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functions were provided by analysis of xlsirts , which are noncoding inter-
spersed repeat transcripts ( Kloc et al., 1993 ). Depletion of xlsirts RNA from
stage IV oocytes using antisense oligos caused the loss of gdf1 RNA but not
nos1 mRNA, from the vegetal cortex ( Kloc and Etkin, 1994 ). However,
these studies used quite high doses of oligos and off-target effects could
not be excluded. More recently, depletion of vegt RNA was also found
to cause delocalization of gdf1 , as well as other mRNAs including bicc1
and wnt11b ( Heasman et al., 2001 ). In this case, nos1 was again not affected,
but the effect on bicc1 and wnt11b mRNAs suggests that RNA-dependent
anchoring may be a more general phenomenon. Importantly, the effects
of antisense DNA oligos were compared with that of MOs, which do
not cause the destruction of target RNAs, and the delocalization effect
was only seen using DNA-based oligos ( Heasman et al., 2001 ). These results
stress that it is the RNA molecules involved in anchoring gdf1 , not the Vegt
protein. Endogenous vegt and xlsirts transcripts have been visualized in asso-
ciation with the cytokeratin cytoskeleton using molecular beacons ( Kloc,
2009; Kloc et al., 2005 ), and depletion of these transcripts can alter the orga-
nization of the cytokeratin network ( Kloc, 2009; Kloc et al., 2005 ).
4.5.2 plin2
Depletion of plin2 also has a similar effect ( Kloc, 2009 ), although the exact
patterns of disorganization were different when vegt , plin2 ,or xlsirts are
depleted. This could suggest transcript-specific, nonredundant roles. Curi-
ously, the effect of vegt depletion on cytokeratin organization could be res-
cued by exogenous vegt , in a localization-independent manner ( Kloc et al.,
2005 ). This result indicates that the presence of the RNA at a sufficient
abundance is needed for cytoskeletal organization, not localization per se .
4.5.3 General roles of structural RNAs
These data reflect a growing body of evidence that RNAs can play general
structural or scaffolding roles in the cell. Recent data have shown that RNAs
associate with and are required for the function of the mitotic spindles in
both egg extracts and cell lines ( Blower et al., 2007; Sharp et al., 2011 ).
Additionally, centrosomes have long been recognized to have a significant
and essential RNA component ( Heidemann et al., 1977 ). Recently, the
roles of unique centrosomal RNAs are being reinvestigated ( Alliegro and
Alliegro, 2008; Blower et al., 2007 ). Since there is a increasing realization
of structural roles for RNA, it has become critical for those using antisense
methods to study localized mRNA function to validate interesting results
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