Name: Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy (Video) | JoVE Journal
Uploaded: 2019-07-29T15:00:00.000+00:00
Duration: 7 min 26 s
Description: Learn circular RT-PCR for mapping primary and processed transcripts in maize mitochondria. This protocol uses RNA normalization to reduce unstable 5' triphosphate effects, enabling accurate transcript discrimination.

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This work was supported by the National Natural Science Foundation of China (grant no. 31600250, Y.Z.), Science and Technology Projects of Guangzhou City (grant no. 201804020015, H.N.), and the China Agricultural Research System (grant no. CARS-04-PS09, H.N.).

1. Primer Design
<ol>
	<li>Design gene-specific primers for reverse transcription (RT) using PCR primer design software (Table of Materials) based on the general rules of primer design17. 
	NOTE: RT primers are highly specific to the target transcripts, and they are generally anchored on the 5&#8217; part of coding sequences (mature mRNAs and precursor RNAs), or ~500&#8211;600 nt downstream of the anticipated 5&#8217; end (18S and 26S rRNAs).</li>
	<li>Design pairs of divergent primers to amplify the circularized transcripts by cRT-PCR. 
	NOTE: The paired divergent primers flank the 5&#8217;-3&#8217; junction of the circularized transcripts, and their positions vary among the target transcripts analyzed (Figure S1). Some transcripts have long UTRs; for example, nad2-1 5&#8217; UTR and rps4-1 3&#8217; UTR are 1,985 and 1,826/1,834 nt, respectively1. If both primers are anchored on coding region, it will be hard to amplify the target transcripts. By contrast, some UTRs are short; for example, the 3&#8217; UTRs of nad2-1 and nad4-1 are 34/35 and 29&#8211;31 nt, respectively1. If the paired primers are located far away from the coding sequences, the PCR will fail. Generally, two pairs of divergent primers are designed for each target transcripts, while multiple pairs may be necessary to map those whose transcript patterns are complicated and/or UTRs are very long.</li>
	<li>Design pairs of convergent primers to prepare RNA probes for Northern blot. 
	NOTE: The paired primers are located on coding region of the target gene, and the size of the PCR product should be in the range of 100 to 1,000 bp. Each forward primer should contain a restriction enzyme site to minimize vector sequences in the resulting probes.</li>
</ol>
2. Preparation of Crude Mitochondrion from Maize Developing Kernels
<ol>
	<li>Sterilize pestles, mortars, glass funnels, tubes, and tips by autoclave, and dry them in oven.</li>
	<li>Perform all procedures at 4 &#176;C or on ice, and pre-cool all solutions.</li>
	<li>Prepare 100 mL of extraction buffer (EB), composed of 0.3 M sucrose, 5 mM tetrasodium pyrophosphate, 10 mM KH2PO4, 2 mM EDTA, 1% [w/v] polyvinylpyrrolidone 40, 1% [w/v] bovine serum albumin, 5 mM L-cysteine, and 20 mM ascorbic acid. Adjust to pH 7.3 with KOH and sterilize by filtration.</li>
	<li>Prepare 100 mL of wash buffer (WB) consisting of 0.3 M sucrose, 1 mM EGTA, and 10 mM MOPS (3-(N-morpholino)propanesulfonic acid). Adjust to pH 7.2 with NaOH and sterilize by filtration. 
	NOTE: It is suggested to prepare EB and WB using DEPC-treated deionized H2O.</li>
	<li>Collect 20 g developing kernels at 11&#8211;20 days after pollination (DAP) to a 50-mL tube placed on ice, and then transfer the kernels to pre-cooled mortars. 
