The mechanism of nuclear pre-mRNA splicing

Nuclear pre-mRNA splicing requires a large number of trans-acting factors that aid proper splice site selection as well as pre-mRNA folding, bringing together the sites at which ligation of exons will occur. These factors are brought together in a stepwise manner to form a dynamic macromolecular machine called the spliceosome.

The spliceosome assembles from 5 small uridine-rich nuclear RNAs (U1, U2, U4, U5 and U6 snRNAs) organized in ribonucleoprotein complexes (snRNPs) and a plethora of non-snRNP proteins (Will and Lührmann, 2006; Shatkin, 1976; Shatkin and Manley, 2000; Jurica and Moore, 2003). In metazoans, about 1% of all introns (called U12-type introns in contrast to U2-type introns of the major spliceosome) are spliced by a distinct spliceosome, called the minor spliceosome or U12-dependent spliceosome, comprising U11, U12, U4atac, U6atac snRNPs, which are functional analogs of U1, U2, U4, U6 snRNPS of the major spliceosome, respectively (Patel and Steitz, 2003; Will and Lührmann, 2005). The U5 snRNP is shared by both spliceosomes. The spliceosome assembles de novo on each intron of the pre-mRNA and catalyzes two transesterification reactions which are required for excision of introns and ligation of exons.

Chemically, the splicing process seems very simple. It involves a two step reaction which produces an excised intron and ligated exons. However, the sites at which the mRNA is cleaved to splice out the intron(s) must be precisely selected since an error of one nucleotide shifts the reading frame and results in a completely different protein product. Correct splice

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site (SS) selection is a major challenge to the splicing machinery, especially in higher eukaryotes, considering that introns have variable sequences and lengths as well as low information content defining exon-intron boundaries. Nevertheless, a set of specific sequences required for splicing can, in most cases, be identified in introns and at the intron/exon boundaries (Fig. 1.2) (Stephens and Schneider, 1992). The 5' SS defines the 5' end of the intron and, in yeast, it is composed of 5'-R|GUAUGU-3' (Lopez and Séraphin, 1999) ('|' defines the exon-intron boundary, 'R' is a purine base and nucleotides in bold indicate at least 90% conservation among yeast introns). In higher eukaryotes, the 5' SS is characterized by the consensus sequence 5'-AG|GURAGU-3' (bold letters indicate invariable nucleotides). The highly conserved yeast branch point (BP) sequence 5'-UACUAAC-3' contains a conserved adenosine (underlined) which is essential for catalysis of the first step of nuclear pre-mRNA splicing. The BP adenosine is located 10 to 155 nucleotides upstream of the yeast 3' SS (Spingola et al., 1999). In human, the BP sequence is normally located 18-40 nucleotides upstream of the 3' SS and comprises a degenerate sequence 5'-YNCURAC-3' ('Y' is a pyrimidine base, 'N' is any nucleotide) (Reed, 1989; Wahl et al., 2009; Zhang, 1998). A 10-15 nucleotide long pyrimidine rich sequence, the polypyrimidine tract (PPT), can often be found in higher Eukarya introns one to five nucleotide upstream of the 3' SS. The PPT is essential for splicing in humans possibly due to the less conserved BP sequence in human introns. The 3' SS follows the sequence 5'-YAG|-3' in most of the introns in vertebrates. The invariant AG dinucleotide defines the end of the intron (Reed, 1989). In addition, splicing is modulated by ESEs/ISEs and ESSs/ISSs, which are short sequences within the pre-mRNA recruiting regulatory proteins that either repress or stimulate spliceosome assembly. These sequences are not only important for constitutive splicing but also play a crucial role in alternative splicing as mentioned above (Black, 2003; Matlin et al., 2005).

Figure 1.2: Conserved sequence elements found in introns from metazoans and budding yeast (S. cerevisiae).

The 5' and 3' exons are shown as boxes. The branch point adenosine is underlined. "Y" – pyrimidine and "R" – purine. The polypyrimidine tract is indicated by "Y(n)".

In metazoans and plants, a separate, less abundant class of introns exists harboring different consensus sequences (Jackson, 1991). In these U12-type introns, the sequences

5'-|AUAUCCUUU-3' and 5'-YAC|-3' represent highly conserved elements at the 5' and 3' SS,

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respectively. Additionally, the U12-type introns lack the PPT and show a higher level of conservation of the 5' SS and BP sequence. The first identified introns of this class had 5' AT (AU for RNA) and 3' AC di-nucleotides instead of the highly conserved GT-AG (GU-AG) present at the 5' and 3' ends of U2-type introns. Consequently, they were originally called 'ATAC'-introns. As previously mentioned, these introns are removed by the minor spliceosome (Burge et al., 1999).

Regardless of the spliceosome type, introns are removed by a conserved mechanism involving two sequential S_N2-type transesterification reactions (Fig. 1.3) (Query et al., 1994;

Will and Lührmann, 2006). First, the oxygen of the 2' OH group of the BP adenosine makes a nucleophilic attack at the phosphodiester bond of the 5' SS exon-intron boundary. This leads to the formation of a free 3' OH group at the 3' terminal nucleotide of the 5' exon and the formation of 5'-2' phosphodiester bond between the 5' SS guanosine and the BP adenosine.

The result is a free 5' exon and a lariat intermediate containing the intron and the downstream exon. In the second step, the 3' OH group of the 5' exon attacks the phosphodiester bond at the 3' SS, thereby joining 5' and 3' exons and excising the intron as a lariat. Subsequently, the lariat intron is debranched and typically degraded, but can also be a source of regulatory RNAs (Carthew and Sontheimer, 2009; Voinnet, 2009), whereas the mRNA is exported from the nucleus into the cytoplasm (Brow, 2002).

Figure 1.3: Schematic representation of the two-step mechanism of pre-mRNA splicing. Boxes and solid lines represent the exons and the intron, respectively. The branch site adenosine is indicated by the letter "A" and the phosphate groups by the letters "p" at the 5' and 3' splice sites. The red arrows indicate the nucleophilic attacks at the phosphodiester bond at the 5' and 3' splice sites during step 1 and 2.

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