RESUMEN
The YTH domain of YTHDC1 belongs to a class of protein "readers", recognizing the N6-methyladenosine (m6A) chemical modification in mRNA. Static ensemble-averaged structures revealed details of N6-methyl recognition via a conserved aromatic cage. Here, we performed molecular dynamics (MD) simulations along with nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) to examine how dynamics and solvent interactions contribute to the m6A recognition and negative selectivity toward an unmethylated substrate. The structured water molecules surrounding the bound RNA and the methylated substrate's ability to exclude bulk water molecules contribute to the YTH domain's preference for m6A. Intrusions of bulk water deep into the binding pocket disrupt binding of unmethylated adenosine. The YTHDC1's preference for the 5'-Gm6A-3' motif is partially facilitated by a network of water-mediated interactions between the 2-amino group of the guanosine and residues in the m6A binding pocket. The 5'-Im6A-3' (where I is inosine) motif can be recognized too, but disruption of the water network lowers affinity. The D479A mutant also disrupts the water network and destabilizes m6A binding. Our interdisciplinary study of the YTHDC1 protein-RNA complex reveals an unusual physical mechanism by which solvent interactions contribute toward m6A recognition.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas de Unión al ARN , Adenosina/análogos & derivados , Espectroscopía de Resonancia Magnética , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
N(6)A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N(6)-methylated adenosine reader domain and report its solution structure in complex with a N(6)-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m(6)A recognition. These findings establish a molecular function for YTH domains as m(6)A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m(6)A recognition.
Asunto(s)
Adenosina/análogos & derivados , Proteínas del Tejido Nervioso/química , Proteínas de Unión al ARN/química , ARN/química , Adenosina/química , Animales , Sitios de Unión , Guanina/química , Metilación , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ratas , Factores de Empalme Serina-ArgininaRESUMEN
Partitioning of chromatids during mitosis requires that chromosome compaction and spindle length scale appropriately with each other. However, it is not clear whether chromosome condensation and spindle elongation are linked. Here, we find that yeast cells could cope with a 45% increase in the length of their longest chromosome arm by increasing its condensation. The spindle midzone, aurora/Ipl1 activity, and Ser10 of histone H3 mediated this response. Thus, the anaphase spindle may function as a ruler to adapt the condensation of chromatids, promoting their segregation regardless of chromosome or spindle length.
Asunto(s)
Anafase , Cromosomas Fúngicos/fisiología , Saccharomyces cerevisiae/fisiología , Huso Acromático/fisiología , Huso Acromático/ultraestructura , Isomerasas Aldosa-Cetosa/genética , Aurora Quinasas , Segregación Cromosómica , Cromosomas Fúngicos/genética , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The YTH (YT521-B homology) domain was identified by sequence comparison and is found in 174 different proteins expressed in eukaryotes. It is characterized by 14 invariant residues within an alpha-helix/beta-sheet structure. Here we show that the YTH domain is a novel RNA binding domain that binds to a short, degenerated, single-stranded RNA sequence motif. The presence of the binding motif in alternative exons is necessary for YT521-B to directly influence splice site selection in vivo. Array analyses demonstrate that YT521-B predominantly regulates vertebrate-specific exons. An NMR titration experiment identified the binding surface for single-stranded RNA on the YTH domain. Structural analyses indicate that the YTH domain is related to the pseudouridine synthase and archaeosine transglycosylase (PUA) domain. Our data show that the YTH domain conveys RNA binding ability to a new class of proteins that are found in all eukaryotic organisms.