	NOTE: Use a ratio of 100 mL of EB to 20 g of maize kernels.</li>
	<li>Add 10&#8211;20 mL of ice-cold EB to each mortar, and grind the kernels completely.</li>
	<li>Add more EB, and filter the ground tissues through two layers of filter cloth (Table of Materials).</li>
	<li>Centrifuge the filtrate at 8,000 x g for 10 min, and discard the pellet.</li>
	<li>Transfer the supernatant to a new tube, and centrifuge it at 20,000 x g for 10 min.</li>
	<li>Pour off the supernatant, and resuspend the pellet in 6 mL WB.</li>
	<li>Aliquot the suspension to five 1.5 mL RNase-free tubes, and centrifuge them at 14,000 x g for 5 min.</li>
	<li>Discard the supernatant, freeze the mitochondrial pellet in liquid nitrogen, and store at -80 &#176;C.</li>
</ol>
3. Extraction of Mitochondrial RNA
<ol>
	<li>Extract mitochondrial RNA with a commercial reagent (Table of Materials) according to the manufacture&#8217;s instructions. 
	CAUTION: This reagent contains phenol and guanidine isothiocyanate. Work with it in a fume hood, and wear lab coat and gloves.</li>
	<li>Dissolve the isolated mitochondrial RNA in DEPC-treated deionized H2O, and estimate RNA concentration and purity with a spectrophotometer (Table of Materials). 
	NOTE: Generally, ~250 &#956;g mitochondrial RNA is obtained from 20 g of 15-DAP maize kernels.</li>
	<li>Prepare one agarose gel composed of 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1mM EDTA), 1.5% agarose (Table of Materials), and 1x nuclear staining dye (Table of Materials).</li>
	<li>Add an appropriate volume of 10x loading buffer (0.5% bromophenol blue, 0.5% xylene cyanol FF, and 50% glycerol), and load mitochondrial RNA/Loading buffer mixture on the 1.5% agarose gel.</li>
	<li>Run the gel in 1x TAE buffer at 5&#8211;6 V/cm for 20&#8211;25 min, and evaluate mitochondrial RNA integrity by imaging the gel with a gel documentation system (Table of Materials). 
	NOTE: The presence of two distinct bands (~3,510 and ~1,970 nt for maize mitochondrial 26S and 18S rRNAs, respectively) is an acceptable standard for intact mitochondrial RNA. To exclude possible degradation, RNA integrity should be investigated during the multiple steps of circularized RNA preparation (Figure 2).</li>
</ol>
4. RNA 5&#8217; Polyphosphatase Treatment
<ol>
	<li>Set up RNA 5&#8217; polyphosphatase (Table and Materials) treatment (Table 1), and incubate at 37 &#176;C for 30&#8211;60 min.</li>
	<li>Recover the 5&#8217; polyphosphatase-treated RNA with an RNA purification kit (Table of Materials) according to the manufacturer&#8217;s instructions.</li>
	<li>Repeat steps 3.3 to 3.5.</li>
</ol>
5. RNA Circularization
<ol>
	<li>Prepare two circularization reactions using the same amounts of 5&#8217; polyphosphatase-treated and non-treated mitochondrial RNAs (Table 2), and incubate both reactions at 16 &#176;C for 12&#8211;16 h.</li>
	<li>Recover the two sets of self-ligated RNAs using the same kit as in step 4.2. 
	NOTE: It should be noted that only a fraction of mitochondrial RNA will be self-ligated, and the recovered RNA will be a mixture of linear and circularized transcripts.</li>
	<li>Repeat steps 3.3 to 3.5.</li>
</ol>
6. Reverse Transcription
<ol>
	<li>Synthesize two sets of cDNAs from the same amounts of circularized 5&#8217; polyphosphatase-treated and non-treated RNAs (200 ng).</li>
	<li>Prepare a primer mixture by adding an equal ratio of 26S-CRT and up to 7 other RT primers. 
