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Graille, M.

Publications and source records attributed to Graille, M..

3 recordsLinked to original sources

Pho92 direct interaction with Not1 recruits the CCR4-NOT complex to m6A containing mRNAs to modulate meiosis in S. cerevisiae

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNAs. In S. cerevisiae, m6A is exclusively deposited during meiosis, where it plays an essential role in meiotic progression. In this context, the absence of Pho92, the sole known m6A-binding protein in budding yeast, leads to the abnormal persistence of m6A-containing mRNAs, ultimately resulting in delayed meiosis. Here, we investigate how m6A recognition by Pho92 regulates gene expression. Using complementary in vivo and in vitro approaches, we identify direct interactions between Pho92 and Not1, the scaffold subunit of the CCR4-NOT deadenylase complex. Structural analyses reveal that a short evolutionarily conserved motif in Pho92 adopts an -helical conformation that docks onto specific regions of Not1. Functionally, these interactions are required for efficient meiotic progression. Altogether, our findings demonstrate that Pho92 couples m6A recognition to the main deadenylase to shape the meiotic transcriptome. More broadly, our work provides mechanistic insights into how epitranscriptomic signals interface with the core mRNA decay machinery to regulate gene expression during cellular differentiation.

molecular biology↗

Identification of an evolutionary conserved binding motif responsible for the recruitment of NMD factors to the UPF1 helicase

The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTC) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1, and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes.

biochemistry↗

The YTHDF1-3 proteins are bidirectionally influenced by the codon content of their mRNA targets

N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNAs and plays critical roles in a broad variety of biological processes. Recognition of m6A by the YTHDF1-3 proteins results in the alteration of the translation efficiency and stability of methylated mRNAs, although their mode of action is still matter of debates. To decode the molecular basis of YTHDF1-3 action in human cells, we performed an unbiased proteomic screen of their full spectrum of interacting proteins using BioID (proximity-dependent biotin identification). Our systematic BioID mapping revealed that each YTHDF protein is a dynamic hub that associates with both mRNA silencing machineries and the translation apparatus. Based on this, we identified a direct interaction between YTHDF2 and the ribosomal protein RACK1, and found that the silencing activity of YTHDF2 is bidirectionally modulated by the codon content of its targeted mRNAs. Using a tethering reporter system that recapitulates this phenomenon, we confirmed that the three YTHDF proteins selectively repress mRNAs enriched in optimal codons, while they activate those enriched in non-optimal codons. Altogether, these results have important implications for understanding the underlying multiplicity of YTHDF1-3 and could reconcile seemingly contradictory data.

cell biology↗