Prp8

IntroductionIntroduction

Prp8 BackgroundPrp8 Background

Prp8's name originates from the discovery that it is intricately involved with pre-m RNA processing within Eukaryotic organisms, and extensive research on the protein has revealed it is indispensable for the catalytic activity of the spliceosome earning it the nickname of 'Master Regulator of the Spliceosome' . UV crosslinking experiments have shown that Prp8 has contact with the 5' splice site, 3' splice site, polypyrimidine tract, and branch point of pre-mRNA transcripts, as well as associating with all five of the snRNAs and their associated proteins . Thus, Prp8 is the only protein to date involved with pre-mRNA splicing that actually contacts all catalytic elements of the spliceosome, indicating that it indeed has a very crucial role in pre-mRNA splicing catalysis, and resides in the catalytic heart of the splicing comlex. Two other unique properties of Prp8 are its size and evolutionary conservation. The protein is approximately 230 - 280 kDa and is approximately 2335 - 2413 amino acid residues in length (depending on the species) .

Pre-mRNA SplicingPre-mRNA Splicing

Figure 1. Movements of the snRNPs and steps in pre-mRNA splicing catalysis. The two rectangles represent exons and the piece initially connecting them is an intron.

Pre-mRNA splicing is carried out by one of the most complex pieces of cellular machinery known to date; the Spliceosome. It is a large, catalytic protein-RNA complex responsible for removing the intronic sequences of pre-mRNA and “splicing” together the exonic sequences to form mature mRNA. The spliceosome is made up of approximately 145 distinct spliceosomal proteins, and five small nuclear RNAs (snRNAs) . The five snRNAs have been subsequently named U1, U2, U4, U5, and U6. The spliceosome carries out a two-step transesterification reaction through a series of chemical steps in order to remove the intronic sequences from the pre-mRNA, which is facilitated by the movements, rearrangements, and dynamic exchanges of the snRNAs and splicing associated proteins .

First, the U1 snRNP recognizes and binds to the 5’ splice site of the pre-mRNA and the U2 snRNP recognizes and binds to the branch site of the intronic sequence, which contains a conserved adenine nucleotide, which is collectively known as complex A . Subsequently, the U4/U6 U5 tri-snRNP moves in and associates with the intronic sequence (now known as complex B). This causes the U4 and U1 snRNPs to be displaced, forming the catalytically active spliceosome (complex B*) and the remaining U2, U5, and U6 snRNP facilitate the two transesterification reactions, dissociate from the now mature mRNA, and are recycled for subsequent use in another splicing reaction .

As stated above, the chemical mechanism pre-mRNA splicing follows is a two-step transesterification reaction. With the release of U1 and U4, following the subsequent rearrangement of the remaining snRNPs, the 2’ hydroxyl of the conserved adenine nucleotide carries out a nucleophilic attack on the 5’ splice site, which cuts the backbone of the mRNA, freeing the first exon . Now the 5’ end of the intron is covalently bonded to the adenine nucleotide, forming a lariat structure . Finally, the 3’ OH of the first exon nucleophilically attacks the 5’ end of the second exon, displacing the intron and splicing the exons together . This is followed by the subsequent release of the lariat structure and the remaining spliceosome dissociates .