Abstract
modulo belongs to the modifier of Position Effect Variegation class of Drosophila genes, suggesting a role for its product in regulating chromatin structure. Genetics assigned a second function to the gene, in protein synthesis capacity. Bifunctionality is consistent with protein localization in two distinct subnuclear compartments, chromatin and nucleolus, and with its organization in modules potentially involved in DNA and RNA binding. In this study, we examine nucleic acid interactions established by Modulo at nucleolus and chromatin and the mechanism that controls the distribution and balances the function of the protein in the two compartments. Structure/function analysis and oligomer selection/amplification experiments indicate that, in vitro, two basic terminal domains independently contact DNA without sequence specificity, whereas a central RNA Recognition Motif (RRM)-containing domain allows recognition of a novel sequence-/motif-specific RNA class. Phosphorylation moreover is shown to down-regulate DNA binding. Evidence is provided that in vivo nucleolar Modulo is highly phosphorylated and belongs to a ribonucleoprotein particle, whereas chromatin-associated protein is not modified. A functional scheme is finally proposed in which modification by phosphorylation modulates Mod subnuclear distribution and balances its function at the nucleolus and chromatin.
Highlights
§ Present address: Institut de Genetique Humaine, UPR 1142 du CNRS, 141 rue de la Cardonille, 34396 Montpellier cedex 5, France
The results showed that the deletion of the two positively charged Nand C-terminal domains is required to abolish the ability of Mod to bind DNA in this assay, whereas truncated mutant proteins of only one of these domains still exhibit some activity
Previous studies indicated that Mod likely fulfils two distinct nuclear functions, because the dominant suppression of Position Effect Variegation (PEV) [12] and recessive Minute-like [17] phenotypes are suggestive for roles in chromatin compaction and regulation of nucleolus activity, respectively
Summary
§ Present address: Institut de Genetique Humaine, UPR 1142 du CNRS, 141 rue de la Cardonille, 34396 Montpellier cedex 5, France. A shared structural feature is the presence of modules thought to represent functional domains [3] Such modules include basic and acidic stretches, which possibly interact with ribosomal proteins [4], and the so-called RNA Recognition Motif (RRM) [5], which could bind either rRNAs at different stages of maturation or snoRNAs. Ribosome biogenesis is tightly regulated in a close relationship with cell growth or differentiation. Mod Phosphorylation, RNA/DNA Binding and Localization associated to condensed chromatin and heterochromatin sites, is abundantly found at the nucleolus Consistent with this localization, Mod displays a modular organization which is often found in nonribosomal nucleolar proteins, including an acidic stretch, two basic regions located at both ends of the molecule, and four reiterated RRMs in the remaining core portion [18, 19]
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