Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May;20(5):1278-88.
doi: 10.1105/tpc.108.058883. Epub 2008 May 27.

MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor

Silin Zhong  1 , Hongying Li, Zsuzsanna Bodi, James Button, Laurent Vespa, Michel Herzog, Rupert G Fray
Affiliations

MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor

Silin Zhong et al. Plant Cell. 2008 May.

Abstract

N6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Two-dimensional TLC Detection of m6A in Arabidopsis Poly(A) RNA. (A) Two-dimensional TLC analysis of in vitro transcribed RNA containing m6A and normal adenosine. (B) Schematic diagram of the relative positions of nucleotide spots. (C) Two-dimensional TLC analysis of total RNA extracted from 2-week-old Arabidopsis seedlings. (D) Two-dimensional TLC analysis of poly(A) RNA from 2-week-old Arabidopsis seedlings. The m6A:A ratio is 1.5%.
Figure 2.
Figure 2.
Seeds of the SALK_074069 Insertion in MTA Arrest at the Globular Stage. (A) Arrangement of At4g10760 showing the location of the SALK_074069 T-DNA insertion in exon 4. Exons are shown in black, and introns are shown in white. (B) Siliques from a plant hemizygous for SALK_074069 (top) and from a wild-type control plant (bottom). Seeds homozygous for the SALK_074069 insertion appear white and fail to develop normally. (C) White seeds become shrivelled and nonviable at maturity (arrows). (D) Arrested embryo of the hemizygous SALK_074069. (E) A phenotypically normal embryo from the same silique as in (D) that has reached the heart stage. (F) Although the arrested embryo has not developed past the globular stage, some cell division has continued. (G) A phenotypically normal embryo from the same silique as in (F). Bars = 100 μm.
Figure 3.
Figure 3.
Complementation of SALK_074069 with 35S:MTA. (A) DNA gel blot analysis of wild-type, hemizygous SALK_074069, and homozygous SALK_074069 lines complemented with the MTA cDNA construct. (B) Silique phenotype of plants hemizygous for SALK_074069 (left) and homozygous for both SALK_074069 and the complementing cDNA transgene (right). (C) TLC analysis of the poly(A) RNA purified from the complemented line. (D) qRT-PCR analysis shows a sevenfold higher expression of the MTA transgene in the complemented line. Error bars show sd from three replicates.
Figure 4.
Figure 4.
Strong MTA Promoter Activity Is Limited to Discrete Developmental Tissues. GUS expression in various organs of transformed plants is shown. (A) Expression in anthers is initially seen only in tapetal cells (arrow). Bar = 1 cm. (B) Close-up of the staining in (A). Bar = 50 μm. (C) Pollen microspore in a mature anther. Bar = 10 μm. (D) Developing seeds (arrow). Bar = 1 mm. (E) Weak expression is seen in the ovule prior to fertilization (arrow). Bar = 25 μm. (F) In young seedlings, GUS expression is concentrated in the apical meristem (arrow). Bar = 5 mm. (G) In roots, GUS expression is observed primarily during lateral root initiation. Bar = 100 μm.
Figure 5.
Figure 5.
m6A Levels in Root, Leaf, and Floral Tissues. (A) Root. (B) Fully expanded leaf. (C) Flower buds. (D) RNA gel blot assay of MTA expression.
Figure 6.
Figure 6.
Disruption of MTA Results in the Loss of m6A from the mRNA of Embryo-Defective Seeds. (A) m6A is readily detectable in the mRNA from wild-type seeds. (B) m6A is not detectable in the mRNA from white embryo-defective seeds of SALK_074069, even after prolonged exposure. (C) m6A is readily detectable in the mRNA from white seeds of the control embryo development mutant emb15. (D) RT-PCR showing the absence of MTA transcript in the poly(A) RNA from SALK_074069 white seeds.
Figure 7.
Figure 7.
MTA Interacts with FIP37. (A) MTA interacts with FIP37 (b) but does not interact with the empty prey vector, pDEST22 (a). FIP37 does not transactivate GUS expression in the presence of the empty bait vector, pDEST32 (c). ProQuest (Invitrogen) controls for assessing the relative interaction strengths are indicated 1 to 5. (B) Proteins of wild-type and transgenic Arabidopsis plants expressing MTA-c-Myc were extracted under nondenaturing conditions and immunoprecipitated using anti-c-Myc antibody. After SDS-PAGE separation, the anti-FIP37 antibody detected a protein of 48 kD (bottom arrow) in the precipitated extracts of plants expressing the MTA-c-Myc fusion but not in those of the wild-type control. (C) Transient expression of fluorescent protein fusions in onion epidermal peels. FIP37-CFP (left) and MTA-YFP (middle) colocalized to the nucleus. YFP and CFP images were superimposed on the bright-field image to show the position of the nucleus (right). Bars = 10 μm.
See this image and copyright information in PMC

References

    1. Adams, J.M., and Cory, S. (1975). Modified nucleosides and bizarre 5′-termini in mouse myeloma messenger-RNA. Nature 255 28–33. - PubMed
    1. Agris, P.F., Vendeixa, F.A.P., and Grahama, W.D. (2007). tRNA's wobble decoding of the genome: 40 years of modification. J. Mol. Biol. 366 1–13. - PubMed
    1. Aloni, Y., Dhar, R., and Khoury, G. (1979). Methylation of nuclear simian virus-40 RNAs. J. Virol. 32 52–60. - PMC - PubMed
    1. Beemon, K., and Keith, J. (1977). Localization of N6-methyladenosine in Rous-sarcoma virus genome. J. Mol. Biol. 113 165–179. - PubMed
    1. Bjork, G.R., Ericson, J.U., Gustafsson, C.E., Hagervall, T.G., Jonsson, Y.H., and Wikstrom, P.M. (1987). Transfer RNA modification. Annu. Rev. Biochem. 56 263–287. - PubMed

Publication types

MeSH terms