Stepwise assembly of the spliceosome

In order to splice out the introns and ligate the adjacent exons, the 5' SS, BP and 3' SS have to be brought into close proximity. Self-splicing group II introns spontaneously adopt a three-dimensional fold that provides an active site where the reactive groups of the introns are juxtaposed (Toor et al., 2008b; Chan et al., 2012). In contrast, considering the limited information contained in the nuclear introns and the fact that the consensus sequences defining the 5' SS, BP and 3' SS are highly degenerated in metazoans, nuclear introns do not contain sufficient secondary and tertiary structure information to assemble in a productive fold that leads to splicing. As a result, the efficient folding of nuclear pre-mRNA introns in a

Introduction

13

manner conductive to splicing depends on many trans-acting factors that are brought together with the pre-mRNA to form the spliceosome. For each round of splicing, the spliceosome is assembled de novo and undergoes several rearrangements of its components generating well defined intermediate complexes that can be isolated in vitro (reviewed in Will and Lührmann, 2011; Wahl et al., 2009).

When introns do not exceed 200-250 nt, the spliceosome assembles across the intron (Fig.

1.5) (Fox-Walsh et al., 2005). In major (U2-dependent) spliceosomes, the assembly starts with the ATP-independent recognition of the 5' splice site by U1 snRNP with the 5' end of the U1 snRNA base-pairing with the 5' splice site of the intron (Ruby and Abelson, 1988;

Seraphin and Rosbash, 1989). Also, in the early assembly phase of the spliceosome, SF1/BBP protein and the 65 kDa subunit of the U2 auxiliary factor (U2AF) bind to the BPS and to the PPT, respectively (Berglund et al., 1998). Additionally, the 35 kDa subunit of U2AF binds to the AG dinucleotide of the 3' SS and, together, all these interactions yield the spliceosomal E complex (Hong et al., 1997; Das et al., 2000).

Subsequent to E complex formation, U2 snRNA engages in base-pairing interactions with the BPS in an ATP-dependent manner, assisted by UAP56 and hPrp5 helicases (Xu et al., 1996; Zhang, 2001; Fleckner et al., 1997). In the short U2-BPS duplex, the branch site adenosine is bulged out offering its 2'-OH as a nucleophile for the first catalytic step (Query et al., 1994). This base-pairing is stabilized by SF3a and SF3b protein complexes from U2 snRNP and by the RS domain of U2AF. Association of U2 snRNP leads to the dissociation of SF1/BBP from the BPS and results in A complex formation (Lim and Hertel, 2004).

In the next step, the pre-formed U4/U6.U5 tri-snRNP particle is recruited to the spliceosome, forming the B complex (Cheng and Abelson, 1987). Although it contains all snRNPs, the B complex is catalytically inactive and requires major compositional and conformational rearrangements. During spliceosome activation, U1 and U4 snRNPs dissociate from the spliceosome, giving rise to the activated spliceosome (B^act complex). hPrp28 and hBrr2 helicases are involved in disrupting the base-pair interactions of U1 and the 5' SS and of U4/U6 di-snRNA, respectively (Staley and Guthrie, 1999; Laggerbauer et al., 1998;

Raghunathan and Guthrie, 1998). Concomitantly, the 5' end of U6 snRNA substitutes U1 and base pairs with the 5' SS. Extensive base-pairing network is formed between U2 and U6, bringing together the 5' SS and the BP sequence for the first step of splicing. Additionally, a central region of U6 snRNA forms an intramolecular stem-loop structure (U6-ISL) that seems to be crucial for catalysis. U5 snRNA also interacts with nucleotides near the 5' SS.

Introduction

14

Figure 1.5: Cross-intron assembly and disassembly of the major spliceosome. Only the stepwise interactions of the spliceosomal snRNPs (colored circles) but not those of the non-snRNP factors are shown. The spliceosomal complexes are named according to the metazoan nomenclature. Exons and introns are represented by boxes and lines, respectively. The stages at which remodeling takes place driven by SF2 RNA helicases and the GTPase Snu114 are indicated.

At this stage, Prp2 plays a role in reorganizing the spliceosome, generating the B*

complex (a catalytically activated spliceosome), which catalyses the first step of splicing (Kim and Lin, 1993; Warkocki et al., 2009). This yields the C complex. Prior to the second catalytic step, the spliceosome is remodeled again by the Prp16 helicase possibly to reposition the splicing intermediates (Schwer and Guthrie, 1992). Also before step 2, U5 contacts exon nucleotides downstream of the 3' SS and aligns 5' and 3' exons for the second catalytic step.

All these events lead to the catalysis of step 2 (reviewed by Umen and Guthrie, 1995; Smith et al., 2008). Finally, the exon junction complex (EJC) is deposited 20 to 25 nt upstream of the exon-exon junction (Le Hir et al., 2000; Bono et al., 2006). The mRNA is then released in the form of an mRNP and transported out of the nucleus (Le Hir et al., 2000; Bono and Gehring, 2011). At the same time, the post-spliceosomal complex (Makarov et al., 2002) is disassembled and the snRNPs are recycled to take part in subsequent splicing events. The released lariat intron (Martin et al., 2002) is debranched by Dbr1 and typically degraded (Chapman and Boeke, 1991).

Introduction

15

Alternative spliceosome assembly pathways exist in metazoans, whose mRNAs contain multiple extremely large introns, from several hundred to several thousand nt (Deutsch and Long, 1999) and rather short exons. When intron length exceeds 250 nt, spliceosomal components assemble across an exon, a process called exon definition (Berget, 1995). During exon definition, U1 snRNP binds to the 5' SS downstream of an exon and stimulates the association of U2AF with the PPT and the 3' SS upstream of the same exon. Then, U2 snRNP is recruited to the BPS also upstream of the exon and ESEs recruit proteins of the SR family which stabilize the exon-defined complex (Hoffman and Grabowski, 1992; Reed, 2000). In a subsequent step, these cross-exon interactions must be substituted by cross-intron interactions.

However, this process is poorly understood. It is suggested that exon exclusion and skipping during alternative splicing events occurs during the transition from a exon to a cross-intron complex (Sharma et al., 2008).