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. 2010 Sep;38(16):5327-35.
doi: 10.1093/nar/gkq266. Epub 2010 Apr 26.

Yeast targets for mRNA methylation

Zsuzsanna Bodi  1 , James D Button, Donald Grierson, Rupert G Fray
Affiliations

Yeast targets for mRNA methylation

Zsuzsanna Bodi et al. Nucleic Acids Res. 2010 Sep.

Abstract

N(6)-Methyladenosine (m(6)A) is a modified base present in the mRNA of all higher eukaryotes and in Saccharomyces cerevisiae, where there is an increase in m(6)A levels during sporulation. The methyltransferase, Ime4, is responsible for this modification and has a role in the initiation of meiosis. However, neither the function, nor the extent of distribution of this nucleotide modification is established. We demonstrate that in S. cerevisiae, substantial levels of internal adenosine methylation are present in the GpA context in mRNA from sporulating cells, which is consistent with the preferred methylation consensus of higher eukaryotes. Based upon our quantification data, every second transcript could contain one m(6)A during meiosis. As methylation is distributed across all mRNA size ranges, it is likely that m(6)A is not limited to a small population of messages. We developed a new antibody based method for identifying m(6)A containing messages, and using this method the transcripts of three key, early regulators of meiosis, IME1, IME2 and IME4 itself, were identified as being methylated. The position of m(6)A in IME2 was narrowed down to a region in the 3'-end. Methylation of these and other targets suggests mechanisms by which IME4 could control developmental choices leading to meiosis.

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Figures

Figure 1.
Figure 1.
Two-dimensional TLC detection of m6A in mRNA from sporulating SK1 yeast. (a) TLC analysis of in vitro transcribed RNA containing methylated and non-methylated adenosine. (b) Schematic diagram of the relative positions of nucleotide spots. (c) TLC analysis of poly(A) RNA from log phase SK1 strains. (d) TLC analysis of poly(A) RNA from sporulating SK1 strain (3 h after meiosis initiation) m6A:A ratio is 1% (e) TLC analysis of total RNA from log phase SK1 strain. (f) TLC analysis of total RNA from sporulating SK1 strain (3 h after meiosis initiation), m6A:A ratio is 0.07%.
Figure 2.
Figure 2.
Quantification of m6A in size fractionated poly(A) RNA from sporulating SK1 cells (3 h after meiosis initiation). Poly(A) RNA was separated on a 1.3% agarose TBE gel and three size fractions cut and extracted. Purified RNA samples were analyzed on an RNA6000 LabChip. Lane 1: RNA6000 Ladder. Lane 2: starting poly(A) RNA, 205 ng. Red boxes indicate the positions of the excised gel slices. Lane 3: mRNA population, fraction 1, after purifying from agarose. Lane 4: mRNA, fraction 2, after purification from agarose. Lane 5: mRNA, fraction 3, after purification from agarose. (25–50 ng of RNA was loaded in each lane). The amount of m6A in each fraction was calculated using 2D TLC as described in ‘Materials and Methods’ section.
Figure 3.
Figure 3.
Immunoprecipitation of m6A containing messages enriches for IME2 transcripts. Quantitative RT–PCR of the anti-m6A antibody-bound fraction showed a 7-fold enrichment of IME2 messages relative to the antibody unbound fraction. The starting mRNA was a mixture of poly(A) RNA (non-methylated) from a log phase culture and poly(A) RNA from sporulating cells. Error bars represent SD from three replicates.
Figure 4.
Figure 4.
Immunoprecipitation of early meiotic messages. The relative abundance of messages in the anti-m6A antibody bound and unbound fractions were estimated, using quantitative RT–PCR. IME2 and IME1 mRNA species were 2-fold enriched, while IME4 had the highest, 3-fold enrichment in the bound fraction relative to the depleted (Ab unbound) fraction. Error bars represent SD from three replicates.
Figure 5.
Figure 5.
Mapping potential m6A positions in the IME2 message. (A) The GAC consensus sites for adenosine methylation are grouped in two clusters in the IME2 message. The first eight GACs are in the 5′ 507-nt region. There are a further 12 GACs in the 3′-region starting at nt 1281, leaving a 774-nt area in the middle of the message free of GACs. The dark grey boxes indicate the UTRs. (B) When the 5′ GAC containing area is protected (light grey box), only the 3′ 12 GACs are accessible to the T1 nuclease. TLC analysis following T1 digestion revealed the presence of m6A (m6A:A ratio is 0.48%). (C) When the 11 GACs in the 3′-region are protected (light grey box), no m6A was detected by TLC analysis following the T1 nuclease digestion.
Figure 6.
Figure 6.
CNN repeat candidates in the IME2 message. The IME2 message contains two groups of CNN repeats, highlighted as red boxes. Additional C-rich regions adjacent to CNN repeats are accentuated by red Cs. Three GACs lie between the CNN repeats (bold and large font). The two GACs indicated in blue are in the region where methylation was detected.
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