A stem–loop structure directsoskarmRNA to microtubule minus ends

Abstract

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem-loop in the oskar 3 ′ UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein-and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.

oskar RNA plays multiple noncoding roles to support oogenesis and maintain integrity of the germline/soma distinction

“…A possible explanation is that the osk mRNA must be concentrated in the oocyte for at least one aspect of its function. Only the central fragment, within which an oocyte entry signal (OES) has been mapped (Jambor et al 2014), conferred oocyte enrichment. Testing larger fragments, containing the central region and either 5 ′ or 3 ′ flanking sequences, showed that the combination of the central and 5…”

“…Some of the UAS-egfp transgenes with osk 3 ′ -UTR sequences (Fig. 2) are from Jambor et al (2014). For the new transgenes, the osk 3 ′ -UTR segments used are provided in the Supplemental Materials.…”

“…In situ hybridization to detect osk or egfp mRNA in ovary wholemount preparations was performed as previously described (Jambor et al 2014). For the K10-TLS 119 of 3b transcripts, the probe was from transcribed UASp vector sequences (Supplemental Materials).…”

mRNA localization in theDrosophilagermline

“…Increasingly data also suggests that localization-incompetent RNA molecules can hitch-hike on transport particles. [76][77][78] The osk 3 0 UTR mediates hitch-hiking, presumably through RNA-RNA interactions, however the full details of how an RNA is recognized, attached and released from a transport particle is yet to be explained.…”

Multiple cis-acting signals, some weak by necessity, collectively direct robust transport of oskar mRNA to the oocyte

“…Sequences contributing to the function of this element have been more-precisely mapped to part of a stem-loop structure which resembles the TLS in having an AU-rich stem. Mutation of the distal part of the stem disrupts transport of a reporter mRNA and compensatory changes restore activity, defining the oocyte entry signal (OES) (Jambor et al, 2014). Despite some structural similarity between the TLS and the OES, the OES is depauperate by comparison: it is not active in isolation and directs transport of a reporter mRNA only in the context of larger regions of the osk 3′UTR (Bullock and Ish-Horowicz, 2001;Jambor et al, 2014).…”