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12.8: Alternative Splicing

For many genes, all the introns in the mRNA are spliced out in a unique manner, resulting in one mRNA per gene. But there is a growing number of examples of other genes in which certain exons are included or excluded from the final mature mRNA, a process called alternative splicing. Some exons may be included in some tissues and not others, or may be sex‑specific, indicating some regulation over the selection of splice sites. 

Alternative splicing of pre‑mRNA means that a single gene may encode more than one protein product (e.g., sex determination in Drosophila melanogaster).

Sex determination in Drosophila melanogaster

The X to autosome ratio (X:A ratio) in the zygote will determine which of two different developmental pathways along which the fly will develop. If the X:A ratio is high (e.g. the female is XX and the X:A ratio is 1.0), the fly will utilize the female pathway; if the ratio is low (e.g. 0.5 since the male is XY), it will develop as a male.

The X:A ratio is determined by "counting" certain genes (or their expression) on the X chromosome (e.g. sisterless a, sisterless b, and runt) for the numerator and counting other genes (such as deadpan) for the denominator. All of the products of these genes are homologous to various calsses of transcription factors, consistent with at least part of the regulation of sex determination being transcriptional. However, as discussed below, alternative splicing plays a key role as well, at least in Drosophila.

The pathways have at least four steps that were defined genetically by mutations that caused, e.g. genetically female flies (high X:A) to develop as males. In each case, the same gene encodes both male and female‑specific mRNAs (and proteins), but the sex‑specific mRNAs (and proteins) differ as a result of alternative splicing. 

In all cases, the default state is male development, and some new activity has to be present to establish and maintain the female pathway. 

  1. The target of the X:A signal is the Sex-lethalgene (Sxl), which serves as a master switch gene. In early development, an X:A ratio of 1 in females leads to the activiation of an embryo-specific promoter of the Sxlgene, whereas Sxlis not transcribed in male embryos. Later in development, Sxlis transcribed in both sexes. Now the high X:A ratio leads to the skipping of an exon in the splicing of pre‑mRNA from the Sex‑lethalgene. This produces a functional Sxl protein in females. In males (default pathway), the mRNA has an early termination codon, and no functional Sxl protein is made.

 

Figure 3.3.19.

  1. A functional Sxl protein inhibits the default splicing of pre‑mRNA from the transformergene, to generate a functional Tra protein in female embryos. In the female‑specific splicing of trapre‑RNA, a 5' splice site (common to both male and female splicing) is connected to an alternative 3' splice site, thereby removing a termination codon and allowing function Tra protein to be made (Fig. 3.3.15). 
  2. The Tra protein promotes female‑specific splicing of pre‑mRNA from the tra2gene, again generating a functional Tra2 protein only in females. 
  3. Tra and Tra2 proteins promote female‑specific splicing of pre‑mRNA from the doublesexgene (dsx). In this case, the male‑specific mRNA has skipped an exon (Fig. 3.3.15). Skipping an exon requires an alteration in the splicing pattern at both the 3' splice site and the 5' splice sites surrounding the exon.
  4. The male‑specific Dsx protein blocks female differentiation and leads to male development. The female‑specific Dsx protein represses expression of male genes and leads to female development.

Some clues about mechanism

  1. Tra and Tra2 are RNA‑binding proteins related to Splicing Factor 2 (SF2). This latter protein has a domain rich in the dipeptide Arg‑Ser, which defines one type of RNA‑binding domain. SF2 is required for early steps in spliceosome assembly. The related Tra and Tra2 proteins are not required for viability, but they do regulate the specific splicing events for pre‑mRNA from dsx. 
  2. Tra2 binds in the female‑specific exon of the dsx transcript, and presumably regulates splice site selection. The binding site for Tra2 within the exon is an example of a splicing enhancer. The mechanisms by which the binding of splicing regulatory proteins (e.g. Tra, Tra2) to splicing enhancers is a very active area of research currently. 
  3. Sxl is another RNA‑binding protein that inhibits the default splicing pattern for tra pre‑mRNA.

 

Figure 3.3.20.