A single m6A modification in U6 snRNA diversifies exon sequence at the 5’ splice site

N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5’ splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5’ exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.

U5 snRNA interactions with exons ensure splicing precision

Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with precision of the message reading frame. Apart from intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron-start. Association between these guanines cannot be explained solely by base-pairing with U1snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon-end. Current U5snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon-termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: 1) Optimal binding register for human exons and U5 - the exon junction positioned at U5Loop1 C39|C38. 2) Common mechanism of base pairing of human U5snRNA with diverse exons and bacterial Ll.LtrB intron with new loci in retrotransposition - guided by base pair geometry. 3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Our statistical analyses show increased U5 Watson-Crick pairs with the 5’exon in the absence of +5G at the intron-start. In 5’exon positions -3 and -5 this effect is specific to U5snRNA rather than U1snRNA of the early spliceosome. Increased U5 Watson-Crick pairs with 3’exon position +1 coincide with substitutions of the conserved -3C at the intron 3’end. Based on mutation and X-ray evidence we propose that -3C pairs with U2 G31 juxtaposing the branchpoint and the 3’intron-end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3’intron-end ensure that the 3’exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6 and U2snRNAs that stabilise the pre-catalytic complex by Watson-Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson-Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA-therapeutics and gene repair by reverse splicing.

Structure of the activated human minor spliceosome

The minor spliceosome mediates splicing of the rare but essential U12-type pre-mRNA. Here we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-Å resolution. The 5′-splice site and branch point sequence of the U12-type intron are recognized by U6atac and U12 small nuclear RNA (snRNA), respectively. Five newly identified proteins stabilize the conformation of the catalytic center. The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome. The RBM48/ARMC7 complex binds the γ-monomethyl phosphate cap at the 5′-end of U6atac snRNA. The U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 snRNP. CRIPT stabilizes U12 snRNP. Our study provides a framework for mechanistic understanding of the function of the minor spliceosome.

Structures of the fully assembledSaccharomyces cerevisiaespliceosome before activation

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo–electron microscopy structures of theSaccharomyces cerevisiaepre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5′ splice site (5′SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5′-exon–5′SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

Post-catalytic spliceosome structure reveals mechanism of 3'-splice site selection

Introns are removed from eukaryotic mRNA precursors by the spliceosome in two transesterification reactions – branching and exon ligation. Following branching, the 5'-exon remains paired to U5 snRNA loop 1, but the mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7Å cryo-EM structure of the yeast P complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognised through non-Watson-Crick pairing with the 5'-splice site and the branch point adenosine. A conserved loop of Prp18 together with the α-finger and the RNaseH domain of Prp8 clamp the docked 3'-splice site and 3'-exon. The step 2 factors Prp18 and Slu7 and the C-terminal domain of Yju2 stabilise a conformation competent for 3'-splice site docking and exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides of the introns and provides insight into the catalytic mechanism of exon ligation.