Synergy of RNA Concentration, RNA Binding Proteins, and RNA Palindrome Drives clustering of oskar mRNA in vivo

Organization of mRNAs into clusters has been 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 (Stau), and intermolecular base-pairing driven by an RNA palindrome influence clustering of oskar (osk) mRNA in Drosophila embryos. We find that these factors collectively optimize osk clustering by promoting its dimerization and subsequent oligomerization. Both processes depend on all three factors, although oligomerization is more sensitive to their perturbation, indicating that the driving force for osk oligomerization is partially distinct from that governing dimerization. Furthermore, expression of Stau nearly doubles the likelihood of osk dimerization whereas disruption of the palindrome reduces it fourfold indicating that the presence of Stau and the palindrome lowers the concentration threshold of osk mRNA required for dimerization. Notably, insertion of the osk palindrome into a reporter mRNA markedly increased its association with the endogenous osk, further supporting the conclusion that the palindrome potently drives intermolecular base pairing. Importantly, this experiment also identified the palindrome as the major contributor to heterotypic clustering between the endogenous osk and the reporter mRNA. Finally, computational analyses identified a subset of early embryonic mRNAs predicted to harbor palindromes similar to those found in osk. Among these, eIF3a mRNA emerged as a candidate whose clustering may likewise be driven by intermolecular base pairing. Together, our findings 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 early Drosophila development.