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

Summary

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.

Abstract

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.

Introduction

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 tra....

Protocol

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. Desi.......

Discussion

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.......

Materials

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 (5000x)	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