Module 6. Alternative splicing

In this investigation, we will focus on tra-RB, the second isoform of the tra gene, and will explore how multiple different mRNAs and polypeptides can be encoded by the same gene. The story of tra-RB is an exciting story of sex, alternative splicing, and poison exons!

Now we can see the RNA-Seq data for males (red) and females (blue). Recall that peaks in RNA-Seq Read Coverage tracks usually correspond to the regions of the genome that are being transcribed. These two samples generally show similar RNA-Seq read coverage along the entire span of the tra gene. However, the adult female sample shows substantially lower RNA-Seq read coverage at around 9,971-10,145 (red box in Figure 3). We can also see the RNA-Seq Exon Junctions track, which shows the location of splice sites supported by the RNA-Seq data (as you saw in Module 4). Recall that the black boxes in the FlyBase Genes track are exons and the thin lines with arrowheads show the locations of the introns. Notice that the diagrams for the first and second RNA-Seq Exon Junctions have the same 5' splice site but different 3' splice sites. Let’s see what we can find out about these splice sites. First, we need to establish the reading frame for the first exon. Zoom in on the 5' end of the transcript around position 9850-9860.

Now zoom in on the location of the 5' splice site at the end of the first exon in both tra-RA and tra-RB (Figure 4). We will also be thinking about the concept of phase here. To review splicing and phase, watch the Splicing and Phase video.

The 3' acceptor site for the first intron in tra-RA is found inside the second exon of tra-RB. This intron is alternatively spliced. Alternative splicing is one way eukaryotes produce different proteins from the same coding regions of DNA. Here the alternative decision is made in a sex-specific manner; male fruit flies have targeted the spliceosome to use the first 3' acceptor site identified by the RNA-Seq Exon Junction data, while female fruit flies have targeted the spliceosome to use the second 3' acceptor site identified. This change in splicing has profound effects — in fact, it drives the programming of male and female characteristics in the developing fly. To review alternative splicing, watch the Genes and Isoforms video.

Now look at the tra-RB isoform:

The Tra protein has an important function in female Drosophila and is itself a splicing factor that regulates splicing. Careful annotation of genes, as we have done here, can provide many insights into biological control mechanisms.

Now that we know that tra is alternatively spliced to make two isoforms, tra-RA and tra-RB, and that males express one isoform while females express the other, let’s try to figure out how alternative splicing affects the polypeptides produced from translating these mRNAs. To do this, we need to produce a gene model for tra-RB and compare it to the gene model for tra-RA that you constructed in Module 5, showing where the start codons and stop codons appear in each isoform.

Use what you learned in Module 5 to construct a gene model for tra-RB. Locate the start codon, splice sites, and the stop codon. Construct the gene model below.