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.

Yolk-Associated RNAs in C. elegans

B. Sharrock and M. Sutherlin

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.