Characterization of RNA content in individual phase-separated coacervate microdroplets

Abstract

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

Bio‐inspired functional coacervates

“…The important factors that regulate the partitioning of various molecules in and out of the coacervate droplets are not well understood. [238,239] To overcome these challenges, we need to develop a better understanding of the molecular mechanisms that govern the properties of functional coacervates. We also need to achieve fine spatiotemporal control over the coacervate systems.…”

Ribozyme-phenotype coupling in peptide-based coacervate protocells

“…The realisation of a phenotype-genotype linkage is essential for true open-ended evolution, otherwise phenotypes with obvious fitness advantages have no way of being propagated to future generations. We envision that recent methodological developments in RNA sequencing from single coacervate microdroplets will enable future selection experiments on populations of protocells with varying RNA genotypes and varying degrees of fitness with respect to environmental pressures such as heat or salt concentration 59 . It should be noted that as a ribozyme developed by in vitro selection, the R3C ligase is already a highly optimized system and offers little potential for further enhancement via selection.…”

Aging RNA granule dynamics in neurodegeneration

“…It is possible that P-body-associated RNA helicases are required for proper resolution of multivalent RNA tangles (Hondele et al, 2019;Majerciak et al, 2021;Linsenmeier et al, 2022), which can age condensates into gels. Together, these studies demonstrate that RNA can promote solid-or gel-like transitions in RNA granules, and sequencing-based technologies like CLIP-seq and others may help identify changes in RNA biology during disease (Van Nostrand et al, 2016;Hallegger et al, 2021;Wollny et al, 2022) (Table 1). Although we do not yet know how exactly aged RNA granules and fibers lead to cell death, the next section will discuss how the persistence of aged granules and fibers may disrupt cellular homeostasis.…”