Specific isoforms of Squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis

Abstract

Heterogeneous nuclear RNA-binding proteins, hnRNPs, have been implicated in nuclear export of mRNAs in organisms from yeast to humans. A germ-line mutation in a Drosophila hnRNP, Squid (Sqd)/hrp40, causes female sterility as a result of mislocalization of gurken (grk) mRNA during oogenesis. Alternative splicing produces three isoforms, SqdA, SqdB, and SqdS. Here we show that these isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in grk mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. We show that this differential nuclear accumulation is correlated with a differential association with the nuclear import protein Transportin. Finally, we provide evidence that grk mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. These results demonstrate the isoform-specific contributions of individual hnRNP proteins in the regulation of a specific mRNA. Moreover, these data suggest a novel role for hnRNPs in localization and translational regulation of mRNAs.

mRNA Localization and ER-based Protein Sorting Mechanisms Dictate the Use of Transitional Endoplasmic Reticulum-Golgi Units Involved in Gurken Transport inDrosophilaOocytes

“…That mislocalized gurken mRNA was able to induce the local synthesis, transport, and processing of full-length Gurken protein was suggested by Serano et al (1995) and Norvell et al (1999). Our results show here that this is sustained by polarized exocytosis through selected tERGolgi units (and post-Golgi compartments) situated in proximity to, and chosen by, gurken mRNA.…”

“…When transfected with Brho alone, Gurken is cleaved but not secreted, and with Star alone, Gurken is neither processed nor secreted (Ghiglione et al, 2002). Because both in squid and K10 mutants Gurken protein is transported all tER-Golgi units along the anterior side and at the ventral/anterior corner (our observations) and is found in the space between the oocyte and the nurse cells (Serano et al, 1995;Norvell et al, 1999;our observations), this suggests that at least Star and Brho are present in all tER-Golgi units, including these away from the D/A corner, and act in the processing and transport of Gurken. Along the same line, Star, Cornichon, and Brho are also likely localized to the tER-Golgi units at the posterior pole, in WT stage 6 -7 and stage 9 -10 merlin oocytes where Gurken protein is synthesized, transported and processed, so it signals to the posterior follicle cells.…”

“…gurken mRNA localizes to an asymmetric ring along the anterior side of the oocyte with a weaker intensity toward the ventral side. The translation and deposition of Gurken protein is nevertheless sustained (Serano et al, 1995;Norvell et al, 1999; Figure 6). As ex- Yolkless is transported in all tER-Golgi units.…”

“…This suggests that mRNA localization can indeed dictate the usage of tERGolgi units located next to it, in the D/A corner for a stage 9 -10 WT oocyte, and at the posterior pole for an equivalent merlin oocyte. To test this prediction further, we used the fs(1)K10 and squid 1 mutants, where gurken mRNA is no longer restricted to the D/A corner, but delocalized along the anterior cortex and at the ventral/anterior corner (Serano et al, 1995;Norvell et al, 1999;Figure 6, A and E). gurken mRNA localizes to an asymmetric ring along the anterior side of the oocyte with a weaker intensity toward the ventral side.…”

“…Mutant fly stocks used are dCOG5-GFP (w;GFP-Fws/Cyo) (Farkas et al, 2003); PDI-GFP (PDI::GFP/GFP-TM3,Sb) (Bobinnec et al, 2003) (Norvell et al, 1999); cni AR55 (b cni AR55 pr cn/Cyo), cni CF5 (b cni CF5 pr cn wx bw/Cyo) and cni AA12 (b cni AA12 /Cyo) (Roth et al, 1995); grk 2B6 (grk[2B6]b cn s L bo/Cyo) and grk 2E12 (w; grk[2E12]b/Cyo) (Neuman-Silberberg and Schupbach, 1993); and fly lines expressing Gurken lacking the transmembrane and the cytoplasmic domain g⌬TC4.1 and the cytoplasmic domain g⌬C100 (Queenan et al, 1999). The stocks carrying the constructs encoding the truncated versions of Gurken proteins were crossed into the grk 2B6 background and females from this cross were mated with male grk 2E12 to yield orange-eyed black flies (g⌬TC4.1; grk 2B6 /grk 2E12 and g⌬C100; grk 2B6 /grk 2E12 ).…”