	NOTE: The final concentration of the primer mixture should be 1 &#956;M.</li>
	<li>Prepare two pre-mixtures by combining the reagents listed in Table 3, incubate at 65 &#176;C for 5 min, and then chill on ice for 2 min.</li>
	<li>Assemble two RT reaction systems (Table 4), and incubate them at 42 &#176;C for 50 min.</li>
	<li>Heat both RT reactions at 70 &#176;C for 5 min, and then chill them on ice.</li>
</ol>
7. Normalization
<ol>
	<li>Prepare two PCR reactions by adding the same volume of template cDNAs derived from 5&#8217; polyphosphatase-treated or non-treated RNAs, divergent primers flanking the 5&#8217;-3&#8217; junction of circularized 26S rRNA (i.e. 26S-CF1 and -CR1), etc. (Table 5).</li>
	<li>Run the two reactions in a thermal cycler (Table of Materials) under conditions described in Table 6.</li>
	<li>Prepare one agarose gel composed of 1x TAE buffer, 1.0% agarose, and 1x nuclear staining dye.</li>
	<li>Add 2 &#956;L 10x loading buffer (0.5% bromophenol blue, 0.5% xylene cyanol FF, and 50% glycerol) to each of the two PCR products, and load them on the gel.</li>
	<li>Run the gel in 1x TAE buffer at 5&#8211;6 V/cm for 30&#8211;40 min, and image it using the same gel documentation system as step 3.5.</li>
	<li>Compare the abundance of the two PCR products using computer software (Table of Materials, Figure 4A), and optimize the normalization by changing the amounts of template cDNAs if necessary.</li>
</ol>
8. PCR Amplification
<ol>
	<li>Prepare pairs of PCR reactions by adding appropriate volume of the normalized cDNAs and a pair of divergent primers flanking 5&#8217;-3&#8217; junction of the target transcripts (Table 7). 
	NOTE: The amount of template cDNAs used for PCR amplification of the target transcripts are determined by the 26S rRNA normalization results.</li>
	<li>Perform the PCR reactions according to the program described in Table 8.</li>
	<li>Repeat steps 7.3 to 7.5.</li>
	<li>Change to nested divergent primers, and verify the first round PCR results by repeating steps 8.1 and 8.2.</li>
	<li>Repeat steps 7.3 to 7.5.</li>
	<li>Recover the prominent bands that could be repeated in two rounds of PCR amplification using a gel DNA recovery kit (Table of Materials).</li>
</ol>
9. Determination of Transcript Termini
<ol>
	<li>Clone the gel-purified PCR products into a blunt-end vector (Table of Materials) using standard techniques.</li>
	<li>Perform colony PCR to select positive clones containing the target inserts, and sequence them commercially. 
	NOTE: Positive clones containing inserts with variable size are usually detected from a single recovered band because many steady-state transcripts in plant mitochondrial have heterogeneous 5&#8217; and/or 3&#8217; ends1,12.</li>
	<li>Align the sequencing data with maize mitochondrial genome using basic local alignment search tool (BLAST) of national center for biotechnology information (NCBI). Choose organism &#8220;maize (taxid:4577)&#8221;, and search database &#8220;Nucleotide collection (nr/nt)&#8221;.</li>
	<li>Find the 5&#8217;-3&#8217; junction of the circularized transcript, and determine the positions of 5&#8217; and 3&#8217; transcript termini.</li>
	<li>Calculate the size of target transcripts.</li>
</ol>
10. Verification of the cRT-PCR Mapping Results by RNA Gel Blot Hybridization
NOTE: RNA gel blot hybridization is performed by using a commercial kit (Table of Materials), which contains the reagents for transcription-labeling of RNA with digoxigenin (DIG) and T7 RNA polymerase, hybridization, and immunological detection. Please refer to the protocols provided in this kit for more details. Make sure that only RNase free equipment is used for the whole procedure.
