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

Abstract

SummaryDyskerin, 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.

SUMO: Glue or Solvent for Phase-Separated Ribonucleoprotein Complexes and Molecular Condensates?

“…In support of this idea, SUMOylation of the snoRNP component NOP58 was shown to facilitate its interaction with Box C/D snoRNA, thereby targeting snoRNPs to the nucleolus (Westman et al, 2010). Similarly, miscibility of Dyskerin (DKC1) within the nucleolar DFC was proposed to rely on SUMO-dependent binding of DKC1 to a SIM in GAR1, a component of the H/ACA snoRNP complex (MacNeil et al, 2021). Altogether, these data suggest that SUMO may contribute to phase separation in the nucleolar compartment.…”

Complex interaction network revealed by mutation of human telomerase ‘insertion in fingers’ and essential N-terminal domains and the telomere protein TPP1

“…FLAG co-IP of FLAG-NLS-hTERT truncations coupled to quantitative real-time polymerase chain reaction (qPCR) of hTR was performed as previously described ( 46 ). Briefly, transfected HEK293 cells were washed with ice-cold PBS 1× and then suspended in 150 μl low-salt cytosolic lysis buffer (25 mM Hepes pH 7.9, 0.5 mM MgCl 2 , 5 mM KCl, 0.5% NP-40 + 1× protease inhibitor + 0.04 U/μl RNaseOUT (Life Technologies)).…”

Dyskerin: an essential pseudouridine synthase with multifaceted roles in ribosome biogenesis, splicing, and telomere maintenance

“…SUMOylation sites were identified in dyskerin (Hendriks and Vertegaal 2016;Becker et al 2013;Hendriks et al 2014;Lamoliatte et al 2017;Hendriks et al 2018), of which several are located in its lysine-rich C-terminal NLS. Dyskerin lacking the C-terminal residues 446-514 accumulates mostly in the cytoplasm (MacNeil et al 2021). The fusion of this cytoplasmic localized truncation variant with SUMO3 restores nuclear accumulation, but not nucleolar localization, possibly due to the loss of NoLS in this C-terminal region along with important residues that might regulate the nucleolar localization of dyskerin, such as K467.…”

“…These studies also identified a short non-covalent SUMO-interaction motif in GAR1. Thus, SUMO plays an important role in the H/ACA RNP biogenesis and its localization to the nucleolus (MacNeil et al 2021). In addition to SUMO, the single-strand-selective monofunctional uracil DNA glycosylase 1 (SMUG1) is involved in the localization of dyskerin.…”