A microexon confers functional divergence of the actin nucleator Arp2/3

Arp2, a subunit within the actin nucleator Arp2/3, encodes two splice variants that differ by five residues in the D-loop, a critical element for catalyzing actin polymerization. It is unknown if this alternative exon, or "microexon," impacts Arp2/3 function. Here, we find that the microexon has been evolutionarily retained for over 600 million years yet varies in sequence. We investigate the unique microexon in Drosophila Arp2 and show that the longer "Arp2L" variant is expressed, though less than the short variant, "Arp2s." We purify recombinant Drosophila Arp2/3 containing Arp2s or Arp2L, and although we do not detect differences in actin polymerization bulk assays, the microexon attenuates inhibition by Arp2/3 inhibitors in vitro, suggesting intrinsic differences. We generate flies expressing only Arp2s or Arp2L, and while both splice variants rescue Arp2-knockout lethality, they functionally diverge in late sperm development due to the microexon sequence, not the length. Overall, our findings demonstrate that the Arp2 microexon alters Arp2/3 function in certain contexts.