Functional roles of protein splicing factors

Abstract

RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors. DExD/H-box RNA helicases are required to mediate structural changes in the spliceosome at various steps in the assembly pathway and have also been implicated in the fidelity control of the splicing reaction. Other proteins also play key roles in mediating the progression of the spliceosome pathway. In this review, we discuss the functional roles of the protein factors involved in the spliceosome pathway primarily from studies in the yeast system.

Figure 1. Two-step transesterification reaction of pre-mRNA splicing

Yeast splice site conserved sequences are shown in red. The splicing reaction takes place in two steps. The first step is cleavage of the 5′ splice site and formation of lariat intron–exon 2 via a 2′–5′ phosphodiester linkage. The second step is cleavage of the 3′ splice site and ligation of the two exons.

Figure 2. Schematic representation of the spliceosome pathway in yeast

The pathway can be divided into four stages: spliceosome assembly, spliceosome activation, catalysis and spliceosome disassembly. Spliceosome assembly involves ordered interactions of snRNPs with pre-mRNA. Spliceosome activation starts with the release of U1 and U4, followed by binding of the NTC. After the two catalytic reactions, the spliceosome is disassembled first by releasing the mature mRNA, and the spliceosome is then dismantled.

Figure 3. RNA–RNA interactions in pre-activated and activated spliceosomes

(A) The pre-activated spliceosome contains five snRNAs. U1 snRNA base-pairs with the 5′ splice site and U2 snRNA base-pairs with the branch site with a stem IIa structure. U2/U6 helix II has been shown to be required for tri-snRNP recruitment in mammalian cells. Loop I of U5 snRNA interacts with two broad regions of exon 1. (B) On the activated spliceosome, base-pairings of the 5′ splice site with U1 are displaced by U6. In addition, the Lsm complex is dissociated from U6 to allow interactions of its 3′ end with the intron sequence in a region downstream from the 5′ splice site.

Figure 4. Spliceosome dynamics during the catalytic steps

Both catalytic steps can be divided into ATP-dependent and ATP-independent steps. Prp2 and Prp16 are required in ATP-dependent steps. Hydrolysis of ATP results in the release of SF3a/b and Yju2/Cwc25 in the first and second step respectively. This allows Yju2/Cwc25 and Slu7/Prp18/Prp22 to bind to the spliceosome and promote the first and second transesterification reactions respectively in an ATP-independent manner. Yellow circles with different colours in the centre represent spliceosomes with different sets of splicing factors binding at the catalytic centre.