LUC7 proteins define two major classes of 5' splice sites in animals and plants

Mutation or deletion of the U1 snRNP-associated factor LUC7L2 is associated with myeloid neoplasms, and knockout of LUC7L2 alters cellular metabolism. Here, we uncover that members of the LUC7 protein family differentially regulate two major classes of 5' splice sites (5'SS) and broadly regulate mRNA splicing in both human cell lines and leukemias with LUC7L2 copy number variation. We describe distinctive 5'SS features of exons impacted by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5'SS with stronger consensus matching on the intron side of the near-invariant /GU, while LUC7L3 enhances splicing of “left-handed” 5'SS with stronger consensus matching upstream of the /GU. We validated our model of sequence-specific 5’SS regulation both by mutating splice sites and swapping domains between human LUC7 proteins. Evolutionary analysis indicates that the LUC7L2/LUC7L3 subfamilies evolved before the split of animals and plants. Analysis of Arabidopsis thaliana mutants confirmed that plant LUC7 orthologs possess similar specificity to their human counterparts, indicating that 5'SS regulation by LUC7 proteins is deeply conserved. Flag-tagged human LUC7L, LUC7L2 and LUC7L3 ORFs were transfected into HEK293 RMCE cells as described above. RNA was extracted 24 hours after transfection using the Qiagen RNeasy Mini kit (cat. no. 74104) according to the manufacturer’s instructions with the optional on-column DNase digestion (cat. No 79254). RNA was eluted in nuclease-free water and quantified using Nanodrop. Illumina-compatible libraries were prepared by MIT BioMicroCenter using NEB II Ultra Directional RNA with poly(A) selection and sequenced on NovaSeq 6000 with 2 x 150 bp reads. Seeds of Wildtype Col0, luc7 single mutants (luc7a-1, luc7a-2, luc7b-1 and luc7rl-1), luc7 double mutants (luc7a-1 luc7b-1, luc7a-2 luc7b-1, luc7a-1 luc7rl-1 and luc7b-1 luc7rl-1) and a luc7 triple mutant (luc7a-2 luc7b-1 luc7rl-1) were surface sterilized with chlorine-gas and then grown on half-strength Murashige Skoog (MS) plates containing 0.8% phytoagar in continuous light at 22°C for 10 days. Seedlings were collected and flash frozen in liquid nitrogen. Total RNA was isolated using RNeasy® Plant Mini Kit (Quiagen, cat. nos. 74904) according to the manufacturer’s instructions. mRNA stranded library preparation and sequencing (PE150) was done by Novogene (Cambridge, United Kingdom) using an Illumina Novaseq6000 system.