biochemical method that is widely used for studying the direct binding of proteins with nucleic acids. EMSA relies on gel electrophoretic shift in bands, which are routinely visualized using chemiluminescence to detect biotin labels6,7, the fluorescence of fluorophore labels8,9, or by autoradiography of radioactive 32P labels10,11. Other purposes of biochemical studies are the identification and characterization of nucleic acid-processing activity of proteins, such as &#160;nuclease-based reactions to assess the cleavage products from nucleic acid substrates12,13,14 and DNA/RNA structure-unwinding assays to evaluate helicase activities15,16,17.
In such enzymatic activity assays, the radioisotope-labeled or fluorophore-labeled nucleic acids are often used as substrates due to their high sensitivity. Analysis of radiographs of enzymatic reactions involving 32P labeled radiotracers has been found to be sensitive and reproducible18. Yet, in an increasing number of laboratories in the world, the usage of radioisotopes is restricted or even prohibited due to the health risks associated with potential exposure. In addition to biosafety concerns, other drawbacks are the required necessary equipment to conduct work with radioisotopes, required radioactivity license, short half-life of 32P (about 14 days), and gradual deterioration of the probe quality due to radiolysis. Thus, alternative non-isotopic methods have been developed (i.e., labeling the probe with fluorophores enables detection by fluorescent imaging19). However, a high-resolution imaging system is needed when using fluorescently labeled probes. Biotin, a commonly used label, readily binds to biological macromolecules such as proteins and nucleic acids. Biotin-streptavidin system operates efficiently and improves detection sensitivity without increasing non-specific background20,21. Besides EMSA, biotin is widely used for protein purification and RNA pull-down, among others22,23,24.
This protocol successfully uses biotin-labeled nucleic acids as substrates for in vitro biochemical assays that include EMSA, in addition to enzymatic reactions in which biotin has not been commonly used. The MEIOB OB domain is constructed and two mutants (truncation and point mutation) are expressed as GST fusion proteins25,26,27, as well as mouse MOV10 recombinant FLAG fusion protein16. This report highlights the effectiveness of this combined protocol for protein purification and biotin-labeled assays for miscellaneous experimental purposes.

