New connections between splicing and human disease

Abstract

The removal by splicing of introns from the primary transcripts of most mammalian genes is an essential step in gene expression. Splicing is performed by large, complex ribonucleoprotein particles termed spliceosomes. Mammals contain two types that splice out mutually exclusive types of introns. However, the role of the minor spliceosome has been poorly studied. Recent reports have now shown that mutations in one minor spliceosomal snRNA, U4atac, are linked to a rare autosomal recessive developmental defect. In addition, very exciting recent results of exome deep-sequencing have found that recurrent, somatic, heterozygous mutations of other splicing factors occur at high frequencies in particular cancers and pre-cancerous conditions, suggesting that alterations in the core splicing machinery can contribute to tumorigenesis. Mis-splicing of crucial genes may underlie the pathologies of all of these diseases. Identifying these genes and understanding the mechanisms involved in their mis-splicing may lead to advancements in diagnosis and treatment.

Figure 1. Splice site consensus sequences for U2-dependent (a) and U12-dependent introns (b)

The two classes of introns found in human genes can be distinguished by their splice site sequences. The boxes show graphical representations of the consensus sequences in which the size of each letter represents the frequency of each base at each position over all introns. The bases are ordered by frequency from top to bottom. U2-dependent introns almost always begin with the dinucleotide GT and end with AG while U12-dependent introns can have either AT and AC termini or GT and AG termini.

Figure 2. The splicing reaction and interactions in the early phase of intron recognition and spliceosome formation

A. The splicing reaction catalysed by the spliceosome occurs in two steps. In the first step, the phosphodiester bond at the 5’ splice site is broken and the 5’ end of the intron is joined to the 2’ hydroxyl group of the branch site adenosine residue. In the second step, the phosphodiester bond at the 3’ splice site is broken and joined to the free 3’ hydroxyl group of the 5’ exon. The products are the ligated exons and the intron in the form of a lariat RNA. B. During the initial steps in splicing, the splice sites and adjacent RNA sequences are bound by a network of interacting factors. A subset of these factors is shown here of which several have been implicated in myeloid malignancies as discussed in the text. Some of the initial interactions include the binding of SR family proteins such as SRSF2 to splicing enhancer elements located within exons which recruit the U2AF65/U2AF35 heterodimer to the 3’ splice site either directly or through additional factors such as ZRSR2. The U2AF65 subunit binds to the pyrimidine rich region of the 3’ splice site while the U2AF35 subunit binds to the AG dinucleotide at the splice junction. SF1 binds to the branch site A residue while U1 snRNP binds to the 5’ splice site through base pairing interactions. The 5’ and 3’ splice site complexes are joined together by protein-protein interactions mediated by factors such as PRPF40B. Subsequent to these steps, the U2 snRNP is recruited by U2AF to the branch site where it base pairs to the intron RNA. U2 snRNP binding is also stabilized by binding of the SF3b complex (which includes the SF3B1 protein) to the RNA upstream of the branch site.

Figure 3. Formation of the spliceosomes

The early steps of spliceosome formation culminate in the base pairing of U1 or U11 and U2 or U12 to the 5’ and 3’ splice sites of U2-dependent or U12-dependent introns respectively (base pairings are indicated by the yellow bars). In the next phase of assembly, tri-snRNP complexes composed of U4, U5 and U6 or U4atac, U5 and U6atac are joined to the forming spliceosome. The base pairs connecting U4 and U6 or U4atac and U6atac are unwound and new pairings are made between U6 and U2 or U6atac and U12 leading to the release of U4 or U4atac, U6 or U6atac also form base pairs to the 5’ splice site displacing U1 or U11 from the complex. U5 interacts with the exons to hold the RNAs in place during the splicing reactions.

Figure 4. Locations of MOPD I mutations in U4atac snRNA

The sequence and predicted secondary structure of U4atac snRNA (in black) is shown in a base pairing configuration with U6atac snRNA (in gray). The sites of MOPD I mutations are boxed and the base changes seen in the patients are indicated. Each patient was homozygous for a single base change or heterozygous for different point mutations on the two alleles.