Worm Breeder's Gazette 9(2): 47
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have obtained preliminary evidence that two RNAs are associated with the yolk proteins of C. elegans in what appears to be a discrete ribonucleoprotein complex. Such a complex could be functionally important in the transport of yolk proteins across cell membranes. We were prompted to look for RNAs in preparations of yolk particles by a 1971 report from Dubois et al. [1]. These investigators isolated and characterized RNAs from subcellular fractions of sea urchin eggs and embryos. A fraction enriched for yolk platelets ( the large, membrane-bounded particles in which yolk proteins are localized in the cytoplasm) yielded two RNA species not found in other fractions. On the basis of electrophoretic mobility, these RNAs were assigned nominal sedimentation coefficients of 9S and 12S. We have prepared yolk platelet fractions from C. elegans homogenates and extracted RNA from them. We then synthesized labelled cDNA from the extracted RNA and hybridized the cDNA to blots of electrophoretically resolved C. elegans total RNA. These experiments are messy, as there is a lot of ribosomal RNA in the yolk platelet preps. But we can see two non-ribosomal bands, corresponding to RNAs of about 500 and 800 nucleotides. Other workers investigating sea urchin yolk have reported that yolk proteins form a discrete complex with a sedimentation coefficient on the order of 25S [2, 3]. This is much smaller than the platelet, and may be a fundamental packing unit of yolk in the platelet. We have isolated an apparently analogous complex from C. elegans, using both immunoabsorption and sedimentation in sucrose gradients. At least under certain conditions, the 500 and 800 nucleotide RNAs observed in platelet preps co-isolate with this complex, suggesting that they are specifically associated with yolk proteins. At this point, however, things get a little sticky. It seems to be easy enough to get a complex that contains all four yolk proteins. But not all complex preps contain the two RNAs. The complex isolated by immunoadsorption in the presence of the non-ionic detergent NP-40, for example, yielded only ribosomal sequences on cDNA analysis. But a complex isolated by sedimentation from a homogenate in 150 mM Tris-HCl (pH 7.5) was markedly enriched for the RNAs in comparison to other yolk-containing fractions of the gradient. We really need to know more about the organization and genesis of the yolk platelet. We have found that platelets dissociate in 0.1% NP-40, or in buffers of low osmolarity, to yield yolk protein complexes (maybe different complexes under different conditions). But these conditions also seem to deplete the complexes of the RNAs. For example, when we isolated complex in 50 mM Tris-HCl instead of 150 mM, we got a much better yield of complex, but no RNAs. This, with the absence of the RNAs from a complex isolated in the presence of NP-40, and in the context of the speculations entertained below, suggests involvement with the platelet membrane. If the RNA-containing complex we are looking for is actually some sort of vesicular structure, it could well be labile to both detergent and low osmolarity. What might RNAs be doing in association with yolk proteins? One possible functional role for RNAs might be in the transport of yolk proteins across membranes, by analogy with the 7S RNA of the signal recognition particle [4] and the as yet uncharacterized RNA(s) involved in the import of nuclear-encoded mitochondrial enzymes [5]. Such transport may be necessary for both the genesis of the yolk platelet itself and the striking segregation of yolk to the intestine late in C. elegans development [6]. Little is known about the genesis of yolk platelets, though in C. elegans, accumulation of yolk in the gonad is not evident distal to the point at which individual oocytes are distinct. Presumably, incorporation of yolk proteins into the membrane-bounded platelets occurs during maturation of the oocyte. Similarly, it's unclear what is going on during the segregation of yolk to the intestine. Segregation occurs well after the embryonic E lineage is complete. Formally, it's possible that yolk is preferentially degraded everywhere except the E cells, or synthesized de novo in the embryonic intestine. Neither of these alternatives makes a lot of biological sense, and the immunofluorescence fields published in 1983 [6] seem best explained by active segregation of yolk within the embryo. This would require that yolk proteins, or possibly intact yolk platelets, cross cell membranes.