Previous studies have shown that orb
autoregulation promotes localized accumulation of Orb protein in subcellular compartments where its activity is required 
. To investigate the mechanisms underlying orb
autoregulation we sought to identify proteins that physically associate with Orb and thus potentially help regulate its expression and/or activity. We have previously shown that the Drosophila
Fragile-X protein (dFMR1) is found in complexes with Orb in Drosophila
ovaries and functions to negatively regulate orb
accumulation and activity 
. Here we show that Rin, the Drosophila
G3BP homologue, is also associated with Orb in ovaries. However, in contrast to dFMR1, Rin functions as a positive regulatory factor, helping to promote Orb accumulation and activity.
Our results show that Rin associates with Orb as part of an RNase-resistant complex. The RNase-resistance aspect of this interaction suggests that their association is mediated by protein-protein interactions rather than, or in addition to, binding to the same mRNA species. While it is possible that Orb and Rin interact directly with each other, an equally plausible scenario is that their association is mediated by one or more proteins found in both Orb and Rin immunoprecipitates. For example, mammalian G3BP has been shown to interact directly with Caprin-1, and the fly Caprin protein is found in both Rin and Orb immunoprecipitates. Two other findings would also seem to argue in favor of an indirect, rather than a direct interaction. First the overlap between Rin and Orb, especially in vitellogenic chambers is quite limited. Second, only a small subset of the proteins associated with Rin or Orb are common to both. It is also possible that the initial association between Orb and Rin could depend upon binding to the same target mRNAs and their subsequent interaction could depend upon a short stretch of RNA that is hidden in the complex and protected from RNase activity. In this case, the limited co-localization observed in egg chambers would imply that only a subset of their mRNA targets are in common.
GB3Ps in mammals are thought to have two functions. The first is repressing the translation of target mRNAs by mechanisms that depend upon their helicase and RNase activities, while the second is in the assembly of stress granules under conditions of environmental stress such as heat shock or drug treatment. For this reason, we anticipated that rin, like dfmr1, would function to negatively regulate orb activity and/or expression. However, we observed exactly the opposite result. Instead of suppressing the DV polarity defects in eggs from Hd19G orb343/+ females, reducing rin activity increases the frequency of DV defects. Conversely, DV polarity defects are suppressed by providing excess rin activity. While it is clear from the phenotypic effects seen in mutants that rin has multiple functions in oogenesis (and these will be independent of orb), the most plausible explanation for the effects of decreasing and increasing rin activity on DV polarity in Hd19G orb343/+ females is that they arise because of changes in the expression/accumulation of Orb protein. Consistent with this explanation, we find that we can change the level of Orb protein by manipulating rin activity. If orb is wild type, reducing the rin dose by half has little if any effect on Orb protein accumulation. However, when orb activity is compromised by the Hd19G orb343 combination, heterozygosity for rin results in Orb protein levels that are less than 5% that of wild type. Conversely, increasing rin activity in a wild type background elevates Orb protein levels almost two-fold over wild type, while in background compromised by the Hd19G orb343 combination adding extra rin restores Orb protein levels to that of wild type. Taken together, these findings argue that rin functions as a positive regulator of orb. At this point it is not clear how rin might control the accumulation of Orb protein. Since Rin and Orb are associated with each other, the simplest model is that Rin helps activate the translation of orb mRNA and thus functions as a co-factor in orb autoregulation. The substantial reduction in Orb protein levels evident in rin3 ovaries is consistent with this idea. This view would also be supported by the DV polarity defects evident in eggs laid by rin mutant females. However, other less direct models (e.g., rin represses the translation of some factor that inhibits orb mRNA translation or rin is required to stabilize Orb protein) can't be excluded at this time. Likewise, rin may have other targets besides orb in the establishment of DV polarity.
Potentially arguing in favor of a role for Rin in the translation of orb
mRNA and/or in the activity of Orb protein is the fact that mutations in genes encoding several of the other proteins found in both Orb and Rin immunoprecipitates also show genetic interactions with orb
. Thus, dfmr1
suppress the DV polarity defects in eggs laid by Hd19G orb343/+
females, while pabp
enhance the polarity defects. We have shown previously that dfmr1
also exerts its effects, at least in part, by altering the expression of Orb protein 
. Further connecting the effects of rin
to translation, we also found that Orb, dFMR1 and Rin fractionate with polysomes in sucrose gradients (data not shown). If the proteins detected in Orb and Rin immunoprecipitates are part of the same complex rather than different complexes, then the gene dose effects we have observed would suggest that they act coordinately to regulate Orb expression and that the relative balance between positive (e.g., Rin) and negative (e.g., dFMR1) factors in the egg chamber helps set the level of Orb accumulation. Further studies will be required to understand precisely how rin
influences Orb protein accumulation and how some of the other factors associated with Rin and Orb like dFMR1, Lig and PABP function in this process.
Although our studies implicate rin as a positive regulator of orb, this is clearly not the only role for rin in the ovary. Instead, the phenotypic effects of rin mutations point to a potentially diverse array of functions, not only in gem cells but also in the surrounding somatic follicle cells. For example, the fragmentation of ring canals, the failure to properly disperse the endoreplicated nurse cell chromosomes, and the dumpless phenotype are not observed in orb mutants. Moreover, ring canal and chromatin dispersal defects suggest that rin has functions in nurse cells, which is a compartment that has only little Orb protein. Likewise, the encapsulation defects could be of somatic origin where rin, but not orb is expressed. Further studies will be required to identify the rin regulatory targets in these and potentially other processes.