Genes contain instructions for building and maintaining an organism. These instructions are encoded in DNA, which is transcribed into RNA. For many genes, this RNA transcript, known as pre-messenger RNA (pre-mRNA), undergoes processing before it can be translated into a functional protein. One precise step in this processing is splicing, where non-coding segments called introns are removed, and the remaining coding regions, or exons, are accurately joined together. This ensures that the final protein sequence is correct.
The Splicing Challenge
The precise recognition of splice sites is important for producing functional proteins. Genes are read in sets of three nucleotides (a reading frame), and any error that adds or removes even a single nucleotide during splicing can shift this frame. Such a frameshift mutation can lead to a different protein sequence, often resulting in a non-functional protein or premature termination of protein synthesis. Human genes contain many introns, making the recognition task immense. Errors in this complex process are linked to a variety of human diseases, underscoring its importance.
Key Recognition Signals
The cellular machinery responsible for splicing relies on specific genetic landmarks to accurately identify where introns begin and end. At the boundary where an intron starts and an exon ends, there is a highly conserved 5′ splice site, or donor site, which typically contains the dinucleotide GU. The 3′ splice site, or acceptor site, which almost always features the dinucleucleotide AG. Within the intron itself, usually 18-40 nucleotides upstream of the 3′ splice site, lies a branch point, which contains an adenosine nucleotide. Directly preceding the 3′ splice site is a region rich in pyrimidine nucleotides, known as the polypyrimidine tract; these short recognition sequences are numerous throughout the genome, making their precise identification a considerable challenge for the splicing machinery.
Components of the Spliceosome
The complex pre-mRNA splicing is carried out by the spliceosome, a large and dynamic molecular machine assembled from small nuclear ribonucleoproteins, commonly referred to as snRNPs, and other proteins. Each snRNP consists of one small nuclear RNA (snRNA) molecule associated with several proteins. The spliceosome utilizes five snRNPs: U1, U2, U4, U5, and U6. These snRNPs play roles in identifying the splice sites and facilitating the biochemical reactions necessary for intron removal. Their coordinated action ensures the accurate and efficient processing of pre-mRNA.
The Recognition Process
The recognition and removal of an intron involve a step-by-step assembly of the spliceosome on the pre-mRNA. The process begins with the U1 snRNP binds to the 5′ splice site through complementary base pairing between its U1 snRNA and the pre-mRNA sequence. Subsequently, the U2 snRNP binds to the branch point adenosine within the intron, also through base pairing, which causes the branch point adenosine to bulge out, preparing it for a later reaction. A complex containing the U4, U5, and U6 snRNPs then joins the spliceosome.
This joining brings necessary components into close proximity, initiating a series of conformational changes within the spliceosome. The U6 snRNA displaces the U1 snRNA at the 5′ splice site, forming a new interaction that is important for catalysis. The U5 snRNP helps to position the exon sequences, ensuring their precise alignment for joining. The snRNAs within the spliceosome, particularly U2 and U6, form the catalytic core, acting like enzymes to facilitate two sequential transesterification reactions. These reactions first cleave the 5′ splice site, forming a lariat intermediate where the 5′ end of the intron is linked to the branch point adenosine, and then cleave the 3′ splice site, releasing the intron lariat and ligating the two exon ends.