In a somewhat Herculean effort, Lécuyer and colleagues performed
in
situ hybridization against 3300 mRNAs expressed in
Drosophila embryos [
71]. Surprisingly, approximately 70% of these mRNAs are localized [
71], showing that mRNA localization is
widely used during
Drosophila development for spatial sorting of
proteins. Although the mechanism by which these thousands of mRNAs are localized has
not been characterized, the localization of
bicoid,
gurken and
oskar mRNA in
Drosophila oocytes has been extensively studied [
72-
75]. In addition, apically localizing mRNAs, such as
even
skipped,
wingless and
hairy has also
been studied in
Drosophila embryos [
76-
79]. In this
review, we will focus our discussion on the mechanism of
oskar mRNA
localization.
oskar mRNA is localized to the posterior of the oocyte in a
microtubule-dependent manner [
4,
73,
74] (). The specific
localization of
oskar mRNA, as well as precise translational
regulation of the message, results in restriction of Oskar protein to the posterior
of the oocyte [
73,
80,
81], which is
essential for establishing the anterior-posterior polarity of the egg [
81,
82]. The importance of this is illustrated by the demonstration that
mis-localization of
oskar mRNA results in an oocyte that lacks
polarity and an embryo that dies during development [
73,
81,
82].
Like many localized mRNAs,
oskar is identified as a target for
localization while still in the nucleus. Splicing at the first intron position of
oskar mRNA appears to be required for its localization [
83]. The position of this intron, but not
the nucleotide sequence, is the critical factor for localization [
83]. The mechanism by which splicing at
this position regulates mRNA localization is unclear, but the Exon Junction Complex
(EJC) proteins have all been shown to be involved in
oskar mRNA
localization [
84-
89]. Although EJC proteins are deposited on all spliced
mRNAs, it is possible that they recruit an unknown factor important for
oskar mRNA localization when they are present at the position
of the first intron. In addition to the presence of this first intron, the
3’UTR of
oskar mRNA is also required for localization [
90]. Various regions in the 3’UTR of
oskar mRNA are thought to mediate distinct steps in its
transport from the nurse cells to the posterior of the oocyte [
90]. However, the identity of specific proteins that bind
to these elements is unknown.
The motor responsible for transporting
oskar mRNA to the oocyte
posterior appears to be Kinesin-1 [
91].
Typically, cargoes that are transported by Kinesin-1 are directly bound by the
Kinesin light chain (Klc) adaptor protein [
66], and this complex then directly interacts with the motor subunit of
the complex, Kinesin heavy chain (Khc) [
66]. However, the transport of
oskar mRNA does not fit
this paradigm. While null mutants in
khc result in
oskar mRNA delocalization around the oocyte cortex [
91,
92], null mutants in
klc do not have a significant
effect on
oskar mRNA localization [
92]. Thus,
oskar mRNA is transported to
the oocyte posterior by a Kinesin complex that lacks the canonical light chain
adaptor. Consistent with this picture, another study found that the number of
oskar mRNP particles displaying fast, directed movement was
reduced by five-fold in
khc null oocytes [
93]. However, a significant number of fast-moving
oskar mRNP particles could still be detected in
khc null mutant [
93]. These results suggest that another, as yet unidentified, motor
participates in
oskar mRNP transport.
Collectively, these findings raise two very important questions. The first is
“How does Khc recognize
oskar mRNA as a target for
localization?”. One possibility is that an unknown protein links Khc to
oskar mRNA. If so, identifying this factor will be a critical
next step. The expectation would be that this protein co-localizes with Khc at the
posterior of the oocyte, and that mutations in this gene resemble
khc null mutants. Another possibility is that a known component
of the
oskar mRNP directly binds to Khc and serves as the bridge
between the motor and the transcript. This scenario does not seem likely because
mutants in known components of the
oskar mRNP result in mRNA that
is either diffusely distributed throughout the oocyte, or in mRNA that is trapped at
the anterior of the oocyte. This is in contrast to the
khc null
phenotype, in which
oskar mRNA is found around the oocyte cortex
[
91,
92]. A final possibility is that Khc directly binds to
oskar mRNA. At present there are no data to support a direct
interaction between Khc and
oskar mRNA. Thus, although there is
ample evidence implicating Khc in
oskar mRNA transport, the
mechanism by which this motor binds
oskar mRNA is still an open
question. The second intriguing question suggested by the live imaging of
oskar mRNPs is whether a motor other than Khc also participates
in
oskar transport.
In addition to transporting
oskar mRNA to the posterior pole,
spatial restriction of Oskar protein is achieved by numerous mechanisms of
translational repression [
80,
94]. Only once the mRNA is delivered to the
posterior is the repression relieved. Additionally, specific mechanisms operate at
the posterior of the oocyte to activate translation of
oskar [
80,
94]. A detailed discussion of this topic is beyond the scope of this
review. However, some recent findings have shed light on the mechanism by which the
translation of
oskar mRNA is regulated.
The first factor identified with a role in translational repression of
oskar mRNA was Bruno [
95]. Bruno was shown to bind to specific BRE (Bruno response element)
sequences in the 3’UTR of
oskar mRNA [
95]. Initially, these elements were thought to function
solely in translational repression. However, a recent report from the MacDonald lab
demonstrated that one of these BREs also functions in activating the translation of
oskar mRNA [
96]. The
precise mechanism by which Bruno regulates translation is not known. However, Bruno
has been shown to associate with the protein Cup [
97]. Cup in turn forms a complex with the transition
initiation factor eIF4E and prevents it from binding eIF4G [
97,
98], an
interaction which is required for translation initiation. Thus, Bruno and Cup may
function together to prevent translation initiation of
oskar mRNA.
Interestingly, Bruno also appears to package
oskar mRNPs into large
aggregates called silencing particles, rendering them inaccessible to the
translation machinery [
99]. Finally, the
Drosophila ortholog of hnRNP I/PTB has also been shown to bind
oskar mRNA and to package the message into higher-order
particles that are translationally silent [
100].
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