RNA Processing:- RNA Catalyzes the Splicing of Introns
There are four classes of introns. The first two, the group I and group II introns, differ in the details of their splicing mechanisms but share one surprising characteristic: they are self-splicing—no protein enzymes are involved. Group I introns are found in some nuclear, mitochondrial, and chloroplast genes coding for rRNAs, mRNAs, and tRNAs. Group II introns are generally found in the primary transcripts of mitochondrial or chloroplast mRNAs in fungi, algae, and plants. Group I and group II introns are also found among the rarer examples of introns in bacteria. Neither class requires a high energy cofactor (such as ATP) for splicing. The splicing mechanisms in both groups involve two transesterification reaction steps . A ribose 2- or 3 hydroxyl group makes a nucleophilic attack on a phosphorus and, in each step, a new phosphodiester bond is formed at the expense of the old, maintaining the balance of energy. These reactions are very similar to the DNA breaking and rejoining reactions promoted by topoisomerases and site-specific recombinases . The group I splicing reaction requires a guanine nucleoside or nucleotide cofactor, but the cofactor is not used as a source of energy; instead, the 3-hydroxyl group of guanosine is used as a nucleophile in the first step of the splicing pathway. The guanosine 3-hydroxyl group forms a normal 3,5-phosphodiester bond with the 5end of the intron (Fig. 26–14). The 3hydroxyl of the exon that is displaced in this step then acts as a nucleophile in a similar reaction at the 3end of the intron. The result is precise excision of the intron and lig ation of the exons. In group II introns the reaction pattern is similar except for the nucleophile in the first step, which in this case is the 2-hydroxyl group of an A residue within the intron (Fig. 26–15). A branched lariat structure is formed as an intermediate. Self-splicing of introns was first revealed in 1982 in studies of the splicing mechanism of the group I rRNA intron from the ciliated protozoan Tetrahymena thermophila, conducted by Thomas Cech and colleagues. These workers transcribed isolated Tetrahymena DNA (including the intron) in vitro using purified bacterial RNA polymerase. The resulting RNA spliced itself accurately without any protein enzymes from Tetrahymena. The discovery that RNAs could have catalytic functions was a milestone in our understanding of bio logical systems.
Most introns are not self-splicing, and these types are not designated with a group number. The third and largest class of introns includes those found in nuclear mRNA primary transcripts. These are called spliceo somal introns, because their removal occurs within and is catalyzed by a large protein complex called a spliceosome. Within the spliceosome, the introns undergo splicing by the same lariat-forming mechanism as the group II introns. The spliceosome is made up of specialized RNA-protein complexes, small nuclear ribonucleoproteins (snRNPs, often pronounced “snurps”). Each snRNP contains one of a class of eukaryotic RNAs, 100 to 200 nucleotides long, known as small nuclear RNAs (snRNAs). Five snRNAs (U1, U2, U4, U5, and U6) involved in splicing reactions are generally found in abundance in eukaryotic nuclei. The RNAs and proteins in snRNPs are highly conserved in eukaryotes from yeasts to humans.
Spliceo somal introns generally have the dinucleotide sequence GU and AG at the 5 and 3 ends, re spectively, and these sequences mark the sites where splicing occurs. The U1 snRNA contains a sequence complementary to sequences near the 5splice site of nuclear mRNA introns , and the U1 snRNP binds to this region in the primary transcript. Addition of the U2, U4, U5, and U6 snRNPs leads to formation of the spliceosome . The snRNPs together contribute five RNAs and about 50 proteins to the spliceosome, a supramolecular assembly nearly as com plex as the ribosome . ATP is required for assembly of the spliceosome, but the RNA cleavage-ligation reactions do not seem to require ATP. Some mRNA introns are spliced by a less common type of spliceosome, in which the U1 and U2 snRNPs are re placed by the U11 and U12 snRNPs. Whereas U1- and U2-containing spliceosomes remove introns with (5)GU and AG(3) terminal sequences, the U11- and U12-containing spliceosomes re move a rare class of introns that have (5)AU and AC(3) terminal sequences to mark the intronic splice sites. The spliceosomes used in nuclear RNA splicing may have evolved from more ancient group II introns, with the snRNPs replacing the catalytic domains of their self-splicing ancestors. Some components of the splicing apparatus appear to be tethered to the CTD of RNA polymerase II, suggesting an interesting model for the splicing reaction. As the first splice junction is synthesized, it is bound by a tethered spliceosome. The second splice junction is then captured by this complex as it passes, facilitating the juxtaposition of the intron ends and the subsequent splicing process . After splicing, the intron remains in the nucleus and is eventually degraded. The fourth class of introns, found in certain tRNAs, is distinguished from the group I and II introns in that the splicing reaction requires ATP and an endonuclease. The splicing endonuclease cleaves the phosphodiester bonds at both ends of the intron, and the two exons are joined by a mechanism similar to the DNA ligase reaction . Although spliceo somal introns appear to be limited to eukaryotes, the other intron classes are not. Genes with group I and II introns have now been found in both bacteria and bacterial viruses. Bacteriophage T4, for ex ample, has several protein-encoding genes with group I introns. Introns appear to be more common in archaebacteria than in eubacteria.
FIGURE 26–15 Splicing mechanism of group II introns. The chemistry is similar to that of group I intron splicing, except for the identity of the nucleophile in the first step and formation of a lariatlike intermediate, in which one branch is a 2,5-phosphodiester bond.
