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Hakai is required for stabilization of core components of the m⁶A mRNA methylation machinery.

ABSTRACT: N⁶-methyladenosine (m⁶A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m⁶A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m⁶A levels and altered m⁶A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m⁶A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m⁶A deposition. Our work adds functional and molecular insights into the mechanism of the m⁶A mRNA writer complex.

SUBMITTER: Bawankar P

PROVIDER: S-EPMC8213727 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Hakai is required for stabilization of core components of the m6A mRNA methylation machinery.

Bawankar Praveen P Lence Tina T Paolantoni Chiara C Haussmann Irmgard U IU Kazlauskiene Migle M Jacob Dominik D Heidelberger Jan B JB Richter Florian M FM Nallasivan Mohanakarthik P MP Morin Violeta V Kreim Nastasja N Beli Petra P Helm Mark M Jinek Martin M Soller Matthias M Roignant Jean-Yves JY

Nature communications 20210618 1

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and ...[more]

PMID: 34145251

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Project description:KAR4 , the yeast homolog of the mammalian mRNA N 6 A-methyltransferase complex component METTL14 , is required for two disparate developmental programs in Saccharomyces cerevisiae : mating and meiosis. To understand KAR4 's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat - ) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4 Δ/Δ and 7 meiosis-specific alleles (Mei - ) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4 Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo - ). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4 , a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4 Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.Author summaryIn yeast, KAR4 is required for mating and meiosis. A genetic screen for function-specific mutations identified 25 alleles that map to different surfaces on a predicted structure of the Kar4 protein (Kar4p). The mating-specific alleles interfere with Kar4p's ability to interact with the transcription factor Ste12p, its known partner in mating. The meiosis-specific alleles revealed an independent function: Kar4p is required for entry into meiosis and initiation of S-phase. During meiosis, Kar4p interacts with all components of the mRNA methyltransferase complex and kar4 Δ/Δ mutants have greatly reduced levels of mRNA methylation. Thus, Kar4p is a member of the yeast methyltransferase complex. Overexpression of the meiotic transcriptional activator IME1 rescued the meiotic entry defect but did not lead to sporulation, implying that Kar4p has more than one meiotic function. Suppression by Ime1p overexpression led to arrest after premeiotic DNA synthesis, but before sporulation. Loss of Kar4's sporulation function can be suppressed by overexpression of a translation regulator, Rim4p. Overexpression of both IME1 and RIM4 allowed sporulation in kar4 Δ/Δ cells.

Project description:N 6 -Methyladenosine (m 6 A) is one of the most abundant modifications found on eukaryotic mRNAs. mRNA methylation regulates a host of biological processes including meiosis, a specialized diploid cell division program that results in the formation of haploid cells (gametes). During budding yeast meiosis, m 6 A levels peak early, before the initiation of the meiotic divisions. High-throughput studies and work from our lab showed that Ygl036wp, a previously uncharacterized protein interacts with Kar4p, a meiotic protein required for mRNA m 6 A-methylation. Ygl036wp has no discernable domains except for several intrinsically disordered regions. However, protein folding prediction tools showed that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m 6 A-methylation in higher eukaryotes. In addition, Ygl036wp has several conserved motifs shared with VIRMA/Virilizer/VIR proteins. Accordingly, we propose to call the gene VIR1 for budding yeast ortholog of VIR MA/Virilizer/VIR 1 . In support, Vir1p interacts with all other members of the yeast methyltransferase complex and is required for mRNA m 6 A methylation and meiosis. Vir1p is required for the stability of proteins comprising the methyltransferase complex, suggesting that Vir1p acts as a scaffold to stabilize the complex. The vir1 Δ/Δ mutant is defective for premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. Consistent with IME1 suppression, vir1 Δ/Δ exhibits a defect in the abundance of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed a defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by additional overexpression of RIM4 . Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation.Author summaryYgl036wp is a previously uncharacterized protein that we propose to name Vir1p (budding yeast ortholog of VIR MA/Virilizer/VIR 1 ). Work from our lab and others initially found an interaction between Vir1p and members of the yeast mRNA methyltransferase complex (Kar4p and Mum2p). We found that Vir1p interacts with all known members of the methyltransferase complex and is required for mRNA methylation. Vir1p is required early in meiosis; vir1 Δ/Δ mutants arrest due to the reduced expression of Ime1p. Lower levels of Ime1p cause severe disruption to the meiotic transcriptome in vir1 Δ/Δ. The vir1 Δ/Δ meiotic defect can be partially suppressed by the overexpression of IME1 ; full suppression requires overexpression of both IME1 and RIM4 . Using recent advances in protein folding predictions, we found that Vir1p is a remote homolog of VIRMA/Virilizer/VIR and shares conserved motifs with the protein from other organisms. Vir1p, like VIRMA/Virilizer/VIR, stabilizes the methyltransferase complex.

Dataset Information

Hakai is required for stabilization of core components of the m⁶A mRNA methylation machinery.

Publications

Hakai is required for stabilization of core components of the m<sup>6</sup>A mRNA methylation machinery.

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