Ecer, A. | bioRxiv Science

bioRxiv2026

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.

molecular biology↗

bioRxiv2023

Intrinsically Disordered Regions Promote Protein Refoldability and Facilitate Retrieval from Biomolecular Condensates

Many eukaryotic proteins contain intrinsically disordered regions (IDRs) that intersperse globular folded domains, in contrast with bacterial proteins which are typically highly globular1, 2. Recent years have seen great progress in identifying biological functions associated with these elusive protein sequence: in specific cases, they mediate liquid liquid phase separation3, perform molecular recognition4, or act as sensors to changes in the environment5. Nevertheless, only a small number of IDRs have annotated functions6 despite their presence in 64% of yeast proteins,7 stimulating some to question what general purpose they may serve8, 9. Here, by interrogating the refoldability of two fungal proteomes (Saccharomyces cerevisiae and Neurosporra crassa), we show that IDRs render their host proteins more refoldable from the denatured state, allowing them to cohere more closely to Anfinsens thermodynamic hypothesis10, 11. The data provide an exceptionally clear picture of which biophysical and topological characteristics enable refoldability. Moreover, we find that almost all yeast proteins that partition into stress granules during heat shock are refoldable, a finding that holds for other condensates such as P-bodies and the nucleolus. Finally, we find that the Hsp104 unfoldase12 is the principal actor in mediating disassembly of heat stress granules and that the efficiency with which condensed proteins are returned to the soluble phase is also well explained by refoldability. Hence, these studies establish spontaneous refoldability as an adaptive trait that endows proteins with the capacity to reform their native soluble structures following their extraction from condensates. Altogether, our results provide an intuitive model for the function of IDRs in many multidomain proteins and clarifies their relationship to the phenomenon of biomolecular condensation.

biochemistry↗