Probing the mRNA processing body using protein macroarrays and "autoantigenomics"

Abstract

Messenger RNA processing bodies (P-bodies) are cellular structures that have a direct role in mRNA degradation. P-bodies have also been implicated in RNAi-mediated post-transcriptional gene silencing. Despite the important roles of P-bodies in cellular biology, the constituents of P-bodies and their organization have been only partially defined. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against these structures. Recent advances in protein macroarray technology permit the simultaneous screening of thousands of proteins for reactivity with autoantibodies. We used serum from patients with anti-P-body autoantibodies to screen a protein macroarray and identified 67 potential autoantigens. Immunoreactive proteins included four known P-body components and three additional primary biliary cirrhosis autoantigens. Y-box protein 1 (YB-1), a 50-kDa RNA-binding protein that was not previously known to be a P-body component, was recognized by serum from four of seven patients. YB-1 colocalized with P-body components DCP1a and Ge-1. In cells subjected to arsenite-induced oxidative stress, YB-1 localized to TIA-containing stress granules. Both YB-1 and the previously identified P-body component RAP55 translocated from P-bodies to stress granules during oxidative stress. During recovery, however, the reappearance of YB-1 in P-bodies was delayed compared with that of RAP55, suggesting that YB-1 and RAP55 may have different functions. This study demonstrates that the combination of human autoantibodies and protein macroarray technology provides a novel method for identifying and characterizing components of mRNA P-bodies.