m⁶A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site

Abstract

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5’ splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIO1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5’ splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3’ splice site usage and reinforces splicing fidelity at elevated temperature. We generalise these findings to reveal two major classes of 5’ splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5’ splice sites crucial to alternative splicing.

FIONA1-mediated methylation of the 3’UTR of FLC affects FLC transcript levels and flowering in Arabidopsis

“…The analysis of the m 6 A consensus in fio1-2 identified the YHAGA motif, which is significantly different to the RRACH motif that has been described in both plants and animals [2,26], and to the RGACH consensus sequence that we identify in this work (Fig 7B ) and the motifs identified by Parker et al [16].…”

“…down mutation caused by a T-DNA insertion in the 5'-region of the FIONA1 gene [9,16] showed a similar phenotype. The fio1-1 mutant was shown to have elevated levels of both CONSTANS (CO) and FLOWERING LOCUS T (FT) mRNA.…”

“…Again, this might be due to the application of different methods or an indication that the reduction of FIO1 activity affects the m 6 A methylome more strongly than does the complete loss. A recent paper characterized the effect of the loss of FIO1 function on global splicing patterns and profound changes were identified [16]. The authors relate these changes to defects in U6 snRNA m 6 A modification, rather than being a direct consequence of loss of FIO1 activity.…”