Regulation of Cell Polarity and RNA Localization in Vertebrate Oocytes - International Review of Cell and Molecular Biology - page 169

These and other related areas have remained understudied for many
years, mostly owing to the inability to perform targeted genetic manipula-
tions during oogenesis in frogs and fish. The often-partial loss-of-function
effects caused by antisense oligos or other inhibitory reagents also hamper
understanding the ultimate functions of localized RNAs. Additionally, stud-
ies on the roles of trans -acting proteins in the RNA localization process have
relied on dominant-negative constructs and blocking antibodies, which are
difficult to control for specificity.
Recently, many of the tools commonly used in Drosophila and mouse
have become widely available in amphibians and fish as well. These include
mutagenesis and forward genetics, as well as tissue-specific transgenesis and
gene deletion. Methods for targeted genome editing in these organisms,
including zinc finger nucleases ( Young et al., 2011 ), TALENs ( Bedell
et al., 2012; Dahlem et al., 2012; Lei et al., 2012 ), and Cas9/CRISPRs
( Hwang et al., 2013; Mali et al., 2013 ), have also become more widespread.
There is great potential for genetic manipulation of RNA localization path-
ways, both to disrupt endogenous localization elements and to insert exog-
enous sequences into the UTRs of candidate genes. Additionally, these
methods will allow for more rigorous loss-of-function approaches to test
the functions of localized RNAs in development, including noncoding
RNAs. Even with these more sophisticated genetic approaches, a critical
role in early oogenesis may mask the function of a localized RNA in late
oogenesis or in early development. It will therefore still be important to
optimize methods for depleting maternal RNAs from full-grown oocytes
prior to fertilization. This has been routine for some time in Xenopus and
a similar method has recently been developed for zebrafish ( Nair

et al., 2013 ).
In sum, advances in whole genome analysis and postgenomics technol-
ogies have and will continue to allow a greater appreciation of the role of
RNA localization in cell biology. In the future, it will remain an exciting
challenge to further elucidate the biochemical mechanisms of RNA local-
ization, the organization of the oocyte that allows such localization, and the
diversity of roles of localized molecules in the cell.

ACKNOWLEDGMENTS
The author would like to thank Mary C. Mullins for contributing images. This work was
supported by The University of Iowa and by NIH Grant GM083999.