Restricting intermolecular interactions among complementary sequences is required for the specificity of homotypic mRNA assembly

We reported that the Drosophila mRNAs often organize into homotypic assemblies that are composed of mRNAs with the same primary sequences. While the formation of these assemblies involves intermolecular interactions among proteins and mRNAs, the role of these interactions in generating the specificity of homotypic mRNA assembly is unknown. Here, we demonstrate that intermolecular RNA-RNA interactions are sufficient to sort a mixture of distinct Drosophila RNAs into homotypic assemblies in vitro in the absence of proteins. To determine whether intermolecular interactions among complementary sequences generate the specificity of homotypic assembly, we computationally identified these sequences transcriptome-wide. Strikingly, we observed that the complementary sequences are common and frequently shared among distinct mRNAs with different primary sequences. These results predict that the formation of heterotypic assemblies, which are composed of distinct mRNAs, should be favored, contrary to our in vivo measurements. To resolve this contradiction, we employed DMS-MaPseq to determine the structuredness of mRNAs within assemblies in vivo. We observed that most of the complementary sequences are structurally embedded and thus inaccessible to intermolecular interactions. We also demonstrate that higher RNA concentration in assemblies does not extensively unwind the RNA structure in vitro. Finally, we show that in vitro a single, structurally exposed complementary sequence is sufficient to disrupt the specificity of homotypic assembly, promoting instead heterotypic assembly. Our data demonstrate that restricting the intermolecular interactions among complementary sequences is critical for the specificity of mRNA homotypic assembly. Thus, we propose a model where a combination of intermolecular base stackings, electrostatic forces, and single nucleotide base pairings modulated by RNA structure, generate the specificity of homotypic assembly. This work is supported by the R35GM142737 NIGMS grant awarded to TT.