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

Podsumowanie

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

Streszczenie

In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' 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' triphosphate. However, the triphosphate at primary 5' 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' 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' triphosphate.

In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' 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.

Wprowadzenie

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' ends^1,2,3,4. The primary transcripts have 5' triphosphate ends, which are derived from transcription initiation. By contrast, the processed transcripts have 5' 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' 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'-phosphate-dependant exonuclease (TEX), and the primary transcripts left are then mapped by primer extension analysis^5,6. The third strategy is to pre-cap the primary transcripts using guanylyl transferase, and then the position of triphosphated 5' termini is determined by primer extension together with ribonuclease or S1 nuclease protection analysis^7,8,9. Different from those depending on the presence/absence of 5' 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 sites^8,10,11. By using these strategies, many primary and processed transcripts have been determined in plant mitochondria.

However, several studies have reported that the 5' triphosphate of primary transcripts were unstable, and they were easily converted to monophosphate for unknown reason^2,4,12,13. This problem hinders a clear discrimination of the two types of transcripts by using techniques depending on the presence/absence of 5' triphosphate, and previous efforts to systematically discriminate between the primary and processed transcripts in plant mitochondria failed^2,12.

In this protocol, we combine cRT-PCR, RNA 5' 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' and 3' extremities of a RNA molecule, and it is usually adapted to map transcript termini in plants^2,12,14,15. RNA 5' polyphosphatase could remove two phosphates from the triphosphated 5' 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' terminus, and it was insensitive to RNA 5' polyphosphatase^1,16. To minimize the influence of unstable triphosphate at primary 5' termini, the 5' 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.

Protokół

1. Primer Design

1. 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’ part of coding sequences (mature mRNAs and precursor RNAs), or ~500–600 nt downstream of the anticipated 5’ end (18S and 26S rRNAs).
2. Design pairs of divergent primers to amplify the circularized transcripts by cRT-PCR.
   NOTE: The paired divergent primers flank the 5’-3’ 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’ UTR and rps4-1 3’ 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’ UTRs of nad2-1 and nad4-1 are 34/35 and 29–31 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.
3. 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.

2. Preparation of Crude Mitochondrion from Maize Developing Kernels

1. Sterilize pestles, mortars, glass funnels, tubes, and tips by autoclave, and dry them in oven.
2. Perform all procedures at 4 °C or on ice, and pre-cool all solutions.
3. 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.
4. 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.
5. Collect 20 g developing kernels at 11–20 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.
6. Add 10–20 mL of ice-cold EB to each mortar, and grind the kernels completely.
7. Add more EB, and filter the ground tissues through two layers of filter cloth (Table of Materials).
8. Centrifuge the filtrate at 8,000 x g for 10 min, and discard the pellet.
9. Transfer the supernatant to a new tube, and centrifuge it at 20,000 x g for 10 min.
10. Pour off the supernatant, and resuspend the pellet in 6 mL WB.
11. Aliquot the suspension to five 1.5 mL RNase-free tubes, and centrifuge them at 14,000 x g for 5 min.
12. Discard the supernatant, freeze the mitochondrial pellet in liquid nitrogen, and store at -80 °C.

3. Extraction of Mitochondrial RNA

1. Extract mitochondrial RNA with a commercial reagent (Table of Materials) according to the manufacture’s instructions.
   CAUTION: This reagent contains phenol and guanidine isothiocyanate. Work with it in a fume hood, and wear lab coat and gloves.
2. 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 μg mitochondrial RNA is obtained from 20 g of 15-DAP maize kernels.
3. 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).
4. 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.
5. Run the gel in 1x TAE buffer at 5–6 V/cm for 20–25 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).

4. RNA 5’ Polyphosphatase Treatment

1. Set up RNA 5’ polyphosphatase (Table and Materials) treatment (Table 1), and incubate at 37 °C for 30–60 min.
2. Recover the 5’ polyphosphatase-treated RNA with an RNA purification kit (Table of Materials) according to the manufacturer’s instructions.
3. Repeat steps 3.3 to 3.5.

5. RNA Circularization

1. Prepare two circularization reactions using the same amounts of 5’ polyphosphatase-treated and non-treated mitochondrial RNAs (Table 2), and incubate both reactions at 16 °C for 12–16 h.
2. 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.
3. Repeat steps 3.3 to 3.5.

