The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene in Fern <i>Asplenium nidus</i> Reveals Important Features

doi:10.4236/ajps.2011.24063

Paper Menu >>

Journal Menu >>

American Journal of Plant Sciences, 2011, 2, 535-538

doi:10.4236/ajps.2011.24063 Published Online October 2011 (http://www.SciRP.org/journal/ajps)

535

The Post-Transcriptional mRNA Editing Analysis

of cox3 Mitochondrial Gene in Fern Asplenium

nidus Reveals Important Features

Sebastian Panarese, Guglielmo Rainaldi

Department of Biochemistry and Molecular Biology, University of BARI “Aldo Moro”, Bari, Italy.

Email: [email protected]

Received July 18th, 2011; revised August 20th, 2011; accepted September 5th, 2011.

ABSTRACT

In the mitochondria and chloroplasts of flowering plants (angiosperms), transcripts of protein-coding genes are altered

after synthesis so that their final primary nucleotide sequence differs from that of the corresponding DNA sequence.

This posttranscriptional mRNA editing consists almost exclusively of C-to-U substitutions (direct) and less frequently of

U-to-C substitution (reverse). Editing occurs predominantly within coding regions, mostly at isolated C residues, and

usually at first or second positions of codons, thereby almost always changing the amino acid fro m that specified by the

unedited codon. Editing may also create initiation and termination codons. The effect of C-to-U RNA editing in plants is

to make proteins encoded by plant organelles more similar in sequence to their non plant homologs, then specific C-to-

U editing events are essential for the production of functional plant mitochondrial proteins. Our attention has been

devoted to the study of the mRNA editing in cox3 mitochondrial gene of fern Asplenium nidus. This study reveals the

extreme importance of both C-to -U and U-to-C substitutions for protein expression .

Keywords: mt DNA, Plant, Monilophytes, Aspleniu m ni d us

1. Introduction

Mitochondrial genomes of land plants have been fully

sequenced and characterized in several species belonging

to the Briophytes (Marchantia polymorpha [1] and Phy-

scomitrella patens [2]) and Spermatophytes (Arabidopsis

thaliana [3], Beta vulga ris [4], Oryza sativa [5], Brassica

napus [6], Zea mays [7], Nicotiana tabacum [8] and

Triticum aestivum [9]).

The comparison of organization, structure and expres-

sion between Spermatophyte mitochondrial genomes

reveals several homogeneous features which can be sum-

marized as follows: i) the presence of repeated sequences,

ii) a heterogeneous structure, iii) the presence of DNA

segments of extra mitochondrial origin (mainly chloro-

plastic) carrying in some cases active genes (usually for

tRNAs) [10-12], iv) the editing of transcription products

of structural genes, v) an incomplete set of tRNA genes.

To gain more knowledge on the mitochondrial bio-

genesis of Monilophytes, in particular respect to editing

process, we chose plants of a filicales family, the fern

Asplenium nidus, available at the Botanical Garden of the

University of Bari.

In our previous investigations [13] we verified high

level of editing process in filicales with to respect Sper-

matophytes plants. In our system, the editing analysis of

cox3 mitochondrial gene confirms this hypothesis.

2. Materials and Methods

2.1. Sources of Mitochondrial DNA

Two alternative procedures for the isolation of organelles

have been developed depending on the tissue used as

starting material: roots or leaves. In the former the soil

contained in the thick network of roots was removed by

hand and washed in distilled water. This step was fol-

lowed by drying the roots on filter paper and weighing

and wrapping them with a double layer of sterile gauze.

After washing several times with sterile water, the roots

were suspended in sterile buffer (Mannitol 0,4 M, Mops

25 mM, EGTA 1 mM, PVP 1% pH 7,8) and homogeni-

zed in a blender with five hits every five seconds at me-

dium speed.

Fractionation, lysis of organelles and DNA extraction

were as reported by Hanson [14].

The isolation of organelles and DNA from green

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene

536 in Fern Asplenium nidus Reveals Important Features

leaves (leaf-procedure) was carried out using the same

protocol with few modifications [14].

The mitochondrial DNA isolated by the two alterna-

tive procedures showed significant different levels of

chloroplast contamination, as judged from the PCR am-

plification of highly conserved chloroplast regions [15].