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			<td height="21" style="height:21px;width:351px;">Equipment</td>
			<td style="width:163px;"></td>
			<td style="width:104px;"></td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Centrifuge</td>
			<td style="width:163px;">Eppendorf, Germany</td>
			<td style="width:104px;">5242R</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemiluminescent Imaging System</td>
			<td>Tanon, China</td>
			<td>5200</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital sonifer</td>
			<td>Branson, USA</td>
			<td>BBV12081048A</td>
			<td style="width:176px;">450 Watts; 50/60 HZ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Semi-dry electrophoretic blotter</td>
			<td style="width:163px;">Hoefer, USA</td>
			<td style="width:104px;">TE77XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tube Revolver&#160;</td>
			<td style="width:163px;">Crystal, USA</td>
			<td style="width:104px;">3406051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">UV-light cross-linker</td>
			<td>UVP, USA</td>
			<td style="width:104px;">CL-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Materials</td>
			<td></td>
			<td style="width:104px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-4 Centrifugal Filter&#160;</td>
			<td>Milipore, USA</td>
			<td>UFC801096</td>
			<td>4 ml/10 K</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nylon membrane</td>
			<td>Thermo Scientific, USA</td>
			<td style="width:104px;">TG263940A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:104px;">430167</td>
			<td style="width:176px;">100 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TC-treated Culture Dish</td>
			<td>Corning, USA</td>
			<td style="width:104px;">430597</td>
			<td style="width:176px;">150 mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Microtubes tubes</td>
			<td style="width:163px;">AXYGEN, USA</td>
			<td style="width:104px;">MCT-150-C</td>
			<td style="width:176px;">1.5 mL&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tubes</td>
			<td style="width:163px;">Corning, USA</td>
			<td style="width:104px;">430791</td>
			<td style="width:176px;">15 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Reagents&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:104px;"></td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin</td>
			<td>SunShine Bio, China</td>
			<td>8h288h28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Anti-FLAG M2 magnetic beads</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">M8823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;">ATP</td>
			<td>Thermo Scientific, USA</td>
			<td>591136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCIP/NBT Alkaline Phosphatase Color Development Kit</td>
			<td>Beyotime, China</td>
			<td>C3206</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CelLyticTM M Cell Lysis Reagent&#160;</td>
			<td>Sigma, USA</td>
			<td>107M4071V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ClonExpress II one step cloning kit&#160;</td>
			<td>Vazyme, China</td>
			<td>C112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chemiluminescent Nucleic Acid Detection Kit</td>
			<td>Thermo Scientific, USA</td>
			<td>T1269950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP</td>
			<td>Sigma-Aldrich, USA</td>
			<td>DNTP100-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DMEM</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">10569044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DPBS buffer</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA</td>
			<td>Invitrogen, USA</td>
			<td style="width:104px;">AM9260G</td>
			<td>0.5 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">EDTA free protease inhibitor cocktail</td>
			<td style="width:163px;">Roche, USA</td>
			<td style="width:104px;">04693132001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Maxi Plasmid Kit&#160;</td>
			<td>Vazyme, China</td>
			<td>&#160; 
			DC202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastPure Gel DNA Extraction Mini Kit</td>
			<td>Vazyme, China</td>
			<td>DC301-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FBS</td>
			<td style="width:163px;">Gibco, USA</td>
			<td style="width:104px;">10437028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FLAG peptide</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Glycerol</td>
			<td style="width:163px;">Sigma, USA</td>
			<td style="width:104px;">SHBK3676</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GST Bulk Kit</td>
			<td style="width:163px;">GE Healthcare, USA</td>
			<td style="width:104px;">27-4570-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer</td>
			<td>Sigma, USA</td>
			<td>SLBZ2837</td>
			<td>1 M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td>Thermo Scientific, USA</td>
			<td>34060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">KoAc</td>
			<td style="width:163px;">Sangon Biotech, China</td>
			<td style="width:104px;">127-08-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamin 3000 Transfection Reagent</td>
			<td>Thermo Scientific, USA</td>
			<td style="width:104px;">L3000001</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MgCl2</td>
			<td>Invitrogen, USA</td>
			<td style="width:104px;">AM9530G</td>
			<td>1 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Invitrogen, USA</td>
			<td>AM9759 
			&#160;</td>
			<td>5 M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">NP-40</td>
			<td style="width:163px;">Amresco, USA</td>
			<td style="width:104px;">M158-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM medium</td>
			<td>Gibco, USA</td>
			<td style="width:104px;">31985062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Gibco, USA</td>
			<td>10010023</td>
			<td>PH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">RNase Inhibitor</td>
			<td style="width:163px;">Promega, USA</td>
			<td style="width:104px;">N251B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Streptavidin alkaline phosphatase</td>
			<td style="width:163px;">Promega, USA</td>
			<td style="width:104px;">V5591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TBE</td>
			<td style="width:163px;">Invitrogen, USA</td>
			<td style="width:104px;">15581044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer&#160;</td>
			<td>Invitrogen, USA</td>
			<td>15567027</td>
			<td>1 M, PH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCI Buffer&#160;</td>
			<td>Invitrogen, USA</td>
			<td>15568025</td>
			<td>1 M, PH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tween-20</td>
			<td style="width:163px;">Sangon Biotech, China</td>
			<td style="width:104px;">A600560</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro biochemical assays using biotin labels to study protein-nucleic acid interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

The protein structure of MEIOB and the expression constructs used in this study are illustrated in Figure 1A. OB folds in MEIOB are compact barrel-like structures that can recognize and interact with single-stranded nucleic acids. One of the OB domains (aa 136-307, construct A) binds single stranded DNA (ssDNA), the truncated protein (aa 136-178 truncations, construct C) and the point mutant form (R235A mutation, construct E) of MEIOB do not have DNA-binding activity<sup cla...

Watch this Scientific Journal Video about In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions at JoVE.com

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

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12.5K Views.  Nanjing Medical University. Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely applicable for studying protein-nucleic acid interactions.

Video: In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes.
The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called &#34;transmitochondrial cybrids.&#34; Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model

Transmitochondrial cybrids are hybrid cells obtained by fusing mitochondrial DNA (mtDNA)-depleted cells (rho0 cells) with cytoplasts (enucleated cells) derived from patients affected by mitochondrial disorders. They allow the determination of the nuclear or mitochondrial origin of the disease, evaluation of biochemical activity, and confirmation of the pathogenetic role of mtDNA-related variants.