The selective partitioning of mRNA in biomolecular condensates

Biomolecular condensates (BMCs) are complex assemblies of RNA and protein that form via the process of liquid-liquid phase separation. They have a diverse range of functionalities, often centered around RNA metabolism, and are found throughout the cell. However, how they selectively partition certain molecules whilst excluding others remains a poorly understood, yet pertinent question in biology. Here we addressed this from an mRNA perspective by developing a simplified, in vitro approach, utilising cytoplasmic RNA in combination with a panel of model BMCs. We identified that the length, GC %, and G-quadruplex frequency of an mRNA were critical in defining how it partitioned in these models. Remarkably, very similar rules persist in defining mRNA selection in cellular P-bodies (PBs) and stress granules (SGs). Supporting this notion, we demonstrated that the transcriptome captured by our in vitro model, DDX4N, was highly comparable to that of PBs. These results strongly suggest that the properties of the mRNA are the fundamental driving force behind how it partitions in BMCs. Moreover, we provide evidence for a novel role of 3’ untranslated region alternative cleavage and polyadenylation (3’UTR-APA), in manipulating the length, GC %, and G-quadruplex content of mRNA in order to selectively distribute mRNA isoforms to BMCs, with profound implications for mRNA metabolism. In addition, we demonstrated that SG and PB proteome components were significantly more likely to exhibit selective isoform partitioning within their respective condensate than the rest of the transcriptome. This raises the intriguing prospect that 3’UTR-APA may also be essential in BMC formation and maintenance