NRAP

Abstract: Background: The nucleolus is the site of rRNA synthesis and processing in eukaryotic cells, but its composition remains poorly understood. Results: We have identified a novel nucleolar RNA-associated protein (Nrap) which is highly conserved from yeast (Saccharomyces cerevisiae) to human, with homologues in mouse, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and other species. In the mouse, we have found that Nrap is ubiquitously expressed and is specifically localized in the nucleolus. We have also identified splice variants in human and mouse, and defined the intron-exon structure of the human Nrap gene. Nrap is inherited into daughter nuclei by associating with the condensed chromosomes during mitosis. RNase treatment of permeabilized cells indicated that the nucleolar localization of Nrap is RNA dependent. The effects of actinomycin D, cycloheximide and 5,6-dichloro-beta-D-ribofuranosylbenzimidazole on Nrap expression and distribution in cultured cells suggest that Nrap is associated with the pre-rRNA transcript. Conclusions: Nrap is a large nucleolar protein (of more than 1000 amino acids), and is a new class of protein with new structural and functional motifs. Nrap appears to be associated with ribosome biogenesis by interacting with pre-rRNA primary transcript.

Abstract: Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific-adapters for CUL3 E3 ubiquitin ligase to regulate protein turn-over through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM, however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive in vivo . Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, NRAP. KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric protein contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease causing factors in vivo , which will be imperative for developing mechanism-based specific therapeutic interventions.