AUF1/hnRNP D is a novel EBER1-interacting protein. (A) Experimental outline of the EBER1-MS2 selection coupled to SILAC and mass spectrometry analysis. (B) Schematics of the four isoforms of AUF1 (Wagner et al. 1998), which originate from alternative splicing and differ by the presence or absence of two regions called Insert A and Insert B. The peptide sequenced by tandem mass spectrometry analysis is part of the RNA recognition motif (RRM) II domain as indicated at the bottom. Q-rich indicates glutamine-rich domain. (C) Confirmation of the specificity of the EBER1-MS2 selection of AUF1. Lysates of BJAB cells containing either EBER1 or EBER1-MS2 were subjected to MS2-mediated selection. RNA was isolated and probed for EBER1 by Northern blotting to determine the pull-down efficiency. Western blots for AUF1 showed that AUF1 is selected only from cell lysates containing EBER1-MS2 and not from lysates containing unmodified EBER1. The four isoforms of AUF1 are indicated; the p40 and p42 isoforms comigrate in the gel. The asterisk indicates a cross-reactive band. Western blot analysis of L22 is shown as a positive control.
