Noncoding RNAs (ncRNAs) and their protein partners play critical roles in an enormous variety of cellular processes. The focus of this application is an abundant class of RNPs that is widespread in animal cells and also present in many bacteria. The major protein, the ring-shaped Ro 60 kDa autoantigen, is a clinically important target of autoantibodies in patients with systemic lupus erythematosus (SLE). Ro assists survival of both animal cells and bacteria after some forms of environmental stress, and mice lacking Ro develop an autoimmune syndrome resembling SLE in patients. In all organisms examined, Ro binds ~100 nt ncRNAs called Y RNAs. In the last funding period, by studying Ro RNPs in the bacterium Deinococcus radiodurans, we uncovered a novel role for ncRNA, that of tethering a protein cofactor to an enzyme to alter its substrate specificity. Specifically, we discovered that the bacterial Ro was tethered by Y RNA to the ring-shaped exoribonuclease polynucleotide phosphorylase (PNPase), forming a double-ringed RNP machine specialized for structured RNA decay. Biochemical and structural analyses support a model in which single-stranded RNA threads through the Ro ring into the PNPase cavity for degradation. Consistent with a conserved role, we have now identified a set of mouse and human mRNAs whose levels depend on Ro. We also identified several ncRNAs that require Ro for their stable accumulation. Our current goals are to define these newly identified roles for Ro and Y RNA in mechanistic detail.
Our first aim i s to dissect the mechanism by which binding of mammalian Ro to specific mRNAs assists their decay.
Our second aim i s to determine the mechanisms by which mammalian Ro assists the biogenesis of specific ncRNAs.
Our third aim i s to determine how the new bacterial RNA degradation machine contributes to survival of the human pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) during treatment with mitomycin C, a nucleic acid crosslinker used to treat human gastric and pancreatic cancers. Together, our studies will elucidate novel pathways that regulate mammalian mRNA levels and ncRNA biogenesis and continue our characterization of a new RNP machine. Our studies will also define mechanisms by which RNA populations are adapted during environmental stress, a poorly studied but vital part of maintaining RNA homeostasis.
The first antibodies detected in people who develop systemic lupus erythematosus are against an RNA-binding protein called the Ro 60 kDa autoantigen. In patients, anti-Ro antibodies are strongly associated with sunlight- sensitive skin lesions in adults and with similar skin rashes and permanent heart defects in babies of mothers with these antibodies. Studying how Ro functions in cells and characterizing Ro-bound RNAs may give clues as to how lupus is initiated and make it possible to design drugs that specifically target either Ro or its associated RNAs.
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