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. 2012 Feb 23:5:120.
doi: 10.1186/1756-0500-5-120.

Conserved requirement for DEAD-box RNA helicase Gemin3 in Drosophila oogenesis

Ruben J Cauchi  1
Affiliations

Conserved requirement for DEAD-box RNA helicase Gemin3 in Drosophila oogenesis

Ruben J Cauchi. BMC Res Notes. .

Abstract

Background: DEAD-box RNA helicase Gemin3 is an essential protein since its deficiency is lethal in both vertebrates and invertebrates. In addition to playing a role in transcriptional regulation and RNA silencing, as a core member of the SMN (survival of motor neurons) complex, Gemin3 is required for the biogenesis of spliceosomal snRNPs (small nuclear ribonucleoproteins), and axonal mRNA metabolism. Studies in the mouse and C. elegans revealed that loss of Gemin3 function has a negative impact on ovarian physiology and development.

Findings: This work reports on the generation and characterisation of gemin3 mutant germline clones in Drosophila adult females. Gemin3 was found to be required for the completion of oogenesis and its loss led to egg polarity defects, oocyte mislocalisation, and abnormal chromosome morphology. Canonical Cajal bodies were absent in the majority of gemin3 mutant egg chambers and histone locus bodies displayed an atypical morphology. snRNP distribution was perturbed so that on gemin3 loss, snRNP cytoplasmic aggregates (U bodies) were only visible in wild type.

Conclusions: These findings establish a conserved requirement for Gemin3 in Drosophila oogenesis. Furthermore, in view of the similarity to the phenotypes described previously in smn mutant germ cells, the present results confirm the close functional relationship between SMN and Gemin3 on a cellular level.

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Figures

Figure 1
Figure 1
Ovarian and egg polarity defects in the absence of Gemin3. (A) Schematic of the sequential stages (S1 to S14) of egg chamber development after budding off the germarium (G) [adapted from [30]]. A mature egg (S14) exhibits asymmetry along its A-P and D-V axes. (B, C) Ovaries of gemin3R/ovoD1 female flies are shrivelled in contrast to wild type, a phenotype resulting from the early developmental arrest of gemin3R homozygous germ cells. Images are of the same magnification. (D-G) Compared to wild type, gemin3 mutant eggs are smaller in size and display a range of DA phenotypes including DAs, which are broader and shorter compared to wild type (E, arrow) as well as forked DAs that are fused at their base (F, arrow). Sometimes eggs display a net-like chorion or eggshell with no apparent D-V polarity. In D to G, images are of the same magnification.
Figure 2
Figure 2
Oocytes are incorrectly positioned on loss of gemin3. Compared to wild type in which the oocyte is always found at the posterior of stage 7 egg chambers, in the absence of gemin3, the oocyte was frequently misplaced. Egg chamber posterior is bottom left. DCP1 (A, B) and eIF4E (C, D) were used to identify the oocyte since they are expressed in all germline cells but accumulate in the oocyte (surrounded by a broken line with arrow pointing to the karyosome).
Figure 3
Figure 3
Defective chromatin organisation in post-stage 5 gemin3 mutant egg chambers. Hoescht-stained stage 8 egg chambers (posterior is top right) showing that the nurse cell chromosomes of gemin3R egg chambers retain a compact 'blob-like' morphology, instead of dissociating and decondensing to be uniformly distributed throughout the nucleus as is typical in wild type. Insert shows Hoescht-stained oocyte nucleus, which is frequently decondensed in the mutant in contrast to the wild type where it is typically condensed into a small round karyosome.
Figure 4
Figure 4
Histone locus, Cajal and U bodies in gemin3 mutant germline clones. (A, B) On staining for histone locus body marker Lsm11 (a component of the U7 snRNP), histone locus bodies were dispersed in multiple discrete loci throughout the wild type nurse cell nuclei. In contrast, in gemin3R mutant nuclei, histone locus bodies appear as single aggregates attached to the condensed chromatin. Note that due to the 3D nature of the nucleus and the single sections demonstrated here, the gemin3R mutant histone locus body aggregates sometimes appear as multiple foci, which in reality are a single structure. (C, D) Wild type polyploid nurse cell nuclei display several small coilin-rich Cajal bodies. In the majority of gemin3R mutant egg chambers, this staining pattern is absent though coilin adopts a diffuse nuclear stain. Note that although coilin foci were absent in mutant nurse cell nuclei, they can still be detected in wild type follicle cells (arrow) that envelop the mutant egg chamber, thus highlighting successful immunostaining. (E, F) TMG-stained snRNPs are concentrated in discrete spherical structures (U bodies) within the cytoplasm of wild type egg chambers. In contrast, snRNP distribution is diffuse and exhibits nuclear predominance in gemin3R germline clones.
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