pre-mRNA splicing

The spliceosome cleaves the introns in a very precise reaction out of the pre-mRNA.

This is possible by conserved sequences within the exon and intron. Studies on many eukaryotic systems showed three conserved consensus sequences: the 5' Splice Site (5'SS), the 3' Splice Site (3'SS), and the Branch Site (BS). The 5'SS is located at the 5' end of an intron and the 3'SS at the 3' end of an intron. The BS is located between 10 to 155 nucleotides upstream of the 3'SS. All three elements are highly conserved in S.cerevisiae^[35,36]and the conserved sequences at the exon-intron border of yeast and humans are shown in gure 3.

12 1. Introduction

Figure 3: Schematic representation of conserved sequences in introns of S. cerevisiae and homo sapiens. The branch site (BS) adenosine is highlighted. Bold letters indicate a 90 % or higher conservation of the respective nucleotide. Y stands for pyrimidines,R for purines, N for a G or GX and X for A or G or U or C. (Yn) denotes the polypyrimidine tract and the numbers indicate the distance between the 5'SS and the BS and between the BS and the 3'SS. (Figure also appeared in the PhD thesis of Peter

Odenwälder)

In nature, two dierent types of splicing occur: self-splicing and splicing by the spliceosome. In the process of self-splicing, RNAs can remove their introns without the help of proteins. For non self-splicing introns, the spliceosome, which is a ri-bonucleoprotein complex, has to process the pre-mRNA splicing.^[37].

The self-splicing introns are sorted into two groups. The group I introns are found commonly in fungal and plant mitochondria, plant chloroplasts, and in nuclear RNA of lower eukaryotes (e.g. Tetrahymena). The nucleotide sequences of group I introns are variable, but all of them form a similar three-dimensional structure. Together with several conserved nucleotides, the structure allows the group I introns for cat-alyzing the removal of the intron by themselves. For group I introns, the splicing reaction is started by binding of a G nucleotide to the intron sequence. This G nucleotide is activated to form an attacking group which will break the 5'SS^[34,38]. Group II self-splicing introns are found in the pre-mRNA of fungal and yeast mi-tochondria, and plant chloroplasts. In the self-splicing process of group II introns, the 5' splice site is cleaved rst. Then the 5'SS is covalently bound to an adenosine residue near the 3'SS of the intron and a structure called lariat is formed. Here, the A nucleotide in the intron sequence is the attacking group. In the last step, the

1. Introduction 13

3'SS is cut, the intron is released, and the ends of the exons are bound covalently together^[34,38].

There are similarities between the group I and group II introns. For both groups the sequence of the intron is important, because the intron is folded into a three-dimensional structure which brings the ends of the intron in contact^[34,38].

Figure 4: Principle of pre-mRNA splicing. The cleavage of the 5'SS and the forma-tion of the lariat structure during the rst step are shown. In the second step, the 3'SS is

cleaved and the exons are ligated. (Adapted from Cooper^[39].)

In contrast to the self-splicing introns, the introns of pre-mRNAs in animal cells do not splice themselves. The steps are similar to the splicing of group II introns, but a macromolecular complex, the spliceosome, is necessary to process the intron removal. The spliceosome consists of a large number of proteins and ve ribonucle-oparticles called snRNPs (small nuclear ribonucleoproteins). The snRNPs consist of a small nuclear Ribonucleic Acid (snRNA), seven Sm proteins, and a variable num-ber of specic proteins^[40]. Because of their high amount of uracil, the spliceosome snRNPs are denoted as the U1, U2, U4/U6, and U5 snRNP. The RNA molecules of the snRNPs are relatively short with less than 200 nucleotides, but are involved in the major part of pre-mRNA splicing.

The removal of an intron proceeds through two sequential phosphoryl-transfer re-actions known as transesterications^[41]. In the rst step, the adenosine of the BS

14 1. Introduction

attacks the 5'SS nucleophilically, generating the free 5' exon and the lariat-3'-exon intermediates. In the second step, the 3' hydroxyl group of the 5' exon attacks the 3'SS. In the end, the exons are ligated and the intron lariat is released from the complex^[40] (see g. 4).

Pre-mRNAs can be spliced in dierent ways in a process called Alternative Splicing.

This occurs commonly in human cells and results in the generation of dierent mRNAs from the same pre-mRNA. These lead to dierent polypeptide chains from one gene. During alternative splicing, particular exons may be included within, or excluded from, the pre-mRNA (see g. 5).

Figure 5: Scheme of the dierent modes of splicing: conventional splicing, exon skipping, mutually exclusive exon splicing, alternative 5'SS splicing, alternative 3'SS

splic-ing, and intron retention.

In general, ve dierent modes of alternative splicing can be distinguished: In the exon skipping case, an exon is spliced out or kept in the sequence. It is the most common mode in mammalian pre-mRNAs^[42]. In the case of mutually exclusive exons, one exon is always kept in the mRNA, while a second one is always spliced out. In the next case, dierent alternative 3'SS or 5'SS can be used. In the mode of intron retention, a sequence can be spliced out as an intron or is retained^[42].

1. Introduction 15