6. Reverse Transcription

1. Synthesize two sets of cDNAs from the same amounts of circularized 5’ polyphosphatase-treated and non-treated RNAs (200 ng).
2. 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 μM.
3. Prepare two pre-mixtures by combining the reagents listed in Table 3, incubate at 65 °C for 5 min, and then chill on ice for 2 min.
4. Assemble two RT reaction systems (Table 4), and incubate them at 42 °C for 50 min.
5. Heat both RT reactions at 70 °C for 5 min, and then chill them on ice.

7. Normalization

1. Prepare two PCR reactions by adding the same volume of template cDNAs derived from 5’ polyphosphatase-treated or non-treated RNAs, divergent primers flanking the 5’-3’ junction of circularized 26S rRNA (i.e. 26S-CF1 and -CR1), etc. (Table 5).
2. Run the two reactions in a thermal cycler (Table of Materials) under conditions described in Table 6.
3. Prepare one agarose gel composed of 1x TAE buffer, 1.0% agarose, and 1x nuclear staining dye.
4. Add 2 μL 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.
5. Run the gel in 1x TAE buffer at 5–6 V/cm for 30–40 min, and image it using the same gel documentation system as step 3.5.
6. 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.

8. PCR Amplification

1. Prepare pairs of PCR reactions by adding appropriate volume of the normalized cDNAs and a pair of divergent primers flanking 5’-3’ 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.
2. Perform the PCR reactions according to the program described in Table 8.
3. Repeat steps 7.3 to 7.5.
4. Change to nested divergent primers, and verify the first round PCR results by repeating steps 8.1 and 8.2.
5. Repeat steps 7.3 to 7.5.
6. Recover the prominent bands that could be repeated in two rounds of PCR amplification using a gel DNA recovery kit (Table of Materials).

9. Determination of Transcript Termini

1. Clone the gel-purified PCR products into a blunt-end vector (Table of Materials) using standard techniques.
2. 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’ and/or 3’ ends^1,12.
3. Align the sequencing data with maize mitochondrial genome using basic local alignment search tool (BLAST) of national center for biotechnology information (NCBI). Choose organism “maize (taxid:4577)”, and search database “Nucleotide collection (nr/nt)”.
4. Find the 5’-3’ junction of the circularized transcript, and determine the positions of 5’ and 3’ transcript termini.
5. Calculate the size of target transcripts.

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.

1. 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.
2. 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.
3. Label RNA probes with DIG-11-UTP by a commercial kit (Table of Materials) according to the manufacturer’s instructions.
4. 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.
5. Add 2–3 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 3.1­–3.3.
6. Denature the RNA sample/Loading buffer mixture at 65 °C for 10 min, and then chill on ice for 1 min.
7. Prepare a denatured agarose gel (2% formaldehyde, 1.2% agarose, and 1x MOPS), and load the RNA sample/Loading buffer mixture to the gel (1–2 μg mitochondrial RNA per well).
8. Run the gel in 1x MOPS at 3–4 V/cm for ~4 h.
9. 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.
10. 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 10–16 h.
11. Fix the RNA to membrane by baking at 120 °C for 30 min.
12. Perform RNA hybridization and immunological detection with a commercial kit (Table of Materials) according to the manufacturer’s instruction.

11. Discrimination of Primary and Processed 5’ Ends

1. Discriminate the primary and processed 5’ ends by comparing the cRT-PCR products obtained from the normalized 5’ polyphosphatase-treated and non-treated RNAs.
   NOTE: To primary transcripts, the abundance of PCR products from 5’ polyphosphatase-treated RNA is much higher than that from non-treated counterpart (Figure 1, ‘Gene A,’ 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, ‘Gene B’).
2. Design RT-qPCR primers based on the cRT-PCR mapping results.
3. Verify the cRT-PCR discrimination results by RT-qPCR.
   NOTE: The two cDNA samples derived from 5’ polyphosphatase-treated and non-treated RNAs are normalized by the RT-qPCR products of 26S mature rRNA.

Wyniki

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...

Dyskusje

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 studies^1,2,3,9. We found that the enrichment of mitochondrial RNA i...

