SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

Abstract

Dyskerin, a telomerase-associated protein and H/ACA ribonucleoprotein complex component plays an essential role in human telomerase assembly and activity. The nuclear and subnuclear compartmentalization of dyskerin and the H/ACA complex is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. The posttranslational modification, SUMOylation, targets a wide variety of proteins, including numerous RNA-binding proteins, and most identified targets reported to date localize to the nucleus. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin, each located within one of two lysine-rich clusters. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS and both lysine-rich clusters represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein involved in rRNA and snRNA biogenesis.