<ol>
	<li>Amplify the DNA fragment used to prepare RNA probe, and clone it to the same vector as in step 9.1, which contains a T7 promoter 17 bp upstream of the insertion site.</li>
	<li>Linearize the construct using proper restriction enzyme, recover the linearized plasmid by a DNA purification kit (Table of Materials), and dissolve it in DEPC-treated deionized H2O.</li>
	<li>Label RNA probes with DIG-11-UTP by a commercial kit (Table of Materials) according to the manufacturer&#8217;s instructions.</li>
	<li>Prepare 500 mL of 10x MOPS buffer (0.2 M MOPS, 50 mM sodium acetate, and 10 mM EDTA) using DEPC-treated deionized H2O. Adjust to pH 7.0 by NaOH, and sterilize by filtration.</li>
	<li>Add 2&#8211;3 volumes of loading buffer (50% formamide, 6.2% formaldehyde, 1x MOPS, 10% glycerol, and 0.1% bromophenol blue) to the mitochondrial RNA prepared in steps 3.1&#173;&#8211;3.3.</li>
	<li>Denature the RNA sample/Loading buffer mixture at 65 &#176;C for 10 min, and then chill on ice for 1 min.</li>
	<li>Prepare a denatured agarose gel (2% formaldehyde, 1.2% agarose, and 1x MOPS), and load the RNA sample/Loading buffer mixture to the gel (1&#8211;2 &#956;g mitochondrial RNA per well).</li>
	<li>Run the gel in 1x MOPS at 3&#8211;4 V/cm for ~4 h.</li>
	<li>Prepare 2 L of 20x SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Treat it with 0.1% DEPC overnight, and sterilize by autoclave.</li>
	<li>Rinse the gel twice in 20x SSC (15 min each time), and transfer gel RNA to a nylon membrane (Table of Materials) by capillary transfer with 20x SSC for 10&#8211;16 h.</li>
	<li>Fix the RNA to membrane by baking at 120 &#176;C for 30 min.</li>
	<li>Perform RNA hybridization and immunological detection with a commercial kit (Table of Materials) according to the manufacturer&#8217;s instruction.</li>
</ol>
11. Discrimination of Primary and Processed 5&#8217; Ends
<ol>
	<li>Discriminate the primary and processed 5&#8217; ends by comparing the cRT-PCR products obtained from the normalized 5&#8217; polyphosphatase-treated and non-treated RNAs. 
	NOTE: To primary transcripts, the abundance of PCR products from 5&#8217; polyphosphatase-treated RNA is much higher than that from non-treated counterpart (Figure 1, &#8216;Gene A,&#8217; and Figure 4A). However, to processed transcripts, a comparable level of PCR products would be amplified from the two sets of mitochondrial RNAs (Figure 1, &#8216;Gene B&#8217;).</li>
	<li>Design RT-qPCR primers based on the cRT-PCR mapping results.</li>
	<li>Verify the cRT-PCR discrimination results by RT-qPCR. 
	NOTE: The two cDNA samples derived from 5&#8217; polyphosphatase-treated and non-treated RNAs are normalized by the RT-qPCR products of 26S mature rRNA.</li>
</ol>

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<li>Choi, B. Y., Acero, M. M., Bonen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+of+wheat+mitochondrial+mRNA+termini+and+comparison+with+breakpoints+in+DNA+homology+among+plants%5BTitle%5D)+AND+%22Plant+Molecular+Biology%22%5BJournal%5D)">Mapping of wheat mitochondrial mRNA termini and comparison with breakpoints in DNA homology among plants.</a> Plant Molecular Biology. 80 (4-5), 539-552 (2012).</li>
<li>Calixte, S., Bonen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmentally-specific+transcripts+from+the+ccmFN-rps1+locus+in+wheat+mitochondria%5BTitle%5D)+AND+%22Molecular+Genetics+and+Genomics%22%5BJournal%5D)">Developmentally-specific transcripts from the ccmFN-rps1 locus in wheat mitochondria.</a> Molecular Genetics and Genomics. 280 (5), 419-426 (2008).</li>
<li>Kuhn, K., Weihe, A., Borner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+promoters+are+a+common+feature+of+mitochondrial+genes+in+Arabidopsis%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Multiple promoters are a common feature of mitochondrial genes in Arabidopsis.</a> Nucleic Acids Research. 33 (1), 337-346 (2005).</li>
<li>Jonietz, C., Forner, J., Holzle, A., Thuss, S., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA+PROCESSING+FACTOR2+is+required+for+5%27+end+processing+of+nad9+and+cox3+mRNAs+in+mitochondria+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22ThePlant+Cell%22%5BJournal%5D)">RNA PROCESSING FACTOR2 is required for 5' end processing of nad9 and cox3 mRNAs in mitochondria of Arabidopsis thaliana.</a> ThePlant Cell. 22 (2), 443-453 (2010).</li>
