Miranda mediates asymmetric protein and RNA localization in the developing nervous system - PubMed (original) (raw)

Miranda mediates asymmetric protein and RNA localization in the developing nervous system

A J Schuldt et al. Genes Dev. 1998.

Abstract

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of the prospero 3' UTR. Staufen protein and prospero RNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein and prospero mRNA during neuroblast divisions, and neither Staufen nor prospero RNA are localized in miranda mutants. Thus Miranda, which localizes Prospero protein, also localizes prospero RNA through its interaction with Staufen protein.

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Figures

Figure 1

Staufen binds to the prospero 3′ UTR in vivo. Injection of the bcd 3′ UTR into the precellularization embryo has been shown to recruit Staufen into ribonucleoprotein particles at the site of injection (Ferrandon et al. 1994). We tested whether the prospero 3′ UTR also interacts with Staufen in this assay. In vitro-transcribed RNA was injected into living embryos expressing two different GFP fusion proteins: GFP–Staufen and Tau–GFP (Brand 1995; Micklem et al. 1997). (a) Full-length prospero 3′ UTR RNA (1.5 kb) recruits Staufen to the site of injection within 5–10 min (arrowhead). (b) A similar result is obtained with a 650-nucleotide segment from the 3′ end of the prospero 3′ UTR (arrowhead). (c) Conversely, an 800-nucleotide fragment from the 5′ end of the prospero 3′ UTR recruits almost no Staufen to the site of injection, even after a 20–30 min incubation (arrowhead). (d) The ribonucleoprotein particles containing GFP–Staufen and the prospero 3′ UTR cluster on the basal side of the nuclei and migrate with them as they move to the cortex of the embryo (arrowheads; 650-nucleotide fragment of the prospero 3′ UTR was injected). (e) Although the RNP particles are distributed over the nuclei, there is no apparent association with astral microtubules (labeled with Tau–GFP).

Figure 2

Staufen, Prospero, and Miranda colocalize throughout the cell cycle. Staufen (in green; a,c,e,g), Prospero (in blue; a,c,e,g), and Miranda (in green; b,d,f,h) colocalize in neuroblasts. At interphase, all three proteins are found on the apical side of the cell (a,b); at prophase, they form a crescent at the basal cortex (c,d), which is maintained at metaphase (e,f) and anaphase (g,h) before segregation into the GMC. Interestingly, Staufen is often also associated with the centrosome on the apical side of the neuroblast at anaphase (arrowhead in g), as is Miranda (see Fig. 5j). DNA is labeled in red in all panels; basal is up, apical down.

Figure 3

Deletion of dRBD5 eliminates Staufen localization. Deletion of the loop in dRBD2 of Staufen has little effect on protein localization throughout the cell cycle (a,c,e,g). At interphase, StaufenΔdRBD2 concentrates on the apical side of the cell (a) before moving to the basal cortex during mitosis (c,e,g). Deletion of dRBD5, however, prevents Staufen localization to either the apical side of the neuroblast at interphase (b), or the basal side at prophase (d), metaphase (f), or anaphase (h). Staufen is labeled in green, Prospero in blue, and DNA in red in all panels; basal is up, apical down.

Figure 4

Staufen interacts with Miranda. (a) miranda encodes a protein of 830 amino acids (open rectangle in a, marked 0–830 amino acids), within which are four potential coiled–coil domains (solid boxes). The carboxy-terminal 103 amino acids are deleted in mirandaRR127 (open rectangle, 0–727 amino acids). By use of Staufen dRBD5 as bait in a yeast two-hybrid screen, we isolated six clones, all of which encode amino acids 506–776 of Miranda (black line). When amino acids 820–1026 of Prospero were used as bait in a similar screen, Shen et al. (1997) isolated amino acids 300–830 of Miranda. Ikeshima-Kataoka et al. (1997) used amino acids 1–1403 of Prospero as bait and recovered a fragment of Miranda encoded by amino acids 547–830. (b) Only yeast expressing both Staufen dRBD5 and Miranda amino acids 506–776 activate expression of lacZ (blue colony). Coexpression of either Staufen dRBD1 with Miranda, Staufen dRBD5 with the pVP16 vector, or Staufen dRBD1 with the pVP16 vector, does not activate transcription (white colonies). (c) 35S-Labeled Staufen dRBD5 interacts in vitro with GST–Miranda amino acids 506–776 (first lane) and GST–Miranda amino acids 506–638 (second lane) but not with GST–Miranda amino acids 639–776 (third lane) or GST alone (fourth lane).

Figure 4

Figure 5

The subcellular localization of Staufen requires Miranda. Miranda localizes normally throughout the cell cycle in staufenD3 mutants (a,d,g,j; Miranda labeled in green, DNA in red). Staufen subcellular localization, however, is absolutely dependent on Miranda (b,e,h,k; Staufen labeled in green, Prospero in blue, DNA in red). In mirandaRR127, Staufen is appropriately localized, but concentrates more strongly at the apical side of the neuroblast at interphase (arrowhead in c; Staufen labeled in green, Prospero in blue, DNA in red), and forms a weaker basal crescent at mitosis (i,l). miranda mRNA (labeled in red, DNA in green) localizes predominantly on the apical side of the neuroblast throughout the cell cycle in both wild-type (m) and staufenD3 embryos (n).

Figure 6

Miranda is required to localize prospero mRNA. prospero mRNA localizes on the apical side of neuroblasts at interphase (arrow in a) and moves to the basal cortex during mitosis (arrowhead in b). In the absence of Miranda, the RNA is evenly distributed in all neuroblasts (c,d,e). The arrowheads in c and d indicate individual neuroblasts. prospero mRNA is labeled in red, DNA in green.

Figure 7

Miranda mediates the asymmetric segregation of Prospero, Staufen, and prospero mRNA. (Top) In wild-type neuroblasts, Miranda forms a complex with Prospero protein, Staufen and prospero mRNA, on the apical side of the cell at interphase. The complex moves to the basal side of the cell during mitosis, where it is anchored at the cortex prior to segregation into the GMC. Miranda is then degraded and Prospero enters the GMC nucleus. (Bottom) In the absence of Miranda, Staufen and prospero mRNA are randomly distributed in the neuroblast cytoplasm at interphase, whereas Prospero enters the nucleus. During mitosis, all three factors are evenly partitioned between the neuroblast and the budding GMC. After cytokinesis, Prospero enters the nucleus of both the neuroblast and the GMC, whereas Staufen and prospero mRNA remain randomly distributed in the cytoplasm of both cells.

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Miranda mediates asymmetric protein and RNA localization in the developing nervous system - PubMed (original) (raw)