Nucleolus is the most prominent nuclear domain (ND) and is where rRNAs (ribosomal RNAs) are synthesized, processed and assembled with ribosomal proteins. There is growing evidence ascribing an essential role for the nucleolus in cellular aging. Perturbations in nucleolus structure and rDNA expression have been associated with aging related diseases such as progeria. However, the molecular mechanisms controlling the differential expression of 300-400 rDNA genes clustered in the short arms of five human chromosomes are yet to be determined. Recent studies indicated that nucleolus-localized noncoding RNAs (ncRNAs) contribute to cellular aging by controlling rDNA expression under conditions that influence aging (such as quiescence). We have now generated preliminary data showing the existence of a novel class of nucleolus-localized ncRNA, SNULs (Single NUcleolus-Localized RNAs) that control rDNA expression. SNULs form distinct non-overlapping ?RNA clouds? within the nucleolus, where they coat rDNA repeat or nucleolus organizer regions (NOR) containing short arms (p-arm) of chromosomes. Specifically, SNULs painted only one of the two alleles of each autosome. Further, loss of function studies revealed that SNULs inhibit rDNA expression. The central hypothesis of the present proposal is that SNULs control rDNA expression by regulating rDNA expression. The objectives of this proposal are to identify the determinants underlying allele-specific chromosome coating of SNULs, and to gain insights into the involvement of SNULs in rDNA expression. Guided by strong preliminary data, this hypothesis will be tested in the following specific aims: 1) Determine how cells achieve allele-specific expression/coating of SNULs. 2) Determine how SNUL family of ncRNAs regulates rDNA expression. In the first aim, PI will characterize SNULs and will test whether SNULs display monoallelic chromosome coating utilizing imprinted mechanism or random monoallelic association. Under the second aim, by performing loss-of-function studies, PI will determine the involvement of SNULs in rDNA expression or nucleolar chromatin organization. Our observation of the coating of autosomes by SNULs supports a ?paradigm shifting? model that ncRNA-coating of chromosomes are not restricted only to sex chromosomes. The proposed research will be of strong biological significance through an increased understanding of how cells restrict monoallelic spreading of ncRNAs on autosomes, and their impact on gene expression. The approach is technically innovative, because it employs state of the art cell biological techniques, including super-resolution imaging and genomic approaches such as targeted iso-sequencing to annotate repeat-rich RNAs. Overall, this exploratory proposal holds a strong chance of unraveling a previously unknown mechanism underlying autosomal coating of ncRNAs, and could lead to deeper exploration of this phenomenon under physiological and pathological settings.
The proposed study of investigating the significance of monoallelic chromosome coating by ncRNA in differential ribosomal DNA (rDNA) expression will gain insights towards the role of ncRNAs in aging, as aberrant rDNA expression contributes to premature aging. The proposed research is relevant to public health because the outcome of this proposal will accelerate future efforts towards recognizing the use of noncoding RNAs as novel therapeutic target to regulate rDNA expression under pathological setting such as progeria. Thus, the project is relevant to NIH?s mission of seeking fundamental knowledge about the proper functioning of a cell, and its further application to prevent disease.