<li>Stoll, B., Stoll, K., Steinhilber, J., Jonietz, C., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+transcript+length+polymorphisms+are+a+widespread+phenomenon+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22Plant+Molecular+Biology%22%5BJournal%5D)">Mitochondrial transcript length polymorphisms are a widespread phenomenon in Arabidopsis thaliana.</a> Plant Molecular Biology. 81 (3), 221-233 (2013).</li>
<li>Mulligan, R. M., Lau, G. T., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Numerous+transcription+initiation+sites+exist+for+the+maize+mitochondrial+genes+for+subunit+9+of+the+ATP+synthase+and+subunit+3+of+cytochrome+oxidase%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Numerous transcription initiation sites exist for the maize mitochondrial genes for subunit 9 of the ATP synthase and subunit 3 of cytochrome oxidase.</a> Proceedings of the National Academy of Sciences of the United States of America. 85 (21), 7998-8002 (1988).</li>
<li>Lupold, D. S., Caoile, A. G., Stern, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+maize+mitochondrial+cox2+gene+has+five+promoters+in+two+genomic+regions%2C+including+a+complex+promoter+consisting+of+seven+overlapping+units%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The maize mitochondrial cox2 gene has five promoters in two genomic regions, including a complex promoter consisting of seven overlapping units.</a> Journal of Biological Chemistry. 274 (6), 3897-3903 (1999).</li>
<li>Yan, B., Pring, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+initiation+sites+in+sorghum+mitochondrial+DNA+indicate+conserved+and+variable+features%5BTitle%5D)+AND+%22Current+Genetics%22%5BJournal%5D)">Transcriptional initiation sites in sorghum mitochondrial DNA indicate conserved and variable features.</a> Current Genetics. 32 (4), 287-295 (1997).</li>
<li>Rapp, W. D., Lupold, D. S., Mack, S., Stern, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Architecture+of+the+maize+mitochondrial+atp1+promoter+as+determined+by+linker-scanning+and+point+mutagenesis%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis.</a> Molecular and Cellular Biology. 13 (12), 7232-7238 (1993).</li>
<li>Rapp, W. D., Stern, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+conserved+11+nucleotide+sequence+contains+an+essential+promoter+element+of+the+maize+mitochondrial+atp1+gene%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">A conserved 11 nucleotide sequence contains an essential promoter element of the maize mitochondrial atp1 gene.</a> The EMBO Journal. 11 (3), 1065-1073 (1992).</li>
<li>Forner, J., Weber, B., Thuss, S., Wildum, S., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+of+mitochondrial+mRNA+termini+in+Arabidopsis+thaliana%3A+t-elements+contribute+to+5%27+and+3%27+end+formation%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Mapping of mitochondrial mRNA termini in Arabidopsis thaliana: t-elements contribute to 5' and 3' end formation.</a> Nucleic Acids Research. 35 (11), 3676-3692 (2007).</li>
<li>Binder, S., Stoll, K., Stoll, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maturation+of+5%27+ends+of+plant+mitochondrial+RNAs%5BTitle%5D)+AND+%22Physiologia+Plantarum%22%5BJournal%5D)">Maturation of 5' ends of plant mitochondrial RNAs.</a> Physiologia Plantarum. 157 (3), 280-288 (2016).</li>
<li>Hang, R. L., Liu, C. Y., Ahmad, A., Zhang, Y., Lu, F. L., Cao, X. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arabidopsis+protein+arginine+methyltransferase+3+is+required+for+ribosome+biogenesis+by+affecting+precursor+ribosomal+RNA+processing%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16190-16195 (2014).</li>
<li>Wang, H. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maize+Urb2+protein+is+required+for+kernel+development+and+vegetative+growth+by+affecting+pre-ribosomal+RNA+processing%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">Maize Urb2 protein is required for kernel development and vegetative growth by affecting pre-ribosomal RNA processing.</a> New Phytologist. 218 (3), 1233-1246 (2018).</li>
