Differential m6A methylomes between two major life stages allows potential regulations in Trypanosoma brucei

https://doi.org/10.1016/j.bbrc.2018.12.043Get rights and content

Highlights

  • m6A methylomes in two life stages.

  • m6A peaks differentially located in two life stages.

  • m6A –containing genes harbor longer half-lives, higher abundance.

  • TRRM2 as a potential m6A binding protein.

Abstract

N6-methyladenosine (m6A) is the most prevalent mRNA modification in higher eukaryotes. Recent studies suggest that m6A has a regulatory role in mRNA degradation and translation initiation or efficiency, involving in cell fate determination in yeast, plants, and stem cells of mammalian. Trypanosoma brucei (T. brucei) regulates gene expression through post-transcriptional fashion, which heavily relies on mRNA cis-motifs. However, internal mRNA modification in T. brucei has not been reported yet. Here we found m6A modification is abundant in T. brucei and presented a transcriptome wide methylome of m6A in both life stages of T. brucei. We identified 355 and 95 peaks in procyclic form and blood stream form trypanosomes respectively. A consensus motif of CAU was shared in both life stages of T. brucei. mRNA abundance of m6A-containing genes is higher in procyclic form and tend to be down-regulated in bloodstream form trypanosomes. Furthermore, m6A-containing transcripts harbor relative longer half-lives, and are enriched in pathways of cell morphology and movement in procyclic form trypanosomes. By m6A-containing RNA pulldown in both life stages, we identified TRRM2 as a potential m6A reader in T. brucei. Uncovering the m6A methylome and its binding proteins may provide a new post-transcriptional regulatory pathway in T. brucei.

Introduction

More than 100 modifications have been found in RNA, among them, m6A is the most abundant in eukaryotic mRNAs. m6A was found in RNA of rat hepatoma cells and Rous sarcoma virus several decades ago. Until the availability of specific m6A antibodies and deep sequencing, it is not able to massively determine m6A sites. The finding that m6A is a substrate of obesity associated protein FTO, which suggest that the methylation is dynamically regulated, deeply encouraged the functional studies.

Using m6A antibody immunoprecipitation followed by deep sequencing, two groups successfully mapped the m6A methylomes, and m6A was found in more than 7000 mRNA and non-coding RNA transcripts, many of which are conserved between human and mice [1,2]. A consensus motif of RRACH (where A is N6 methylated; R is A or G; H is A, C or U) was identified in these m6A containing sequences, and distribution analysis indicates that m6A is enriched around stop codon [1,2] or precisely, the last exon [3]. During heat shock response, m6A is accumulated in 5‘UTR of heat shock induced transcripts [4], which may recruit eukaryotic initiating factor eIF3E and initiate translation in an eIF4E independent manner [5].

The level of m6A is dynamically regulated by groups of “writers” and “erasers”. METTL3, a m6A “writer”, forms heterodimer with METTL14 [6], and WTAP is a key regulator of this complex [7]. Knockdown of METTL3 or METTL14 in mouse embryonic stem cells sharply reduced m6A levels and impaired self-renewal capability of these stems cells [8]. Reduced expression or mutation of METTL3 and METTL14 also resulted in reductions of m6A levels in endometrial cancer cells, which promotes cancer proliferation and tumorigenicity through AKT pathway [9]. FTO and ALKBH5 are m6A “erasers”. FTO associated m6A demethylation involved in a lot of physiology and pathophysiology processes or disease progression, such as adipogenesis, ultraviolet induced DNA damage response, acute myeloid leukemia (AML) cell transformation, and leukemogenesis [10]. ALKBH5, which is highly expressed in mouse testes, targets both mRNA and nuclear RNAs. ALKBH5 knockout mice exhibit spermatogenesis defect [11]. Other m6A binding proteins are m6A “readers”: YTH domain containing proteins. Human YTHDF2 destabilizes m6A associated mRNA [12], while YTHDF1 recruits eIF3 and enhances target mRNA translation [13].

T. brucei is an early diverged unicellular parasite, the cause of African sleeping sickness and nagaga of live stocks, affecting a large region of sub-Saharan African. Its life cycle includes at least two stages, a bloodstream form (BSF) which circulates in human blood, and a procyclic form (PCF) which proliferates in transmission vector tsetse fly [14]. mRNA in T. brucei is polycistronically transcribed, and thus the regulation of gene expression is post-transcription dependent [14]. Each mRNA is then trans-spliced with a 39 nt spliced-leader [14]. The first four nucleotide of spliced-leader are hyper-modified [15]. m6A is widely distributed in higher eukaryotes from human, mice, flies to plants, and it is also found in yeast and bacterium [16,17] and proved to be a key posttranscriptional regulator [18]. However, the existence of m6A in trypanosomes was not reported, despite its ability of modifying mRNA in splice leader and its posttranscriptional regulation of gene expression. Here we report that m6A is widely distributed in mRNA and intergenic region of trypanosome brucei, especially in PCF. m6A is preferentially located in CAU consensus motif in two life stages of T. brucei, which is distinct from mammals, bacterium, or plants. Distribution of m6A in different transcripts in different life stages should be another regulatory mechanism of T. brucei.

Section snippets

Cell culture, RNA extraction and slot blot assay

BSF Lister 427 strain cells were cultured in HMI-9 medium with 10% fetal bovine serum (Gibco) in 5% CO2 at 37 °C. PCF cells were cultured in SDM79 medium with 10% fetal bovine serum at 27 °C, with gentle agitation. 107 BSF or PCF trypanosomes were centrifuged down at 1300 g for 10 min at 4 °C, lysed with 1 ml Trizol (Ambion). Human 293 T cells was obtained from American type culture collection (ATCC), and cultured in 37 °C with 5% CO2. Cells were lysed with Trizol in 6-well plates directly. RNA

m6A is abundant in both life stages of trypanosome brucei

Though m6A is the most prevalent internal modification in many species, from mammals to bacterium, its presence in T. brucei, an early diverged eukaryote, is not known. We first detected the levels of m6A in T. brucei by slot-blot. Different amounts of total RNA harvested from log-phase cultured BSF or PCF trypanosomes was transferred to positively charged nylon membrane. RNA from cultured human 293 T cells was used as positive control. After cross-linking, the membrane was detected with m6A

Conflicts of interests

The authors declared no conflict of interests.

Acknowledge

This work was supported by the National Natural Science Foundation of China (Grant No. 81171601), Natural Science Foundation of Guangdong Province (Grant No. 2014A030313216) to GX and major science and technology projects of Hainan Province to WY.

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