A bifunctional ARHGEF17-AS1 locus encodes MSEP, a protein that supports mitotic spindle organization.

Bifunctional genomic loci can produce both coding and noncoding outputs, yet the proteins encoded by many such loci remain unexplored. Here, we identify MSEP, a previously uncharacterized ∼54-kDa protein translated from an open reading frame embedded within the antisense RNA locus ARHGEF17-AS1. MSEP localizes to the mitotic spindle and spindle poles, and disruption of its start codon increases spindle abnormalities. MSEP interacts with the spindle-assembly factor TPX2, and its depletion is accompanied by reduced TPX2 abundance. MSEP undergoes cell-cycle-dependent phosphorylation that is regulated by Aurora A and WEE1. Depletion of MSEP is also associated with diminished Aurora A abundance at spindle poles. Genetic uncoupling of MSEP translation from ARHGEF17-AS1 RNA expression supports largely distinct protein- and RNA-mediated pathways that converge on spindle-pole integrity. Together, these results support a bifunctional-locus model in which ARHGEF17-AS1 RNA and its encoded protein, MSEP, contribute to mitotic spindle integrity through distinct, parallel pathways.