In vivo clustering of oskar mRNA is driven by RNA concentration, RNA binding proteins, and an RNA palindrome.

mRNA organization into clusters is observed in many cellular contexts, yet the features that govern this process in vivo remain poorly understood. Using super-resolution microscopy, single-mRNA imaging, and genetic perturbations, we investigated how mRNA concentration, the double-stranded RNA-binding protein Staufen, and intermolecular base-pairing driven by an RNA palindrome influence clustering of oskar mRNA in Drosophila embryos. We find that these factors collectively optimize oskar clustering by promoting its dimerization and subsequent oligomerization. Both processes depend on all three factors, although oligomerization is much more sensitive to their perturbation, indicating that the driving force for oskar oligomerization is partially distinct from that governing dimerization. Moreover, oskar palindrome is a potent driver of heterotypic mRNA clustering, further supporting its in vivo role in mediating intermolecular base pairing. Finally, computational analyses identified a subset of candidate mRNAs in the early embryo that are predicted to harbor oskar-like palindromes. Among these, eIF3a mRNA emerged as a potential candidate whose clustering may likewise be driven by intermolecular base pairing. These preliminary observations raise the possibility that mRNA clustering driven by palindrome-mediated intermolecular base pairing may be more widespread than previously appreciated and may represent an important mechanism for controlling mRNA spatial organization during Drosophila development.