It is well known now that eukaryotic genomes contained gene sequences that are expressed (exons) and intervening sequences that are unexpressed, introns. How are introns removed from the genetic code, and how are intron sequences recognized?

Transcription of DNA occurs wholly. So, when messenger RNA is built by RNA polymerase from the DNA template, the introns are included in this sequence. After mRNA is capped, and elongation of transciption begin intron splicing can occur. After introns have been spliced, the product is mature mRNA, which is then polyadenylated.

Intron-exon junctions studies show that there is a high degree of homology between eukaryotes, the consensus sequence being an invariant GU sequence at the 5′ intron boundary and an AG invariant sequence at the 3′ boundary. These two sequences are all that is required to define a splicing boundary (as we’ll see later).

There are actually 4 different types of introns, but I will focus on the first two types, which work via two general mechanisms, known as Group 1 and Group 2 (creative, I know). Type 1 introns are self splicing and are employed in nuclear, mitochondrial and chloroplast rRNA, tRNA and mRNA. Type 2 introns are also self splicing, and are used in genes in mitochondria and chloroplasts of fungi algae and plants. Type 3 introns require a spliceosome and small nuclear Ribonucleo Proteins (snRNPs) and are responsible for solely eukaryotic splicing. Type 4 introns require ATP and an endonuclease.

As can be seen in the figure, in Type 1 introns, the 3′ OH group of a free guanosine acts a nucleophile, attacking the 5′ phosphate at the splice site, displacing it from the exon. The 3′ OH at the exon, usually a U, acts as a nucleophile, atacking the phosphodiester bond, removing the other end of the intron, and reforming the phosphodiester bond with itself.

In Type II introns, a similar mechanism is used, except that an internal 2′ OH of an adenoside is used as the nucleophile, attacking the 5′ splice site of the exon to produce a lariat structure. In the final step, the 3′ OH of the 5′ exon attacks the phosphodiester bond of 3′ uracil, to remove the lariat.

In type III introns, a lariat structure is formed, but the snRNPs are used to form the secondary structures and to mediate the reaction, though it still occurs without free energy input. There are 6 snRNP subunits that make up the splicesome.

Intron sequences are recognized by short sequence elements within exons, known as exonic sequence/splicing enhancers (ESEs). But the mechanism by which they function is poorly understood. The fact that the splicing process occurs contemporaneously with RNA polymerase transciption elongation allows for the immediate splicing upon the recognition of an exon-intron junction sequence, and may account for the high accuracy of this system. Additionally, some evidence suggests that the C-terminal domain of RNAP II itself helps to tether the exon and splicing machinery together.

This entry was posted on Tuesday, April 22nd, 2008 at 8:35 pm and is filed under biochemistry. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.