Translational control of the oogenic program by components of OMA ribonucleoprotein particles in Caenorhabditis elegans.
Bottom Line: Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3'-untranslated regions (3'-UTRs).Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2.We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs.
Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota Minneapolis, Minnesota 55455.Show MeSH
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Mentions: Mass spectrometry is extremely sensitive, and >250 different proteins were identified by at least two peptides in both OMA-1 RNP purifications (Figure S8, File S2). Many of these proteins, like CGH-1 and CAR-1, have RNA-related functions and could be important components of OMA-1 RNPs in vivo. Other copurifying proteins likely represent abundant contaminants (e.g., UNC-54/myosin and VIT-1/vitellogenin; see Materials and Methods). We focused on the subset of proteins that copurify with OMA-1 from fog-1(ts) females after RNase treatment based on the expectation that close associations with OMA-1 (be they direct or indirect) might be at least partially resistant to RNase treatment. Many proteins, including some with RNA-related functions (e.g., CGH-1, EDC-3, and CEY-4), were depleted from OMA-1 RNPs by RNase treatment, leaving a much smaller pool of candidates (133 different proteins; Figure S8). Importantly, the eIF4E-binding protein IFET-1 continued to copurify with OMA-1 in the presence of RNase A (File S2). Prior work established that IFET-1 interacts with OMA-1in vitro and represses the translation of OMA target 3′-UTR reporters in vivo (Li et al. 2009; Guven-Ozkan et al. 2010; Oldenbroek et al. 2013). Next, proteins identified in negative controls or as abundant contaminants were excluded from consideration, leaving a smaller list of OMA-1-associated proteins (51 different proteins; Figure S8). This step eliminated CAR-1, but again retained IFET-1 (File S2). It is difficult to eliminate all contaminants identified by mass spectrometry using a limited number of negative controls (Mellacheruvu et al. 2013), so we examined the biological functions of the remaining proteins in more detail (Table 1, File S2).
Affiliation: Department of Genetics, Cell Biology and Development, University of Minnesota Minneapolis, Minnesota 55455.