Long term objectives include establishing the biological significance of the diverse nuclear ribonucleoprotein (RNP) particles which exist in eukaryotic cells and characterizing the functional/evolutionary relationships among certain structural domains that many of the RNP proteins share. One such domain contains repeating elements of glycine, arginine and phenylalanine. Interestingly, the arginines within this domain are dimethylated (DMA). Although the function of this """"""""Gly/DMA/Phe"""""""" domain remains obscure, it has been found in several highly-conserved, essential nucleolar proteins (fibrillarin, nucleolin, others). The longest such domain (80 amino acids) is that at the amino terminus of fibrillarin. The goal of the immediate project is to examine the function of the fibrillarin Gly/DMA/Phe domain in vivo. It is hypothesized that this domain functions in both nuclear localization of fibrillarin and subsequent pre-rRNA processing in the nucleolus. The testing of this hypothesis has been divided into three specific aims: (1) determination of the involvement of the domain in nuclear localization, (2) determination of whether the domain possesses a nucleolar targeting signal, and (3) identification of specific interactions for which the domain is required within the nucleolus. The approach to accomplishing these aims will be to transiently express in cultured mammalian cells various fibrillarin cDNA constructs altered with regard to the methylated domain and to study the effect of the alterations on both nuclear localization and nucleolar RNP association. If evidence of specific targeting signals is found, the minimum element(s) within the methylated domain responsible for the targeting will be defined. The results should clearly reveal the extent to which the methylated domain of fibrillarin functions in nuclear/nucleolar targeting and should begin to address the function of the domain in pre- RNA processing as well. this work has two-fold significance: It is the first study aimed at determining the in vivo relevance of the Gly/DMA/Phe domain which is found in several key nuclear proteins, but whose function is completely unknown. Secondly, fibrillarin is often the target of the autoimmune response in individuals with scleroderma, but the mechanism by which this and other autoimmune responses occur is not understood. Examination of the in vivo role of this autoantigen in human cells, as proposed here, may give insights as to why certain nuclear RNP proteins are often associated with autoimmunity.