Using as a template the mtDNA prepared from the

roots, where the copy number of plastid DNA are re-

duced [16], no amplified products could be detected. The

results of these experiments are reported in Figure 1.

The leaf procedure was used mainly for the isolation

of total RNA employed for reverse transcription and

cDNA synthesis and for the investigation concerning

editing of co x 3 gene transcripts.

2.2. Total RNA Extraction from A. Nidus

RNeasy Plant Mini Kit (Qiagen) was used for the purifi-

cation of total RNA from fresh or frozen plant tissues.

An accurate photometric detection of starting material

was essential in order to obtain optimal RNA yield and

purity. A maximum amount of 100 mg plant material

was generally processed.

2.3. RT-PCR

RT-PCR experiments were carried out in a single step by

means of SuperScript One-Step RT-PCR with Platinum

Taq. Components for both cDNA synthesis and PCR

1 2 3 4 5 6 7 8

Figure 1. Multiple test of amplification performed on nu-

cleic acids extracted with the two different procedures. The

panels show two groups of three amplifications carried out

with the same pairs of universal primers (BA48557-A49291,

BA49317-A49855, BA49873-A50272, [14] for the detection

of cpDNA in the nucleic acid fractions isolated by the two

procedures. Lanes 1-3: template isolated by the leaf proce-

dure. 5-7: template (arrows) isolated by the root procedure.

4 and 8: 1 Kb DNA ladder (3000, 2000, 1500, 1200, 1031,

900, 800, 700, 600, 500, 400, 300, 200, 100 bp).

were combined in a single tube, using gene-specific

primers and target RNAs from total RNA. Reverse tran-

scription automatically followed PCR cycling without

additional steps. The system consisted of two major

components: RT/Platinum Taq Mix and 2X Reaction

Mix where the RT/Platinum Taq Mix contained a mix-

ture of SuperScript II Reverse Transcriptase and Plati-

num Taq DNA Polymerase for optimal cDNA synthesis

and PCR amplification respectively.

The following cycling conditions were established:

cDNA synthesis pre-denaturation (1X) at 50˚C for 15-30’;

94˚C for 2’; PCR amplification (40X) at 94˚C for 20”;

50˚C - 60˚C for 30”; 68˚C - 72˚C for 1’/kb; final exten-

sion (1X) at 72˚C for 7’.

2.4. DNA Cloning in Ta Vectors

The cloning of dsDNA fragments was carried out using

pGEM-T Easy Vector Systems and following the manu-

facturer instructions.

2.5. DNA Sequencing and Determination of

Editing Rates

Sequencing of individual cDNA of 23 clones was per-

formed automatically with the Big Dye1 Terminator Cy-

cle Sequencing Kit (Applied Biosyste ms).

ClustalW program was used to determine editing sites,

aligning DNA and cDNA sequences.

Sequence of cox3 gene were deposited in GeneBank

database (accession number: FR669448).

3. Results and Discussion

3.1. Analysis of Editing Sites on Cox3

Transcripts

The Figure 2 shows the comparison of mitochondrial

sequence of cox3 gene with the corresponding cDNA.

Table 1 shows editing data about some cox3 mitochon-

drial genes of diffe rent plant species.

The analysis of results obtained in this investigation

shows some specific features for the A. nidus mitochon-

Table 1. Comparison of editing value in cox3 mitochondrial

transcripts betw een some land plants.

Editing C-UEditing U-C Site Tot % Gene bp

P. patens 1 1 0.1798

B. vulgaris 4 4 0.5798

B. napus 7 7 0.9798

A. thaliana 8 8 1.0798

T. aestivum 12 1 13 1.6798

O. sativa 1 1 0.1843

A. nidus 19 13 32 4.0798

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene 537

in Fern Asplenium nidus Reveals Important Features

Figure 2. Alignment of genome sequence (upper) with

cDNA counterpart of cox3 mitochondrial gene of A. nidus.

