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together, these data suggest that velo1 likely uses an alternate mechanism for
vegetal localization.
3.2. Early pathway RNAs
3.2.1 Localization of early pathway transcripts to the
mitochondrial cloud
nanos homolog ue 1 ( nos1/xcat2 ) was initially isolated through cDNA library
screening of intermediate filament-associated (detergent-insoluble fraction)
RNAs from Xenopus oocytes ( Mosquera et al., 1993 ). Nos1 localization dur-
ing oogenesis is markedly different than gdf1 , first becoming restricted to
germ plasm within the mitochondrial cloud in stage I oocytes and then tran-
slocating with the cloud material to the vegetal cortex by stage II ( Forristall
et al., 1995 ). In full-grown oocytes, nos1 is in scattered clusters or islands in
the cortex at the vegetal pole apex. In contrast to gdf1 , nos1 does not become
unanchored from the cortex during oocyte maturation ( Forristall et al.,
1995; Mosquera et al., 1993 ), rather it remains cortical and is aggregated into
forming vegetal cleavage furrows in early cleavage blastomeres in a pattern
consistent with germ plasm ( Forristall et al., 1995 ).
Injected nos1 transcripts localize to the mitochondrial cloud during
stage I or to the vegetal cortex during stage IV independently of microtu-
bules and microfilaments ( Chang et al., 2004; Kloc et al., 1996; Zhou
and King, 1996 ). Exogenous nos1 RNA appears stable in these experiments,
arguing against selective RNase protection in the mitochondrial cloud body
as potential localization mechanism. Evidence from live imaging and FRAP
studies suggest that nos1 localizes to the mitochondrial cloud body by a selec-
tive entrapment mechanism, independent of motor activity ( Chang et al.,
2004 ). Injected nos1 message (as well as dazl ) forms labeled particles that
are freely diffusible. Live imaging showed a progressive immobilization in
the mitochondrial cloud, followed by aggregation into denser clusters over-
lapping with dense ER. Igf2bp3 was excluded from the mitochondrial cloud,
suggesting that nos1 localizes regardless of its binding ( Chang et al., 2004 ).
Similar results were seen with dazl localization in those studies, suggesting a
general mechanism for early pathway RNAs.
Recent evidence suggests that ATP-dependent metabolic activity is also
required for mitochondrial cloud localization (MCLS) of nos1 , along with
weak involvement of Kif3b ( Heinrich and Deshler, 2009 ). It is unclear
though whether this energy dependence is needed for RNA movement
or anchoring or for more general processes occurring among the organelles
in the mitochondrial cloud (i.e., organelle trafficking).
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