BIOLOGICAL OVERVIEW

Miranda was identified in a search for proteins that interact with Prospero. Prospero was used as bait to screen a Drosophila library of cDNAs using a yeast two-hybrid system (Shen, 1997). In the yeast two-hybrid system two hybrid proteins are used. The first hybrid protein, termed the 'bait,' is a hybrid between the DNA binding domain of yeast transcription factor LexA, and in this case, the Prospero protein. The bait lacks a transcriptional activation domain. The second hybrid protein is derived from a Drosophila cDNA library (containing sequences that may code for proteins interacting with the bait) tagged with a transcriptional activation domain. Protein interaction between the bait and the second hybrid protein gains the ability to activate transcription of a reporter gene. Miranda, the protein identified as interacting with Prospero protein, is named after Prospero's daughter and companion in exile, characters in Shakespeare's play, The Tempest.

Like Prospero and Numb, Miranda protein is asymmetrically localized during mitosis. At this stage, Miranda colocalizes with Prospero to the basal cell membrane of each neuroblast At the end of telophase, both proteins are segregated into the ganglion mother cell (GMC), the basal daughter of the neuroblast. Shortly after cell division in the GMC, Prospero is released from the membrane and translocated into the nucleus, whereas Miranda becomes undetectable. Therefore, the distribution of Miranda may be controlled by rapid delocalization or degradation in a cell-cycle dependent manner (Shen, 1997).

In embryos lacking Miranda, Prospero fails to become localized and is equally distributed to both progenies of the neuroblast. It is concluded that Miranda is required for the asymmetric localization of Prospero. In embryos homozygous for a null allele of inscuteable, both Miranda and Prospero are unable to form crescents or they form crescents that are randomly localized along the cell membrane. Therefore Miranda crescent formation and localization requires inscuteable. Although Numb is found to interact with Miranda, Numb is localized normally in Miranda deficient embryos. It is still possible that Miranda is sufficient for Numb asymmetric localizaton. It is also possible that the maternal contribution of Miranda is sufficient for Numb localization (Shen, 1997).

It is likely that Miranda is degraded in a cell-cycle-dependent manner. There are four potential destruction boxes in the Miranda protein. The destruction box is a 9-amino-acid motif conserved among the N termini of A- and B-type cyclins (King, 1996). The destruction boxes are responsible for the cell-cycle-dependent degradation of A- and B-type cyclins by an ubiquitin-dependent pathway in anaphase during mitosis (Yamamoto, 1996), whereas Miranda staining disappears later, shortly after mitosis (Shen, 1997).

GENE STRUCTURE

The short transcript has a 90-nucleotide internal deletion and codes for a protein of 800 amino acids (Shen, 1997).

Bases in 5' UTR - 323

Bases in 3' UTR - 161

PROTEIN STRUCTURE

Amino Acids - 830 (from the long transcript) and 800 (from the short transcript)

Structural Domains

The Miranda protein shows no significant homology with any known protein in computer databases. The central portion of the Miranda protein is predicted to contain several coiled-coil structures, which have been implicated in mediating protein-protein interactions. There are four potential destruction boxes in the Miranda protein and therefore, Miranda may be degraded in a cell-cycle dependent fashion (Shen, 1997).

date revised: 28 August 97  

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