Nucleotides underlined correspond to sequencing primers.

drial cox 3 gene:

1) for the transcript of the gene, direct editing events

(19 C-U; 2,3%) and reverse editing events (13 U-C; 1,7%)

are almost equivalent;

2) the editing events have a high relevance: they sup-

press four stop codons within the cox3 gene;

3) 25 amino acids changes and 4 suppressions of stop

codons (3 TAA-1 TAG) are found. These stop codons

are corrected by U to C editing to sense codons of the

amino acid Q (Figure 3);

4) in particularly critical positions, editing events of

both types generate transcripts which can be translated as

functional proteins;

5) the initial codon of cox3 is an ACG codon wh ich is

Figure 3. Amino acids changes after editing events in A.

nidus cox3 mitochondrial transcript.

not changed to AUG by a direct editing event.

This observation can be considered in agreement with

the finding of Dong F. G. [17] who found that the radish

(Raphanus sativus L.) mitochondrial cox2 gene contains

an ACG at the predicted translation initiation site which

is not converted to AUG codon in the mRNA, although

15 other RNA editing sites were identified. So our find-

ing confirms that in plant mitochondria ACG codon can

be utilized as initiation codon;

6) cox3 editing events have a higher frequency on A.

nidus transcripts compared with the same genes studied

on other angiosperm genomes (Table 1);

7) reverse editing events (U-C) are considerably more

frequent in cox3 transcripts of A. nidus than in other

plant species, where they are nearly absent (Table 1);

8) as shown in Figure 4, for 17 codons changes occur

in first position, for 13 codons in second positio n and for

only one codon in third position.

Figure 4. Distribution of editing events across codon posi-

tions.

The Post-Transcriptional mRNA Editing Analysis of cox3 Mitochondrial Gene

in Fern Asplenium nidus Reveals Important Features

538

REFERENCES

[1] K. Oda, K. Yamato, E. Ohta, H. Fukuzawa, T. Kohchi, S.

Nakayama, K. Ishizaki, M. Fujisawa and K. Yamato,

“Gene Organization Deduced from the Complete Se-

quence of Liverwort Marchantia polymorpha Mitochon-

drial DNA. A Primitive Form of Plant Mitochondrial

Genome,” Journal of Molecular Biology, Vol. 223, No. 1,

1992, pp. 1-7. doi:10.1016/0022-2836(92)90708-R

[2] K. Terasawa, M. Odahara, Y. Kabeya, T. Kikugawa, T.

Sekine, M. Fujiwara and N. Sato, “The Mitochondrial

Genome of the Moss Physcomitrella patens Sheds New

Light on Mitochondrial Evolution in Land Plants,” Mo-

lecular Biology and Evolution, Vol. 24, No. 3, 2006, pp.

699-709. doi:10.1093/molbev/msl198

[3] M. Unseld, J. R. Marien feld, P. Brandt and A. Brennicke,

“The Mitochondrial Genome of Arabidopsis thaliana

Contains 57 Genes in 366,924 Nucleotides,” Nature Ge-

netics, Vol. 15, 1997, pp. 57-61. doi:10.1038/ng0197-57

[4] T. Kubo, S. Nishizawa, A. Sugawara, N. Itchoda, A. Es-

tiati an d T. Mikami, “The Complete Nucl eotide Seque nce

of the Mitochondrial Genome of Sugar Beet (Beta vul-

garis L.) Reveals a Novel Gene for tRNA(Cys) (GCA),”

Nucleic Acids Research, Vol. 28, No. 13, 2000, pp.

2571-2576. doi:10.1093/nar/28.13.2571

[5] Y. Notsu, S. Masood, T. Nishikawa, N. Kubo, G. Akiduki,

M. Nakazono, A. Hirai and K. Kadowaki, “The Complete

Sequence of the Rice (Oryza sativa L.) Mitochondrial Ge-

nome: Frequent DNA Sequence Acquisition and Loss

dur- ing the Evolution of Flowering Plants,” Molecular

Genetics and Genomics, Vol. 268, No. 4, 2002, pp.

434-445. doi:10.1007/s00438-002-0767-1

[6] H. Handa, “The Complete Nucleotide Seque nce and RNA

Editing Content of the Mitochondrial Genome of Rape-

seed (Brassica napus L.): Comparative Analysis of the

Mitochondrial Genomes of Rapeseed and Arabidopsis

thaliana,” Nucleic Acids Rese arch, Vol. 31, No. 20, 2003,

pp. 5907-5916. doi:10.1093/nar/gkg795

[7] S. W. Clifton, P. Minx, C. M. Fauron, M. Gibson, J. O.