<li>Maloney, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+in+maize+mitochondrial+26S+rRNA+of+a+short+5%27-end+sequence+possibly+involved+in+transcription+initiation+and+processing%5BTitle%5D)+AND+%22Current+Genetics%22%5BJournal%5D)">Identification in maize mitochondrial 26S rRNA of a short 5'-end sequence possibly involved in transcription initiation and processing.</a> Current Genetics. 15 (3), 207-212 (1989).</li>
<li>Green, R. M., Sambrook, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGreen%2C+RM+%22Molecular+Cloning%3A+A+Laboratory+Manual%22">Molecular Cloning: A Laboratory Manual</a>. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor. New York. (2012).</li>
<li>Canino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arabidopsis+encodes+four+tRNase+Z+enzymes%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Arabidopsis encodes four tRNase Z enzymes.</a> Plant Physiology. 150 (3), 1494-1502 (2009).</li>
<li>Gobert, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+Arabidopsis+organellar+protein+has+RNase+P+activity%5BTitle%5D)+AND+%22Nature+Structural%2C+Molecular+Biology%22%5BJournal%5D)">A single Arabidopsis organellar protein has RNase P activity.</a> Nature Structural, Molecular Biology. 17, 740-744 (2010).</li>
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</ol>

The authors have nothing to disclose.

In a previous study, total and mitochondrial RNAs from cell suspension culture of Arabidopsis were used to map mitochondrial transcript termini by cRT-PCR, and similar results were obtained12. However, only enriched mitochondrial RNA was used to map mitochondrial transcript termini in many other studies1,2,3,9. We found that the enrichment of mitochondrial RNA i...

In plant mitochondria, many mature and precursor RNAs are accumulated as multiple isoforms, and the steady-state transcripts can be divided into two groups based on the difference at their 5&#39; ends1,2,3,4. The primary transcripts have 5&#39; triphosphate ends, which are derived from transcription initiation. By contrast, the processed transcripts have 5&#39; monophosphate generated by post-transcriptional processing. Discrimination and mapping of the two types of transcripts are important to unravel the molecular mechanisms underlying transcription and transcript end maturation.
To distinguish between the primary and processed transcripts in plant mitochondrion, four major strategies have been developed. The first strategy is to pre-treat the mitochondrial RNAs with tobacco acid pyrophosphatase (TAP), which converts 5&#39; triphosphate to monophosphate and enables primary transcripts to be circularized by RNA ligase. The transcript abundances of TAP-treated and non-treated RNA samples are then compared by rapid amplification of cDNA ends (RACE) or circular RT-PCR (cRT-PCR)2,3,4. In the second strategy, processed transcripts are firstly depleted from mitochondrial RNAs using terminator 5&#39;-phosphate-dependant exonuclease (TEX), and the primary transcripts left are then mapped by primer extension analysis5,6. The third strategy is to pre-cap the primary transcripts using guanylyl transferase, and then the position of triphosphated 5&#39; termini is determined by primer extension together with ribonuclease or S1 nuclease protection analysis7,8,9. Different from those depending on the presence/absence of 5&#39; triphosphate, the fourth strategy combines in vitro transcription, site-directed mutagenesis, and primer extension analysis to characterize the putative promoters and determine the transcription initiation sites8,10,11. By using these strategies, many primary and processed transcripts have been determined in plant mitochondria.
However, several studies have reported that the 5&#39; triphosphate of primary transcripts were unstable, and they were easily converted to monophosphate for unknown reason2,4,12,13. This problem hinders a clear discrimination of the two types of transcripts by using techniques depending on the presence/absence of 5&#39; triphosphate, and previous efforts to systematically discriminate between the primary and processed transcripts in plant mitochondria failed2,12.
