RESUMEN
mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch-TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch-TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress-induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp-1, suggesting gain-of-function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.
Asunto(s)
Caenorhabditis elegans/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Enfermedades Neurodegenerativas/genética , Tirosina/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Modelos Animales de Enfermedad , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Modelos Moleculares , Degeneración Nerviosa , Enfermedades Neurodegenerativas/metabolismo , Fosforilación , Conformación Proteica , Dominios ProteicosRESUMEN
High-throughput splicing assays have demonstrated that many exonic variants can disrupt splicing; however, splice-disrupting variants distribute non-uniformly across genes. We propose the existence of exons that are particularly susceptible to splice-disrupting variants, which we refer to as hotspot exons. Hotspot exons are also more susceptible to splicing perturbation through drug treatment and knock-down of RNA-binding proteins. We develop a classifier for exonic splice-disrupting variants and use it to infer hotspot exons. We estimate that 1400 exons in the human genome are hotspots. Using panels of splicing reporters, we demonstrate how the ability of an exon to tolerate a mutation is inversely proportional to the strength of its neighboring splice sites.