Allen, H. Sun, M. Thompson, W. B. Barbazuk, S.

Kanuganti, C. Tayloe, L. Meyer, R. K. Wilson and K. J.

Newton, “Sequence and Comparative Analysis of the

Maize NB Mitochondrial Genome,” Plant Physiology,

Vol. 136, 2004, pp. 3486-3503.

doi:10.1104/pp.104.044602

[8] Y. Sugiyama, Y. Watase, M. Nagase, N. Makita, S.

Yagura, A. Hirai and M. Sugiura, “The Complete Nucle o-

tide Sequence and Multipartite Organization of the To-

bacco Mitochondrial Genome: Comparative Analysis of

Mitochondrial Genomes in Higher Plants, Molecular Ge-

netics and Genomics, Vol. 272, No. 6, 2005, pp. 603-615.

doi:10.1007/s00438-004-1075-8

[9] Y. Ogihara, Y. Yamazaki, K. Murai, A. Kanno, T.

Terachi, T. Shiina, N. Miyashita, S. Nasuda, C.

Nakamura, N. Mori, S. Takumi, M. Murata, S. Futo and

K. Tsunewaki, “Structural Dynamics of Cereal Mitochon-

drial Genomes as Revealed by Complete Nucleotide Se-

quencing of the Wheat Mitochondrial Genome,” Nucleic

Acids Research, Vol. 33, No. 19, 2005, pp. 6235-6250.

doi:10.1093/nar/gki925

[10] A. Kanno, M. Nakazono, A. Hirai and T. Kameya, “A

Chloroplast Derived trnH Gene Is Expressed in the Mito-

chondrial Genome of Gramineous Plants,” Plant Molecu-

lar Biology, Vol. 34, No. 2, 1997, pp. 353-356.

doi:10.1023/A:1005828728036

[11] J. Fey and L. Marechal-Drouard, “Expression of the Two

Chloroplast-Like tRNA (Asn) Genes in Potato Mitochon-

dria: Mapping of Transcription Initiation Sites Present in

the trnN1-tr nYnad2 Cluster and Upstream of trnN2,”

Current Genetics, Vol. 36, No. 1-2, 1999, pp. 49-54.

doi:10.1007/s002940050471

[12] F. Damiano, L. R. Ceci, L. Siculella and R. Gallerani,

“Transcription of Two Sunflower (Helianthus annuus L.)

Mitochondrial tRNA Genes Having Different Genetic

Origins,” Gene, Vol. 286, No. 1, 2002, pp. 25-32.

doi:10.1016/S0378-1119(01)00801-0

[13] S. Panarese, G. Rainaldi, C. De Benedetto and R. Galler-

ani, “Sequencing of a Segment of a Monilophyte Species

Mitochondrial Genome Reveals Features Highly Similar

to those of Seed Plant mtDNAs,” The Open Plant Science

Journal, Vol. 2, 2008, pp. 15-20.

doi:10.2174/1874294700802010015

[14] M. R. Hanson, M. L. Boeshore, P. McClean, M. O’Con-

nell and H. Nivison, “The Isolation of Mitochondria and

Mitochondrial DNA, Methods in Enzymology, Vol. 118,

1986, pp. 437-447. doi:10.1016/0076-6879(86)18091-8

[15] P. Taberlet, L. Gielly, G. Pautou and J. Bouvet, “Univer-

sal Primers for Amplification of the Non-Coding Regions

of Chloroplast DNA,” Plant Molecular Biology, Vol. 17,

No. 5, 1991, pp. 1105-1109. doi:10.1007/BF00037152

[16] K. Isono, Y. Niwa, K. Satoh and H. Kobayashi, “Evi-

dence for Transcriptional Regulation of Plastid Photo-

synthesis Genes in Arabidopsis thaliana Roots,” Plant

Physiology, Vol. 114, No. 2, 1997, pp. 623-630.

doi:10.1104/pp.114.2.623

[17] F. G. Dong, K. G. Wilson and C. A. Makaroff, “The

Radish (Raphanus sativus L.) Mitochondrial cox2 Gene

Contains an ACG at the Predicted Translation Initiation

Site,” Current Genetics, Vol. 34, No. 2, 1998, pp. 79-87.

doi:10.1007/s002940050369