36 other proteins were identified in the AIN-2::GFP IP sample by at least two different peptides but were absent from the control GFP IP sample. GLD-1 and ACO-1, two RNA-binding proteins involved in translational inhibition (Gourley et al., 2003; Jan et al., 1999; Lee and Schedl, 2001), were also found to be in the AIN-2 IP sample, suggesting that miRNA may collaborate with RNA-binding proteins to repress the translation of target mRNAs.
We did not observe significant reduction of mir-48 and mir-84 levels in ain-1(lf);ain-2(rf) animals. Moreover, there was no accumulation of miRNA precursors in the double mutant.
Proteins associated with either an AIN-1::GFP or an AIN-2::GFP protein, each expressed from an integrated functional transgene, were precipitated with an anti-GFP antibody (Ding et al., 2005) and analyzed on a mass spectrometer using the Multidimensional Protein Identification Technology (Mud-PIT) (Washburn et al., 2001). ALG-1 and ALG-2, the two Argonaute proteins that share functions in the miRNA pathway (Grishok et al., 2001), were the most abundant proteins detected in either AIN-1-or AIN-2-associated complexes but were absent in the control samples (Figure 2F). Conversely, we also performed CoIP using GFP-tagged ALG-1 and ALG-2 proteins (Figure 2G). AIN-1 was found to coprecipitate with both in a western blot analysis using a newly raised anti-AIN-1 antibody. The miRISC-specific ALG-1 and ALG-2 were the only two Argonaute proteins detected in either AIN-1 or AIN-2 CoIP samples, indicating that AIN-1 and AIN-2 are likely to be specifically involved in the miRNA pathway. Meanwhile, AIN-1 was not detected in the AIN-2 IP sample, and vice versa, indicating that AIN-1 and AIN-2 are in distinct complexes and supporting the idea that AIN-1 and AIN-2 are redundant components of miRISCs.
36 other proteins were identified in the AIN-2::GFP IP sample by at least two different peptides but were absent from the control GFP IP sample. GLD-1 and ACO-1, two RNA-binding proteins involved in translational inhibition (Gourley et al., 2003; Jan et al., 1999; Lee and Schedl, 2001), were also found to be in the AIN-2 IP sample, suggesting that miRNA may collaborate with RNA-binding proteins to repress the translation of target mRNAs.