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The involvement of mRNA degradation and DNA damage response factors in mRNA localization during Drosophila oogenesis

Final Report Summary - RDDDLOC (The involvement of mRNA degradation and DNA damage response factors in mRNA localization during Drosophila oogenesis)

Project context and objectives

Intracellular mRNA localisation and localised translation are an efficient means for the spatial control of protein expression and play key roles in diverse processes, such as cell fate determination, cell polarity, directed cell motility and body axis determination. The importance of this mechanism is well illustrated in Drosophila oogenesis, in which the localisation of three distinct mRNAs in the oocyte - grk, bcd and osk - determine the body axes.

Some studies have implicated mRNA processing factors, such as RNAi-related factors or P-body components, in the localisation and translation control of osk and grk mRNAs. The overall aim of this project was to study this issue further with the following distinct aims and tasks.

Work performed and main results

1. To determine the pathway by which the DNA damage checkpoint activation in RNAi-associated mutants induces karyosome defects, premature ooplasmic streaming and RNA mislocalisation

The piRNA class of small RNAs (called 'rasiRNAs' in the application), which suppress retrotransposons and thus sustain genomic stability, are required for normal development of the Drosophila female germline. Previous work from others and unpublished data from our group revealed that the female's mutant for piRNA biogenesis factors (e.g. spn-E, aub, armi) have faulty germline maintenance and present several oocyte defects, especially during mid-oogenesis: a) karyosome defects; b) absence/impairment of a cytoplasmic actin mesh; c) abnormal cortical microtubule arrays; d) premature cytoplasmic streaming; and e) defects in grk and osk mRNA localisation and/or translation, which cause mis-patterned eggs. Mutations in the DNA double-strand break (DSB) repair pathway (spn mutants) also give these phenotypes. The phenotype of the piRNA and DSB repair mutants is likely caused by a DNA damage response, because the osk and grk defects were largely rescued by the suppression of the ATR-Ckh2 pathway (e.g. mnk/Chk2 mutants or caffeine treatment). This explains why piRNA mutants and DSB repair mutants share the phenotype.

Several of the piRNA mutant defects are also observed in mutants for spire and capu, which are positive actin regulators, and lark/RMB4, which is a RNA binding protein that is part of the Ago2 RISC complex. The osk and grk mRNA defects of capu and spire mutants are caused by the absence of the cytoplasmic actin mesh, which stops cytoplasmic movements, thus allowing the localisation and anchoring of the mRNAs.

Given the common features between piRNA mutants and capu and spire mutants, we hypothesised that the DNA damage response in the piRNA mutants induces defects in osk and grk mRNAs by impairing the cytoplasmic actin mesh.

We made several substantial findings.

First, although lark mutants share several features of the piRNA mutants, they have normal karyosome and normal retrotransposon repression. Therefore, lark is not required for piRNA biogenesis.

Second, we were able to partially rescue all feature defects of piRNA mutants (on germline maintenance and in the oocyte: karyosome, cytoplasmic movements, cytoplasmic actin mesh, microtubule arrangement, osk and grk mRNA localization/translation and egg patterning) by ectopically expressing an active form of the positive actin regulator Spire. The rescues were modest, however, but so were the Spire expression levels. We have not succeeded in achieving higher Spire expression during mid-oogenesis and, therefore, we could not test whether high levels of ectopic Spire can rescue the defects of piRNA mutants better. Nevertheless, the partial rescue and the fact that spire mutants phenocopy the piRNA and DSB repair mutants, support the hypothesis that actin defects underlie the piRNA and DSB repair phenotypes, possibly by the down-regulation of Spire. We will further test the hypothesis by checking whether endogenous Spire is down-regulated in the piRNA mutants.

Third, the DNA damage response in the piRNA mutants seems to cause the karyosome and cytoplasmic actin mesh abnormalities because these are largely rescued in a mnk/Chk2 mutant background.

Fourth, the mid-oogenesis defects of hypomorphic orb/CPEB mutants are largely rescued by the ectopic expression of active Spire. This strongly suggests that the phenotype of orb mutants is due to abnormalities in the actin mesh and challenges the previous model that Orb/CPEB affects osk mRNA directly, by regulating its poly-A tail.

In conclusion, persistent DNA damage in the female germline (e.g. by transposon up-regulation in piRNA mutants) activates a DNA damage response that compromises oogenesis at two levels. Firstly, it blocks germline maintenance and cyst formation in the germarium. Secondly, during mid-oogenesis, it impairs the cytoplasmic actin mesh (possibly by down-regulating Spire), limiting grk and osk mRNA localisation and/or translation and, consequently, disrupting patterning.

2. To determine the relationship between P-bodies and transport RNPs in the Drosophila oocyte

Localising mRNAs must be translationally silent during transport. The deleterious consequence of ectopic, leaky expression is illustrated by osk mRNA in the Drosophila oocyte, which causes mispatterned, bicaudal embryos when in excess or mislocalised to the anterior.

P-bodies, cytoplasmic granules where mRNA is degraded or translationally silent, have been implicated in osk mRNA localisation and translational control. First, P-body components and osk mRNA are enriched at the oocyte posterior, although Osk-induced pole plasm is itself rich in mRNA silencing and degradation agents. Second, mutants for P-body components show osk mRNA mislocalisation (dcp1) or premature translation (me31B). We therefore hypothesised that P-body components may be part of the transport mRNPs.

We made significant findings in this part of the project.

Firstly, osk mRNA particles and P-bodies have distinct overall behaviours, with the former displaying frequent fast movement while the latter are mainly static. Despite this, osk mRNPs frequently dock to the surface of P-bodies and show episodic co-movement.

Secondly, P-bodies re-localise with osk mRNA in genetic backgrounds in which the mRNA mislocalises to the anterior or centre of the oocyte (BicD, stau, tmII and grk). As osk mRNA is very poorly translated in such mutants, P-bodies must be recruited by osk mRNA instead of osk protein or pole plasm.

Thirdly, P-bodies are still at the oocyte posterior in osk[84] and vasa[PD] mutants, in which osk mRNA localises posteriorly but does not induce pole plasm. This result reinforces the notion that P-bodies are directly recruited by osk mRNA.

Altogether, our data shows that osk mRNPs recruit P-body components. It remains to be shown whether P-bodies are functionally involved in osk mRNA transport.

We also analysed the spatial relationship between bcd mRNPs and P-bodies. Although we found that P-bodies (as defined by Tral) were slightly enriched at the anterior of stage 9 oocytes, at the bcd mRNA localisation region, it was not clear whether the two really co-localised. However, we found that bcd mRNPs co-localised with Exu particles, a helicase that is required for bcd mRNA localisation and which is a P-body component.

This work could not establish a clear spatial/physical link between bcd mRNPs and 'conventional' P-bodies. Nevertheless, bcd mRNPs and Exu particules are spatially correlated, which indicates a direct interaction and, thus, a direct role of Exu on bcd mRNA localisation.