In this protocol, we combine cRT-PCR, RNA 5&#39; polyphosphatase treatment, RT-qPCR, and Northern blot to systematically distinguish the primary and processed transcripts stably accumulated in maize (Zea mays) mitochondrion (Figure 1). cRT-PCR allows simultaneous mapping of 5&#39; and 3&#39; extremities of a RNA molecule, and it is usually adapted to map transcript termini in plants2,12,14,15. RNA 5&#39; polyphosphatase could remove two phosphates from the triphosphated 5&#39; termini, which makes the primary transcripts available for self-ligation by RNA ligase. Previous studies showed that mature 26S rRNA in maize had processed 5&#39; terminus, and it was insensitive to RNA 5&#39; polyphosphatase1,16. To minimize the influence of unstable triphosphate at primary 5&#39; termini, the 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by mature 26S rRNA, and the primary and processed transcripts are then differentiated by comparing the cRT-PCR products obtained from the two RNA samples. The cRT-PCR mapping and discrimination results are verified by Northern blot and RT-qPCR, respectively. Finally, alternative primers are used to amplify those transcripts detected in Northern blot but not by cRT-PCR. By using this cRT-PCR-based strategy, most steady-state transcripts in maize mitochondrion have been differentiated and mapped1.

<table><tbody><tr><td>Acetic acid</td><td>Aladdin, China</td><td>A112880</td><td>To prepare 1x TAE buffer</td></tr><tr><td>Applied Biosystems 2720 Thermal Cycler</td><td>Thermo Fisher Scientific, USA</td><td>4359659</td><td>Thermal cycler for PCR amplification</td></tr><tr><td>Ascorbic acid</td><td>Sigma-aldrich, USA</td><td>V900134</td><td>For preparation of extraction buffer</td></tr><tr><td>Biowest Agarose</td><td>Biowest, Spain</td><td>9012-36-6</td><td>To resolve PCR products and RNAs</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-aldrich, USA</td><td>A1933</td><td>For preparation of extraction buffer</td></tr><tr><td>Bromophenol blue</td><td>Sigma-aldrich, USA</td><td>B8026</td><td>For preparation of loading buffer for agarose gel electrophoresis and Northern blot</td></tr><tr><td>DEPC</td><td>Sigma-aldrich, USA</td><td>V900882</td><td>Deactivation of RNase</td></tr><tr><td>DIG Northern starter kit</td><td>Roche, USA</td><td>12039672910</td><td>For DIG-RNA labeling and Northern blot. This kit contains the reagents for transcription-labeling of RNA with DIG and T7 RNA polymerase, hybridization and chemiluminescent detection.</td></tr><tr><td>EDTA</td><td>Sigma-aldrich, USA</td><td>V900106</td><td>For preparation of extraction buffer and 1x TAE buffer</td></tr><tr><td>EGTA</td><td>Sigma-aldrich, USA</td><td>E3889</td><td>For preparation of wash buffer</td></tr><tr><td>Gel documentation system</td><td>Bio-Rad, USA</td><td>Gel Doc XR+</td><td>To image the agarose gel</td></tr><tr><td>Glycerol</td><td>Sigma-aldrich, USA</td><td>G5516</td><td>For preparation of loading buffer for agarose gel electrophoresis</td></tr><tr><td>GoldView II (5,000x)</td><td>Solarbio,. China</td><td>G8142</td><td>DNA staining</td></tr><tr><td>Hybond-N+, Nylon membrane</td><td>Amersham Biosciences, USA</td><td>RPN119</td><td>For Northern blot</td></tr><tr><td>Image Lab</td><td>Bio-Rad, USA</td><td>Image Lab 3.0</td><td>Image gel, and compare the abundance of PCR products.