Question

How is an intron spliced out of a pre mRNA transcript to give rise to a mature mRNA? Explain with detail.

Answer 1

Most genes in higher eukaryotes are composed of exons and introns. The introns must be excised and the exons linked to form the final mRNA in a process called splicing. The consensus sequence at the 5’ splice site in eukaryotes is AGGUAAGU. At the 3’ end of an intron, the consensus sequence is a stretch of 10 pyrimidines (U or C), followed by any base and then by C, and ending with the invariant AG. Introns also have an important internal site located between 20 and 50 nucleotides upstream of the 3’ splice site; it is called the branch site. Parts of introns other than the 5’ and 3’ splice sites and the branch site appears to be less important in determining where splicing takes place. The length of introns ranges from 50 to 10,000 nucleotides.

The process: In brief

Transesterification Reactions

Several small RNAs and proteins that form a large complex called a spliceosome. Splicing begins with the cleavage of the phosphodiester bond between the upstream exon (exon 1) and the 5’ end of the intron. The attacking group in this reaction is the 2 -hydroxyl group of an adenylate residue in the branch site. A 2’, 5’ -phosphodiester bond is formed between this A residue and the 5 terminal phosphate of the intron (this adenylate residue is also joined to two other nucleotides by normal 3’, 5’-phosphodiester bonds). A branch is generated at this site, and a lariat intermediate is formed. The 3 -OH terminus of exon 1 then attacks the phosphodiester bond between the intron and exon 2. Exons 1 and 2 become joined, and the intron is released in lariat form. Again, this reaction is a transesterification. Splicing is thus accomplished by two transesterification reactions rather than by hydrolysis followed by ligation. The first reaction generates a free 3 -hydroxyl group at the 3 end of exon 1, and the second reaction links this group to the 5 -phosphate of exon 2. The number of phosphodiester bonds stays the same during these steps, which is crucial because it allows the splicing reaction itself to proceed without an energy source such as ATP or GTP.

The process: In detail

Small Nuclear RNAs in Spliceosomes Catalyse the Splicing of mRNA Precursors

The nucleus contains many types of small RNA molecules with fewer than 300 nucleotides, referred to as snRNAs (small nuclear RNAs) essential for splicing mRNA precursors. These RNA molecules are associated with specific proteins to form complexes termed snRNPs (small nuclear ribonucleoprotein particles OR "snurps."); Spliceosomes are large (60S), dynamic assemblies composed of snRNPs, composed of proteins called splicing factors, and the mRNA precursors being processed. Splicing begins with the recognition of the 5 splice site by U1 snRNP which base pairs to the 5’ splice site of the pre-mRNA making the spliceosome assembly. U2 snRNP then binds the branch site in the intron by base-pairing between a highly conserved sequence in U2 snRNA and the pre-mRNA. A preassembled U4-U5-U6 complex joins this complex of U1, U2, and the mRNA precursor to form a complete spliceosome. First, U5 interacts with exon sequences in the 5’ splice site and subsequently with the 3’ exon. Next, U6 disengages from U4 and undergoes an intramolecular rearrangement that permits base-pairing with U2 and displaces U1 from the spliceosome by interacting with the 5’ end of the intron. The U2 and U6 snRNAs form the catalytic center of the spliceosome. U4 serves as an inhibitor that masks U6 until the specific splice sites are aligned. These rearrangements result in the first transesterification reaction, generating the lariat intermediate and a cleaved 5 exon. Further rearrangements of RNA in the spliceosome facilitate the second transesterification. These rearrangements align the free 5 exon with the 3 exon such that the 3 -hydroxyl group of the 5 exon is positioned to nucleophilically attack the 3 splice site to generate the spliced product. U2, U5, and U6 bound to the excised lariat intron are released to complete the splicing reaction.