Splice sites (exon-intron junctions) are located by comparing genomic DNA with the cDNA prepared from the corresponding mRNA. Sequences present in genomic DNA but not in the cDNA are introns, and the splice sites may be inferred. Correct splicing only requires 3040 nucleotides at at each end of an intron, while the middle portion may be removed without detrimental effects.
Splicing of pre-mRNAs occurs via two transesterification reactions. Introns are removed as a lariat-like structure in which the 5' G of the intron is joined in an unusual 2',5'-phosphodiester bond to an adenosine near the 3' end of the intron. This A residue is called the branch point because it forms an RNA branch in the lariat structure. In each transesterificaiton reaction, one phosphoester bond is exchanged for another.
Five U-rich small nuclear RNAs (snRNAs) desginated U1,2,4,5,6 particiapate in pre-mRNA splicing. Between 107 to 210 nucleotides, these snRNAs associate with 6-10 proteins in small nucelear ribonucleoprotein particles (snRNPs) in eukaryotic nuclei.
5' end of U1 snRNA base-pairs with the 5' splice site of a pre-mRNA. The 5 snRNPs assemble on the pre-mRNA to form a ribonucleoprotein complex called the spliceosome. U1 and U2 bind to pre-mRNA, while U4 and U6 bind to each other and then to U5. The U4,5,6 complex binds to the U1,2-pre-mRNA complex. This yields a spliceosome.
U1 and U4 are then released following spliceosome rearrangement, and the new spliceosome mediates the first transesterificaiton reaction where a 2',5'-phosphodiester bond is formed between the 2' hydroxyl on the branch point A and the phosphate at the 5' end of the intron. Following another snRNP rearrangment, the second transesterification reaction ligates the two exons together in a standard 3',5'-phosphodiester bond.
The intron is released as a lariat associated with snRNPs; this complex rapidly disassociates and the snRNPs can catalyze another splicing reaction, while the intron is degrade by a debranching enzyme and nuclear RNases.
Complementary Mutation Experiment
The observation that the sequence near the 5’-end of U1 snRNA is complementary to the consensus 5’ splice site sequence of pre-mRNAs led to the hypothesis that the U1 snRNP is the cellular molecule that recognizes 5’ splice sites during splicing. An experiment that proved that this base pairing is indeed required for RNA splicing was the complementary mutation experiment. It involves making a mutation to eliminate a function, followed by reversing the mutation restores function. This identifies conserved sequences needed for functionality. For example, the 5′ splice site was formed with a point mutation and splicing activity was lost; the mutation was reversed and splicing activity was restored. Obviously, splicing is regulated at this sequence.