Splicing Factor Interactions in Drosophila melanogaster

I. Introduction

In pre-mRNA transcripts there are segments of protein-coding sequences called exons, as well as a large number of intervening non-coding sequences called introns. RNA splicing is the process by which the cell removes introns from the primary transcripts of protein-coding mRNAs. These introns must be excised prior to the translation of the mRNA. The splicing reaction occurs in two steps. First is a cleavage at the 5' site, the splice donor site. The next step is the cleavage at the 3' site, the splice acceptor site, where the exons are ligated. A series of transesterification reactions in which hydroxyl groups are displaced and phosphodiester bonds are formed takes place within spliceosomes. Spliceosomes are composed of a variety of splicing factors necessary to facilitate this process. Although RNA splicing is a universal process, many of the splicing factors that make it possible are still unknown. Manipulating RNA splicing is an effective technique for learning about genes whose functions are unclear or undefined. This experiment aims to determine whether the functions of certain genes present in the Drosophila melanogaster genome are related to RNA splicing factors that are essential in the organism's development.
One gene that plays a vital role in the physical development of D. melanogaster is the Ultrabithorax (Ubx) gene. The function of Ubx is to turn specific genes on and off in order to regulate efficiently the synthesis of other developmentally important proteins. The Ubx
pre-mRNA is spliced cotranscriptionally to produce six functionally distinct mRNA isoforms (see Figure 1 on the following page). In the Ubx pre-mRNA, the first microexon can be spliced to one of two competing splice donor sites at the end of E5' to include or to exclude the B element. This splicing regenerates a donor site at the exon-exon boundary, such that the next splicing event again has to choose between two competing donor sites. These alternative splicing decisions are regulated both temporally and spatially (see Figure 2). The Ubx mRNAs are subsequently translated into Ubx proteins that are important for proper development. The homeodomain must be included for the UBX to be functional. Specifically Ubx proteins control the development in the third thoracic segment, where the halteres are located. Proper haltere development requires UBX protein. In contrast, wings develop on the second thoracic segment where UBX is not expressed. Therefore, a loss-of-function mutation causes a transformation of the third thoracic segment into the second thoracic segment.

Figure 1: Ubx is spliced into six different protein isoforms. These isoforms contain
common 5' and 3' exons (white), and three variable regions consisting of a b element (black), microrexon I (checkered), and microexon II (hatched). The homeodomain (HD) is shaded in gray.
In the investigation of Ubx pre-mRNA processing, the activity of the SR protein B52 has been brought to attention. SR proteins are characterized by a C-terminal serine/arginine rich domain and an N-terminal RNA recognition motif (RRM). These proteins interact with pre-mRNA by enhancing the binding of snRNP proteins and regulating the activities of other SR proteins. B52, for example, an SR protein that acts on numerous pre-mRNAs in many different tissues by regulating the choice between two competing donor splice sites. Previous experiments have shown that B52 is one of the proteins that regulates the Ubx alternative splicing mechanism.
B52 is not the only protein involved in Ubx splicing. Minutes are mutations found throughout the fly genome whose functions remain undetermined. They are characterized by short, fine bristles, delayed development, and reduced viability. Although relatively little is known about the function of Minutes, several have been found to encode ribosomal proteins and other proteins involved in translation. This suggests that the Minutes may have a role in general cellular processes. Mutations in the known splicing factor suppressor of white apricot (su(w^a)) result in a thin bristle phenotype like that of the Minutes, implying that some of the Minutes may encode splicing factors.
Drosophila carrying a B52^ED mutation were used to test specifically whether Minutes work with B52 to regulate Ubx splicing. This antimorphic allele of B52 contains a mutation in the RNA binding domain which produces a change in the protein's function. B52^ED is a recessive lethal and results in RNA processing defects.

Figure 2: Ubx Splicing Model
In this project, flies carrying the antimorphic allele B52^ED were crossed with particular Minutes. A synthetic lethal screen shows whether the Minute gene products interact with B52 in the splicing machinery. If the progeny of the crosses survive to maturity, then the product of the mutated gene (i.e. a Minute) is not necessarily involved in splicing, although the assessment of the Ubx haltere phenotype may reveal a Minute's subtle role in splicing. On the other hand, if the progeny of such a cross die before adulthood, the mutated gene (i.e. a Minute) may act with B52 to regulate Ubx splicing.
Even if the mutation of the gene in combination with the mutant B52 does not kill the fly, the observation of certain phenotypic changes may also indicate a role for the Minute in splicing. Since the role of B52 in Ubx splicing specifically affects the third thoracic segment of D. melanogaster, it is necessary to observe the phenotypic traits of this region. Due to the Ubx gene's role in haltere development, an increase or decrease in the number of bristles on the haltere of the fly will indicate changes in the Ubx splicing pattern. Since wildtype halteres do not have bristles, bristles on the haltere imply a loss-of-function of the UBX protein.
In order to observe alterations to phenotype due to modification of the Ubx splicing pattern, two different mutations of Ubx, Ubx ¹⁹⁵ and Ubx ^9.22, were tested using a synthetic lethal screen. Since the screen was being performed in a heterozygous Ubx mutant background, mutations in the splicing factors affected both the Ubx and the wildtype transcripts. Ubx ¹⁹⁵ contains a stop codon in the second microexon which restricts the synthesis of the functional proteins to that of isoform IV (see Figure 3). In the Ubx¹⁹⁵background, the presence of mutant splicing factors will alter the Ubx isoform concentrations by changing splicing of the mutant and wildtype transcripts. In the presence of mutant splicing factors, the splicing pattern of Ubx¹⁹⁵ can be altered to make more or less isoform IV, which will subsequently change the extent of the Ubx phenotype because haltere development is acutely sensitive to the amount of isoform IV present. In the Ubx^9.22mutant, however, the presence of a mutant splicing factor can only change the Ubx isoform concentration by changing splicing of the wildtype transcript. Ubx ^9.22 contains a deletion which removes part of the 3' exon including the homeodomain. Since Ubx^9.22cannot make any functional protein, a mutation in a splicing factor cannot modify the Ubx^9.22splicing pattern. However, it can modify splicing of the wildtype chromosome in these heterozygotes. Therefore, the use of both Ubx¹⁹⁵and Ubx^9.22 provides two different levels of sensitivity to the screen.

Figure 3: Ubx mutant alleles used in screens for trans-acting regulators of Ubx alternative splicing. Exons are indicated as in Figure 1. Ubx¹⁹⁵ contains a stop codon in the second microexon, and Ubx^9.22 contains a deletion that removes parts of the third intron and E3', including most of the homeodomain (HD, shaded in gray).