Friday, December 6th Lecture Notes
In eukaryotes, the cell must process its primary transcripts to produce functional, translatable mRNAs
For many eukaryotic genes, protein expression is regulated at the level of RNA processing, particularly by alternative splicing
Some genes products are processed in a single, reproducible pattern:
All introns are removed
The exons flanking each intron are spliced together with a phosphodiester bond
Overview of the Splicing Reaction
Splicing occurs in two steps:
The 5’ splice site is cut, with the 5’ end of the intron forming a covalent bond to a branch site in the intron
The 3’ splice site is then cut, and the lariat-shaped intron is removed
A new phosphodiester bond is formed joining the ends of the two exons
Both reactions are catalyzed by a molecular machined called the spliceosome
However, many eukaryotic gene products undergo alternative splicing (two or more different patterns of exon usage)
In humans, it is estimated that 94% of all protein-coding genes exhibit some degree of alternative splicing
Alternative splicing permits a single pre-mRNA to produce different isoforms of the same protein
The protein troponin plays a role in muscle contraction
The gene has five potential exons, and its pre-mRNA can undergo two different patterns of splicing:
Exons 1-2-3-5 can be spliced to produce the a isoform
Exons 1-2-4-5 can be spliced to produce the B isoform
Important Message:
Alternative splicing of troponin allows a single gene to synthesize two protein isoforms that are similar but structurally and functionally distinct
Individual muscle cells can vary the ratio of a and B troponin they produce, fine-tuning the properties of the cell
When alternative splicing occurs, the spliceosome chooses to cut at some potential splice sites, while skipping over other potential splice sites
In a troponin, the downstream end of exon 2 (5’ splice site) is cut and joined to the upstream end of Exon 3 (3’ splice site)
In B troponin, the downstream end of Exon 2 (5’ splice site) is cut and joined to the upstream end of Exon 4 (3’ splice site)
Several factors influence which splice sites are actually used during alternative splicing:
Some splice sites preferentially pair with one another due to specific features of their RNA sequences
A given cell can turn splice sites “on-or-off” by expressing proteins that will bind to the pre-mRNA near that splice site
Splice repressor proteins direct the spliceosome away from a potential splice site
Splice activator proteins direct the spliceosome to use a potential splice site
Variations in Splice Site Sequence
Both 5’ and 3’ splice sites are marked by somewhat variable base sequences
In general, the spliceosome can use a splice site if its sequence is > 60% consensus
The spliceosome can preferentially pair a particular 5’ splice site to one particular 3’ splice site solely because they match in terms of their base sequences
Another important factor in alternative splicing can be the presence of 2 or more core promoters
If a gene is transcribed from multiple promoters, it will produce multiple isoforms with different 5’ exons
Like humans, the fruit fly Drosophila uses an XY system of chromosomal sex determination
Females are XX; males are XY
The X and Y chromosomes of flies are not homologous to the X and Y chromosomes of humans (they contain completely different sets of genes)
In addition, the mechanism of sex determination in flies is different from humans or other mammals
In humans, the presence or absence of a Y chromosome determines whether the embryo develops male or female characteristics
In Drosophila, it is the ratio of X chromosomes to autosomes (non-sex chromosomes) that determines gender
A female fruit fly has 2 X chromosomes and 2 copies of each autosome
1.0 ratio
A male fruit fly has 1 X chromosome and 2 copies of each autosome
0.5 ratio
Unlike humans, an XXY fly develops a female anatomy
Alternative splicing plays a central role in the sex determination of Drosophila
There are four key genes:
Doublesex (dsx)
Sex-lethal (Sxl)
Transformer (tra)
Transformer-2 (tra-2)
All of these genes are autosomes (equal in number between males and females)
The doublesex gene (dsx) encodes a zinc finger transcription factor
This transcription factor has distinct isoforms in males and females; it is the isoform of Doublesex protein expressed by the fly that causes the formation of male or female organs
The male and female isoforms of Dsx protein result from alternative splicing
The male isoform is Exon 3/5; the female isoform is exons ¾
Exon 4 gets spliced to an alternative 3’ UTR with a different polyA signal sequence
The gender-specific splicing of the dsx gene product is determined by three splice regulatory proteins:
Sex-lethal (Sxl) encodes a splice repressor protein
Transformer (tra) and transformer-2 (tra-2) encode splice activator proteins
Female Development
In a female embryo, Sxl protein binds to both its own Sxl pre-mRNA and the tra and tra-2 pre-mRNAs
In each case, this splice repressor protein prevents the inclusion of a male-specific exon in the final mRNA
The TraF and Tra2F proteins bind to the dsx pre-mRNA
These splice activator proteins attract the spliceosome to Exon 4, causing it to be included in the female isoform of Dsx protein
Male Development
In males, there is no expression of the Sxl protein
Rather, the spliceosome includes a male-specific exon in the Sxl mRNA
This male-specific exon contains an in-frame stop codon
Hence, even though the male mRNA is longer than the female mRNA, it produces a short non-functional isoform of the Sxl protein
The failure to produce functional Sxl protein also results in the inclusion of male-specific exons in the tra and tra-2 mRNAs
These exons also contain premature stop codons, and result in the production of short, functionless proteins
In the absence of functional Tra and Tra-2 proteins, Exon 1 of the dsx mRNA is spliced to Exon 3, producing the male isoform
How does the female embryo come to have Sxl protein while the male embryo does not?
It depends upon gender-specific transcription
The Sxl gene has two different core promoters
The Pe or early promoter is only used during the earliest stages of embryonic development
As the embryo matures, transcription switches to the Pm or maintenance promoter
It has a different +1 site and produces a different Exon 1
Transcription from the Pe promoter depends on the relative concentrations of activator TFs Sisterless-a (Sis-a) and Sisterless-b (Sis-b) and the repressor TF Deadpan (dpn)
The dpn gene is on an autosome, so the amount of Dpn protein is constant in the two sexes
But the sis-a and sis-b genes are on the X chromosome, so females have 2 copies of each and males only have 1 copy of each
Hence, females make twice as much activator, and are able to transcribe from the Pe promoter
Males cannot
The use of two different Sxl promoters introduces another form of alternative splicing for the Sxl pre-mRNA:
Transcripts from the early Pe promoter do not require the Sxl protein to skip over the male-specific exon
Transcripts from the maintenance Pm promoter contain a different Exon 1, which does require functional Sxl protein (splice repressor) to skip over the male-specific exon
When Sxl is transcribed from the Pe promoter in female embryos, the spliceosome skips over the male-specific exon without any need for Sxl protein
When Sxl transcription switches to Pm promoter, the pre-mRNA has a different Exon 1; but with Sxl protein present, the male exon is still excluded from the mRNA
Male embryos do not use the Pe promoter
When they begin to use Pm, they have no Sxl protein and the male-specific exon is included in the mature mRNA
Mini Study Guide
mRNAs can be selectively spliced so that each gene may have multiple isoforms
Splicing is carried out by the Spliceosome, which recognizes sequences at splice junctions and removes the intron as a lariat structure
Splicing is regulated by splice activators and splice repressors
Genes can also have alternative start sites with alternative first exons
Understand how sex determination occurs in Drosophila