12.6: Splicing of introns in pre‑mRNAs

1. Splice Sites

The sequence at the 5' and 3' ends of introns in pre-mRNAs is very highly conserved. Thus one can derive a consensus sequence for splice junctions.

5' exon...AG'GURAGU.................YYYYYYYYYYNCAG'G....exon

The GU is the 5' splice site (sometimes called the donor splice site) and the AG is the 3' splice site (or acceptor splice site). GU is invariant at the 5' splice site, and AG is (almost) invariant at the 3' splice site for the most prevalent class of introns in pre-mRNA.

Effects of mutations at the splice junctions demonstrate their importance in the splicing mechanism. Mutation of the GT at the donor site in DNA to an AT prevents splicing (this was seen in a mutation of the b‑globin gene that caused b0 thalassemia.) A different mutation of the b‑globin gene that generated a new splice site caused an aberrant RNA to be made, resulting in low levels of b‑globin being produced (b+ thalassemia).

2. The intron is excised as a lariat

The 2'‑OH of an A at the "branch" point forms a phosphoester with the first G of the intron to initiate splicing. Splicing occurs by a series of phosphoester transfers (also called trans‑esterifications). After the 2'-OH of the A at the branch has joined to the initial G of the intron, the 3'‑OH of the upstream exon is available to react with the first nucleotide of the downstream exon, thereby joining the two exons via the phosphoester transfer mechanism.

c. Intron lariat is the equivalent of a "circular" intermediate.

Figure 3.3.16. Splicing of precursor to mRNA excises the intron as a lariat structure. The chemical reactions are two phosphoester transfers. The first transfer is initiated by the 2’ hydroxyl of the adenine ribonucleoside at the branch point, which attacks the 5’ phosphoryl of the 5’ splice site. This generates a 3’ hydroxyl at exon 1 and joins the A at the branch point to the U at the 5’ splice site, producing a lariat in the intron. The second transfer is initiated by the attack of the newly exposed 3’ hydroxyl of exon 1 on the 5’ phosphoryl of exon 2. The latter reaction joins the two exons and releases the intron as a lariat.

The sequence at the branch point is only moderately conserved in most species; examination of many branch points gives the consensus YNYYRAG. It lies 18 to 40 nucleotides upstream of the 3' splice site.

3. Small nuclear ribonucleoproteins (or snRNPs) form the functional splicesome on pre‑mRNA and catalyze splicing.

a. "U" RNAs and associated proteins. Small nuclear RNAs (snRNAs) are about 100 to 300 nts long and can be as abundant as 105 to 106 molecules per cell. They are named U followed by an integer. The major ones involved in splicing are U1, U2, U4/U6, and U5 snRNAs. They are conserved from yeast to human. The snRNAs are associated with proteins to form small nuclear ribonucleoprotein particles, or snRNPs. The snRNPs are named for the snRNAs they contain, hence the major ones involved in splicing are U1, U2, U4/U6, U5 snRNPs.

One class of proteins common to many snRNPs are the Sm proteins. There are 7 Sm proteins, called B/B’, D1, D2, D3, E, F, G. Each Sm protein has similar 3-D structure, consisting of an alpha helix followed by 5 beta strands. The Sm proteins interact via the beta strands, and may form circle around RNA.

Figure 3.3.17). The right panel shows interactions of the Sm proteins through their beta-strands to make a ring with an inner portion large enough to encircle an RNA molecule. From Angus I. Lamond (1999) Nature 397, 655 - 656 “RNA splicing: Running rings around RNA.”

A particular sequence common to many snRNAs is recognized by the Sm proteins, and is called the “Sm RNA motif”.

b. Use of antibodies from patients with SLE. Several of the common snRNPs are recognized by the autoimmune serum called anti‑Sm, initially generated by patients with the autoimmune disease Systemic Lupus Erythematosis. One of the critical early experiments showing the importance of snRNPs in splicing was the demonstration that anti-Sm antisera is a potent inhibitor of in vitrosplicing reactions. Thus the targets of the antisera, i.e. Sm proteins in snRNPs, are needed for splicing.

c. The snRNPs assemble onto the pre-mRNA to make a large protein-RNA complex called a spliceosome (Figure 3.3.17). Catalysis of splicing occurs within the spliceosome. Recent studies support the hypothesis that the snRNA components of the spliceosome actually catalyze splicing, providing another example of ribozymes.

Figure 3.3.17. Spliceosome assembly and catalysis

d. U1 snRNP: Binds to the 5' splice site, and U1 RNA forms a base‑paired structure with the 5' splice site.

e. U2 snRNP: Binds to the branch point and forms a short RNA-RNA duplex. This step requires an auxiliary factor (U2AF) and ATP hydrolysis, and commits the pre-mRNA to the splicing pathway.

f. U5 snRNP plus the U4, U6 snRNP now bind to assemble the functional spliceosome. Evidence indicates that U4 snRNP dissociates from the U6 snRNP in the spliceosome. This then allows U6 RNA to form new base-paired structures with the U2 RNA and the pre-mRNA that catalyze the transesterification reaction (phosphoester transfers). One model is that U6 RNA pairs with the 5' splice site and with U2 RNA (which itself is paired to the branch point), thus bringing the branch point A close to the 5' splice site. U5 RNA may serve to hold close together the ends of the exons to be joined.

4. Trans‑splicing

All of the splicing we have discussed so far is between exons on the same RNA molecule, but in some cases exons can be spliced to other RNAs. This is very common in trypanosomes, in which a spliced leader sequence is found at the 5' ends of almost all mRNAs. A few examples of transsplicing have been described in mammalian cells.