Several studies indicate a functional relationship between Vasa and both the small interfering RNA and micro-RNA processing pathways. One essential component in both of these pathways is the RNase III endonuclease Dicer,(91)
which, in mice, colocalizes with Vasa in nuage.(89)
Furthermore, ectopically expressed Vasa and Dicer protein interact in COS cell lysates and this interaction requires the C-terminal portion of Vasa.(89)
The C-terminal RNaseIII region of Dicer is sufficient to interact with Vasa and the remaining N-terminal ATPase/helicase-PAZ domain region appears independent of Vasa.(89)
PIWI proteins represent a subgroup of Argonautes required for germ line stem cell maintenance and fertility in several animals.(92) Drosophila
PIWI is a polar granule component that interacts with Vasa and PIWI-mutant flies have normal Vasa protein expression and abdominal patterning of the embryo, but exhibit a severe deficiency in pole cell formation.(93)
Over-expressing PIWI results in a dose-dependent increase in Vasa protein levels and pole cell formation. These data suggest that a PIWI-mediated piRNA pathway regulates the levels of Vasa and Oskar proteins and possibly other genes involved in the germ line determination pathway in Drosophila
A similar interaction is found in mouse, where Vasa protein binds to both recombinant and endogenous MIWI and MILI, which are mouse PIWI homologs.(81,94)
Indeed, MILI and Vasa knockout mice have similar phenotypes and defects in spermatogenesis indicative of cooperative molecular functions. (14,94)
In MIWI knockout mice, Vasa protein does not localize to the nuage structures.(94)
However, it is still unknown whether MIWI is required for nuage and ultrastructural studies in MIWI knockout mice are needed. Exactly how these specific interactions influence Vasa, MIWI or MILI function is unclear.
Recent work has identified Maelstrom as a nuage component that interacts with both mouse Vasa and MIWI, is required for spermatogenesis and also is involved in silencing transposable elements.(95,96)
, Maelstrom protein localizes to nuage in a Vasa-dependent manner. In maelstrom
mutant oocytes, a higher molecular weight Vasa protein species is evident indicating that Maelstrom is required for proper Vasa modification or processing.(87)
Although still not definitive, the consistent association in multiple animals of vasa and members of the RNAi pathway argues strongly that they have a functional relationship. This may not be surprising since both are postulated to be involved in translational regulation, and the engagement of each mRNA with the ribosome is likely a continuously evaluated process that responds with incremental increases, decreases, and rates of translation. Researchers often have difficulty in dissecting overlapping pathways by classic genetic means, so an alternative approach is to make use of blossoming in vitro
cell free lysate assays both for mRNA translational activity, as well as for mRNA stability as a result of miRNAs.(97,98)
In the context of vasa function in the RNAi pathway, it is hard to ignore the intersection here that the small RNAs are 20–30 bases in length, and that vasa is capable of unwinding 20–25 bases of dsRNA.
Vasa evolution resulted in divergent N-termini while retaining the conserved helicase domain
Comparative phylogenetic data suggests that the Vasa gene family originated from a duplication in a PL10-related DEAD-box gene early in metazoan evolution.(7)
While animals have both vasa and PL10 genes, plants and fungi have only PL10 genes and lack vasa genes (). At some point after this gene duplication, vasa genes acquired CCHC Zn-knuckle domains in the region N-terminal of the conserved DEAD box. The number of Zn-knuckle domains found Vasa sequences vary from 1 to 8. However, vertebrates and insects may have both independently lost these Zn-knuckle domains (, ).
CCHC Zn-knuckles can be categorized as a “classical zinc-finger” based its zinc chelation topology of a short β-hairpin followed by an α-helix.(99)
They can bind single and double-stranded DNA or RNA and may be involved in transcriptional regulation.(100,101)
Most examples of CCHC Zn-knuckles come from human retroviruses which all require Zn2+
binding for proper nucleocapsid protein folding.(102–104)
The CCHC Zn-knuckle domains of retroviral nucleocapsid proteins interact with specific structures in the viral RNA genome during packaging.(105–109)
A non-viral CCHC Zn-knuckle example includes the C. elegans
Lin28 protein, which has 2 CCHC Zn knuckles that are crucial for its localization to P-granules and stress granules.(110)
While the exact functional significance of these Zn-knuckle domains is unknown, their RNA-binding properties point to a role in Vasa’s RNA target specificity. Zn-fingers are versatile and can also target binding to other proteins.(99)
Do the CCHC Zn-knuckles impart additional functional dimensions to Vasa proteins? While the presence of the 9 conserved DEAD-box sequence motifs in Vasa allow inference into their RNA helicase catalytic activities, the highly divergent N-terminal regions are more cryptic. The presence of Vasa Zn-knuckles correlates, in many animals, with an expanded expression pattern (and possible functional role) outside of the germ line. The presence or absence of Zn-knuckles may reflect differences in RNA or protein target binding. These different target interaction properties may be important to potential expanded functions outside of the germ line. One exception to this notion is the presence of Zn-knuckles in the 4 Vasa homolog Germ-line helicase (GLH 1-4) genes in C. elegans
, whose expression is restricted to the germ line.(16–18)
Alternatively, it is possible that the loss of Zn-knuckles in insect and vertebrate vasa genes coincided with the emergence of Zn-knuckle containing cofactor proteins which now confer the target specificity. However, no such Vasa cofactors have been identified yet in either insects or vertebrates.