</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-aldrich, USA</td><td>V900041</td><td>For preparation of extraction buffer</td></tr><tr><td>KOH</td><td>Aladdin, China</td><td>P112284</td><td>For preparation of extraction buffer</td></tr><tr><td>L-cysteine</td><td>Sigma-aldrich, USA</td><td>V900399</td><td>For preparation of extraction buffer</td></tr><tr><td>Millex</td><td>Millipore, USA</td><td>SLHP033RB</td><td>To sterile extraction and wash buffers by filtration</td></tr><tr><td>Miracloth</td><td>Calbiochem, USA</td><td>475855-1R</td><td>To filter the ground kernel tissues</td></tr><tr><td>MOPS</td><td>Sigma-aldrich, USA</td><td>V900306</td><td>For preparation of running buffer for Northern blot</td></tr><tr><td>NanoDrop</td><td>Thermo Fisher Scientific, USA</td><td>2000C</td><td>For RNA concentration and purity assay</td></tr><tr><td>NaOH</td><td>Sigma-aldrich, USA</td><td>V900797</td><td>For preparation of wash buffer</td></tr><tr><td>pEASY-Blunt simple cloning vector</td><td>TransGen Biotech, China</td><td>CB111</td><td>Cloning of the gel-recovered band. It contains a T7 promoter several bps upstream of the insertion site.</td></tr><tr><td>Phanta max super-fidelity DNA polymerase</td><td>Vazyme, China</td><td>P505</td><td>DNA polymerase for PCR amplification</td></tr><tr><td>Polyvinylpyrrolidone 40</td><td>Sigma-aldrich, USA</td><td>V900008</td><td>For preparation of extraction buffer</td></tr><tr><td>Primer Premier 6.24</td><td>PREMIER Biosoft, USA</td><td>Primer Premier 6.24</td><td>To design primers for reverse transcription and PCR amplification</td></tr><tr><td>PrimeScript II reverse transcriptase</td><td>Takara, Japan</td><td>2690</td><td>To synthesize the first strand cDNA</td></tr><tr><td>PureLink RNA Mini kit</td><td>Thermo Fisher Scientific, USA</td><td>12183025</td><td>For RNA purificaion</td></tr><tr><td>RNA 5&#039; polyphosphatase</td><td>Epicentre, USA</td><td>RP8092H</td><td>To convert 5&#039; triphosphate to monophosphate</td></tr><tr><td>RNase inhibitor</td><td>New England Biolabs, UK</td><td>M0314</td><td>A component of RNA self-ligation and 5&#039; polyphosphatase treatment reactions, and it is used to inhibite the activity of RNase.</td></tr><tr><td>Sodium acetate</td><td>Sigma-aldrich, USA</td><td>V900212</td><td>For preparation of running buffer for Northern blot</td></tr><tr><td>Sodium chloride</td><td>Sigma-aldrich, USA</td><td>V900058</td><td>To prepare 20x SSC</td></tr><tr><td>SsoFas evaGreen supermixes</td><td>Bio-Rad, USA</td><td>1725202</td><td>For RT-qPCR</td></tr><tr><td>T4 RNA Ligase 1</td><td>New England Biolabs, UK</td><td>M0437</td><td>For RNA circularization</td></tr><tr><td>Tetrasodium pyrophosphate</td><td>Sigma-aldrich, USA</td><td>221368</td><td>For preparation of extraction buffer</td></tr><tr><td>TIANgel midi purification kit</td><td>Tiangen Biotech, China</td><td>DP209</td><td>To purify DNA fragments from agarose gel</td></tr><tr><td>Tris</td><td>Aladdin, China</td><td>T110601</td><td>To prepare 1x TAE buffer</td></tr><tr><td>TRIzol reagent</td><td>Invitrogen, USA</td><td>15596026</td><td>To extract mitochondiral RNA.</td></tr><tr><td>Universal DNA purification kit</td><td>Tiangen Biotech, China</td><td>DP214</td><td>To recover linearized plastmids from the restriction enzyme digestion reaction</td></tr><tr><td>Xylene cyanol FF</td><td>Sigma-aldrich, USA</td><td>X4126</td><td>For preparation of loading buffer for agarose gel electrophoresis</td></tr></tbody></table>

discrimintion and mapping of the primary and processed transcripts in maize mitochondrion using a circular rt-pcr-based strategy

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

Estimation of mitochondrial RNA circularization efficiency
In a previous study, both total and mitochondrial RNAs were used for cRT-PCR mapping of mitochondrial transcript termini in Arabidopsis (Arabidopsis thaliana), and the two types of RNAs gave similar mapping results12. Initially, we also used total RNAs for cRT-PCR mapping of mitochondrial transcript termini in ma...

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

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Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy (Video) | JoVE Journal

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

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