Unstructured mRNAs form multivalent RNA-RNA interactions to generate TIS granule networks

Abstract

The TIS granule network is a constitutively expressed membraneless organelle that concentrates mRNAs with AU-rich elements and interacts with the major site of protein synthesis, the rough endoplasmic reticulum. Most known biomolecular condensates are spherelike, but TIS granules have a mesh-like morphology. Through in vivo and in vitro reconstitution experiments we discovered that this shape is generated by extensive intermolecular RNA-RNA interactions. They are mostly accomplished by mRNAs with large unstructured regions in their 3′UTRs that we call intrinsically disordered regions (IDRs). As AU-rich RNA is a potent chaperone that suppresses protein aggregation and is overrepresented in mRNAs with IDRs, our data suggests that TIS granules concentrate mRNAs that assist protein folding. In addition, the proximity of translating mRNAs in TIS granule networks may enable co-translational protein complex formation.Ma et al., page 2 Biomolecular condensates form through weak interactions of multivalent molecules. Proteinprotein interactions occur between repeated modular domains or between intrinsically disordered regions (IDRs). Protein IDRs lack a defined three-dimensional structure but often contain low-complexity sequence elements that provide the basis for multivalent intermolecular interactions (1). In addition, protein-RNA and RNA-RNA interactions contribute to the multivalency of phase separation systems (2-7). For example, it has been demonstrated that RNA can phase separate without protein and that RNA can promote or inhibit phase separation (3,8). It has also been shown that protein-RNA interactions can influence the identity and material properties of condensates in vitro and in vivo (2,4,6). However, the contribution of specific mRNAs to phase separation is largely unknown, as it has only been studied for few mRNAs (2,4).The TIS granule network is a membraneless organelle that forms a mesh-like compartment that is intertwined with the endoplasmic reticulum (ER). It is present in all cell types studied under physiological conditions. For mRNA to localize to TIS granules, they require several AU-rich elements in their 3′ untranslated regions (3′UTRs) (9). Whereas most known biomolecular condensates are sphere-like (1), TIS granules form a tubule-like meshwork. Here, we set out to investigate how the characteristic three-dimensional organization of TIS granules is determined.During these studies, we examined the influence of 47 human in vitro transcribed 3′UTRs, with sizes ranging from 500-5000 nucleotides, on phase separation of an RNA-binding protein. We observed that mRNAs that form strong local intramolecular interactions generate sphere-like condensates. In contrast, extensive intermolecular RNA-RNA interactions drive the generation of mesh-like condensates and are accomplished by mRNAs with large unstructured regions that are often single-stranded and AU-rich. Taken together, the TIS granule network represents a cytoplasmic compartment that concentrates unstructured, AU-rich mRNAs with a high propensit...

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“…We transcribed six 5 kb fragments and two 1 kb fragments from the genome in vitro ( Figure 1F, Figure 1_figure supplement 1B) (28). In silico methods predict that these RNA fragments contain secondary structure elements ( Figure 1F, Figure 1_figure supplement 1B) (23,29,30). Similar to synthetic RNA substrates that form a secondary structure ( Figure 1D), the N protein formed asymmetric gel-like condensates with viral RNA fragments in vitro ( Figure 1F, Figure 1_figure supplement 1B,C).…”

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