Spliceosomal snRNA modifications and their function

Abstract

Spliceosomal snRNAs are extensively 2'-O-methylated and pseudouridylated. The modified nucleotides are relatively highly conserved across species, and are often clustered in regions of functional importance in pre-mRNA splicing. Over the past decade, the study of the mechanisms and functions of spliceosomal snRNA modifications has intensified. Two independent mechanisms behind these modifications, RNA-independent (protein-only) and RNA-dependent (RNA-guided), have been discovered. The role of spliceosomal snRNA modifications in snRNP biogenesis and spliceosome assembly has also been verified.

Figure 1

Major spliceosome assembly and catalysis of pre-mRNA splicing. The thick lines represent the intron and the boxes are exons. The 5' splice site (5'-SS), the 3' splice site (3'-SS) and the branch point adenosine (BP) are indicated in the pre-mRNA. The conserved residues at the 5' and 3' splice sites and the branch site are shown. The headed thin lines are snRNAs with their names in the ellipses. The short thick lines between RNA strands represent Watson-Crick base-pairing interactions. The lightning symbols depict non-Watson-Crick base-pairing interactions. The 2'-OH groups of branch point adenosine and the cut-off 5' exon are pictured in the activated spliceosome. The small arrows near those 2'-OH group indicate the nucleophilic chemical reactions also known as trans-esterification reactions.

Figure 2

Pseudouridines and 2'-O-methylated residues in human spliceosomal snRNAs. Primary and secondary structures of human major spliceosomal snRNAs (U1, U2, U4, U5 and U6) are shown. Pseudouridines (Ψ) are surrounded by rectangles; 2'-O-methylations are circled. The thick lines indicate the nucleotides participating in RNA-RNA interactions or involved in catalysis during pre-mRNA splicing. The gray boxes highlight the Sm-binding sites. The 5' caps (2,2,7 trimethylated guanosine cap for U1, U2, U4, U5 and γ-methylated guanosine cap for U6) are also depicted.

Figure 3

Schematic depiction of the two most abundant modified nucleotides in spliceosomal snRNA. (Top) Pseudouridine is a rotational isomer of uridine, in which the N-C glycosidic bond is broken to form an C-C bond. This results in the presence of an extra hydrogen bond donor (d), while the number of hydrogen bond acceptors (a) is unchanged. (Bottom) Schematic representation of a 2'-O-methylated ribose.

Figure 4

Schematic depiction of box H/ACA and C/D RNAs. (A) Secondary structure of a eukaryotic pseudouridylation guide box H/ACA RNA. The RNA adapts a Hairpin-Hinge-Hairpin-Tail structure. Present within the hinge region is the box H (5'-ANANNA-3'), the box ACA (5'-ACA-3') motif typically lies three nucleotides from the 3'-end of the RNA. A CAB box (5'-ugAG-3'), responsible for Cajal body localization, may be present in the apical loop of either hairpin. Pseudouridylation is targeted to substrate RNAs by complementary base-pairing interactions between the internal loop (pseudouridylation pocket) and nucleotides adjacent to the target uridine. The thick lines denote substrate RNAs. (B) Secondary structure of a box C/D RNA. Boxes C, D, C’ and D’ are shown. The 2'OMe represents the target 2'-O-methylation site that is always the fifth nucleotide from box D or D’. The thick line represents substrate RNA.

Figure 5

Shown are primary and secondary structures of human minor spliceosomal snRNAs, U11, U12, U4atac and U6atac. U5 snRNA is shared by both the major and minor spliceosomes. Pseudouridines (Ψ) are surrounded by rectangles; 2'-O-methylations are circled. Pseudouridines within U12 and U4atac are believed to function analogously to their homologous modifications within U2 and U4 snRNAs, respectively. The thick lines indicate the nucleotides participating in RNA-RNA interactions or involved in catalysis during pre-mRNA splicing. The gray boxes highlight the Sm-binding sites.