Small RNAs in Germ Cell Development - Current Topics in Developmental Biology - page 93

2008a,b ). In contrast to the miRNAs, whose processing relies on Dicer and
on Drosha/DGCR8, the endo-siRNA precursors are processed by Dicer
and subsequently are loaded onto Argonaute proteins and following specific
RNA recognition results in cleavage of the target ( Claycomb et al ., 2009;
Grishok et al ., 2001; Hutvagner and Zamore, 2002; Ketting et al ., 2001;
Knight and Bass, 2001 ). In addition to the proteins listed above, the
biogenesis and function of endo-siRNAs require the dsRNA-binding
proteins Loqs-PD and R2D2 in Drosophila ( Hartig et al ., 2009; Liu et al .,
2003; Pham et al ., 2004; Tomari et al ., 2004; Zhou et al ., 2009 )( Fig. 4.2 B).
In addition to the mechanism described above for Drosophila and mouse,
dsRNAs in C. elegans are typically synthesized by RNA-dependent RNA
polymerases (RdRPs). Dicer further processes the dsRNA fragments to
generate primary endo-siRNAs, including the 26G RNAs, which are
loaded onto a “primary Argonaute” ( Gent et al ., 2010, 2009; Pavelec
et al ., 2009 ). It is thought that primary endo-siRNAs recruit the RdRPs
EGO-1 or RRF-1 to the cleaved target mRNA, leading to de novo synthesis
of the secondary endo-siRNAs, the 22G short antisense RNAs that can
directly bind to specialized secondary AGO proteins (SAGOs) ( Liu et al .,
2004b; Meister et al ., 2004; Okamura et al ., 2004; Pak and Fire, 2007; Ruby
et al ., 2006; Sijen et al ., 2007; Yigit et al ., 2006 ).
3.2. endo-siRNA function in germ cell
differentiation—Oogenesis
Investigating the possible role of endo-siRNAs in early germline development
of Drosophila , the effect of lack of dicer-2 and ago2 proteins whose functions are
required for endo-siRNAs function was analyzed. Interestingly, whereas a
profound effect on dsRNA processing was observed, the function of both
proteins appeared to be dispensable for germline specification andmaintenance
in this organism ( Chung et al ., 2008; Lee et al ., 2004; Okamura et al ., 2008a ).
In C. elegans , mutants lacking the homologue of the plant RdRP EGO1 or
other RdRPs that are required for endo-siRNA precursor synthesis display
severe defects in oogenesis and spermatogenesis leading to infertility ( Smardon
et al ., 2000 ). The RNAi response of the mutants in the germline is reduced,
correlating with defects in mitosis, premature meiosis, and failure in meiotic
recombination, culminating in the production of defective oocytes.
Similarly, examining the role of endo-siRNAs in mouse oogenesis by
analyzing knockouts in the dicer or ago2 genes revealed a critical role the
corresponding proteins play in spindle organization in meiosis ( Murchison
et al ., 2007; Tang et al ., 2007; Watanabe et al ., 2008 ). In principle, this
phenotype could result from lack of endo-siRNAs or reflect a requirement
for miRNAs, both of which depend on Dicer function. Since the lack of
DGCR8, whose function is required specifically for miRNA biosynthesis
( Wang et al ., 2007 ), was nonconsequential for oogenesis, the defects observed