Materiały

Name	Company	Catalog Number	Comments
Acetic acid	Aladdin, China	A112880	To prepare 1x TAE buffer
Applied Biosystems 2720 Thermal Cycler	Thermo Fisher Scientific, USA	4359659	Thermal cycler for PCR amplification
Ascorbic acid	Sigma-aldrich, USA	V900134	For preparation of extraction buffer
Biowest Agarose	Biowest, Spain	9012-36-6	To resolve PCR products and RNAs
Bovine serum albumin	Sigma-aldrich, USA	A1933	For preparation of extraction buffer
Bromophenol blue	Sigma-aldrich, USA	B8026	For preparation of loading buffer for agarose gel electrophoresis and Northern blot
DEPC	Sigma-aldrich, USA	V900882	Deactivation of RNase
DIG Northern starter kit	Roche, USA	12039672910	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.
EDTA	Sigma-aldrich, USA	V900106	For preparation of extraction buffer and 1x TAE buffer
EGTA	Sigma-aldrich, USA	E3889	For preparation of wash buffer
Gel documentation system	Bio-Rad, USA	Gel Doc XR+	To image the agarose gel
Glycerol	Sigma-aldrich, USA	G5516	For preparation of loading buffer for agarose gel electrophoresis
GoldView II (5,000x)	Solarbio,. China	G8142	DNA staining
Hybond-N+, Nylon membrane	Amersham Biosciences, USA	RPN119	For Northern blot
Image Lab	Bio-Rad, USA	Image Lab 3.0	Image gel, and compare the abundance of PCR products.
KH2PO4	Sigma-aldrich, USA	V900041	For preparation of extraction buffer
KOH	Aladdin, China	P112284	For preparation of extraction buffer
L-cysteine	Sigma-aldrich, USA	V900399	For preparation of extraction buffer
Millex	Millipore, USA	SLHP033RB	To sterile extraction and wash buffers by filtration
Miracloth	Calbiochem, USA	475855-1R	To filter the ground kernel tissues
MOPS	Sigma-aldrich, USA	V900306	For preparation of running buffer for Northern blot
NanoDrop	Thermo Fisher Scientific, USA	2000C	For RNA concentration and purity assay
NaOH	Sigma-aldrich, USA	V900797	For preparation of wash buffer
pEASY-Blunt simple cloning vector	TransGen Biotech, China	CB111	Cloning of the gel-recovered band. It contains a T7 promoter several bps upstream of the insertion site.
Phanta max super-fidelity DNA polymerase	Vazyme, China	P505	DNA polymerase for PCR amplification
Polyvinylpyrrolidone 40	Sigma-aldrich, USA	V900008	For preparation of extraction buffer
Primer Premier 6.24	PREMIER Biosoft, USA	Primer Premier 6.24	To design primers for reverse transcription and PCR amplification
PrimeScript II reverse transcriptase	Takara, Japan	2690	To synthesize the first strand cDNA
PureLink RNA Mini kit	Thermo Fisher Scientific, USA	12183025	For RNA purificaion
RNA 5' polyphosphatase	Epicentre, USA	RP8092H	To convert 5' triphosphate to monophosphate
RNase inhibitor	New England Biolabs, UK	M0314	A component of RNA self-ligation and 5' polyphosphatase treatment reactions, and it is used to inhibite the activity of RNase.
Sodium acetate	Sigma-aldrich, USA	V900212	For preparation of running buffer for Northern blot
Sodium chloride	Sigma-aldrich, USA	V900058	To prepare 20x SSC
SsoFas evaGreen supermixes	Bio-Rad, USA	1725202	For RT-qPCR
T4 RNA Ligase 1	New England Biolabs, UK	M0437	For RNA circularization
Tetrasodium pyrophosphate	Sigma-aldrich, USA	221368	For preparation of extraction buffer
TIANgel midi purification kit	Tiangen Biotech, China	DP209	To purify DNA fragments from agarose gel
Tris	Aladdin, China	T110601	To prepare 1x TAE buffer
TRIzol reagent	Invitrogen, USA	15596026	To extract mitochondiral RNA.
Universal DNA purification kit	Tiangen Biotech, China	DP214	To recover linearized plastmids from the restriction enzyme digestion reaction
Xylene cyanol FF	Sigma-aldrich, USA	X4126	For preparation of loading buffer for agarose gel electrophoresis