Gaining diversity by AS
In the last decade, the sequencing of the genomes of various organisms, ranging from bacteria and viruses up to the human genome, has become routine. The knowledge of the exact genetic information shows one remarkable fact: the number of protein-coding genes in higher eukaryotic organisms is much smaller than the number of proteins produced.
Studies on expressed sequences and protein content in human cells suggested that there should be between 100.000 and 150.000 protein-coding genes. However, results from sequencing experiments showed that the human genome only contains around 30,000 genes.
The questions arising from these facts are:
Why is there a big discrepancy between gene and protein numbers? What is the mechanism that “causes” the complexity of higher eukaryotic organisms even with a relatively low number of genes?
There must be a way to regulate and diversify the function of genes of differentially specialised cell types. Evolution’s solution for the given problem is alternative splicing (AS), which is the most important posttranscriptional regulatory mechanism that causes proteome diversity and functional complexity. Alternative splicing means that during RNA splicing different combinations of exons are joined to create a diverse array of mRNAs from a single pre-mRNA. Resulting mature mRNAs are either non-functional or might give rise to proteins with different activities and functions.
More than 70% of the human protein-coding genes are alternatively spliced. This explains the fact that the relatively small number of ~ 30,000 genes can lead to a proteome (the total of all proteins in a cell or organism) of several hundred thousands of proteins. Another example is the fruit fly’s (Drosophila melanogaster) genome (14,000 genes) containing about 5,000 genes less than the one of a simple primal nematode (19,000 genes). An outstanding example of an alternatively spliced gene is the so called Dscam gene of the fruit fly. This one gene can be processed into about 38,000 spliced variants.
Alternative splicing patterns
Alternative splicing joins different pieces of the mRNA together to make different mRNAs from the same gene. There are many ways in which this can happen to generate functionally distinct mRNAs and proteins:
Different variants of alternative splicing events in higher eukaryotes.
The different alternative splicing events described above can occur anywhere in the pre-mRNA.
Use of cassette alternative exons
The most common type of alternative splicing events (around one third) involves cassette type alternative exons. In this case an exon is either included or excluded from the mRNA. Splicing of a cassette exon can result in the complete inclusion or loss of a specific functional protein domain.
Use of alternative splice sites
In this case alternative 5’- or 3’- splice sites in exon or introns sequences are chosen leading to the inclusion or exclusion of a part of an exon or intron. The use of an alternative splice site can lead to subtle changes in the protein activity and therefore to a fine tuning of the protein function.
Intron retention
This is when an intron is not removed from the mRNA. This can lead to the incorporation of a protein sequence or a change in the reading frame.
Mutual exclusion of exons
In this splicing event, either one or other of two exons is included in the final mRNA – both mutually exclusive exons are not found together in the mRNA.
Use of alternative promoters, poly-A sites and terminal introns
Primary RNA transcripts are - additionally to splicing - processed on their 5’- and 3’- ends (5’ capping and 3’ polyadenylation). These modifications are necessary for the protection of mRNAs and also for regulation of translation. Alternative splicing causing an alteration at any of the two RNA ends can thus lead to changes in protein production. On the other hand changes in the 5’ region of the new transcript influences subsequent alternative splicing events further downstream on the same RNA.
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