Micellization: A new principle in the formation of biomolecular condensates

Abstract

Phase separation is a fundamental mechanism for compartmentalization in cells and leads to the formation of biomolecular condensates, generally containing various RNA molecules. RNAs are biomolecules that can serve as suitable scaffolds for biomolecular condensates and determine their forms and functions. Many studies have focused on biomolecular condensates formed by liquid-liquid phase separation (LLPS), one type of intracellular phase separation mechanism. We recently identified that paraspeckle nuclear bodies use an intracellular phase separation mechanism called micellization of block copolymers in their formation. The paraspeckles are scaffolded by NEAT1_2 long non-coding RNAs (lncRNAs) and their partner RNA-binding proteins (NEAT1_2 RNA-protein complexes [RNPs]). The NEAT1_2 RNPs act as block copolymers and the paraspeckles assemble through micellization. In LLPS, condensates grow without bound as long as components are available and typically have spherical shapes to minimize surface tension. In contrast, the size, shape, and internal morphology of the condensates are more strictly controlled in micellization. Here, we discuss the potential importance and future perspectives of micellization of block copolymers of RNPs in cells, including the construction of designer condensates with optimal internal organization, shape, and size according to design guidelines of block copolymers.

Keywords: NEAT1; architectural RNA (arcRNA); biomolecular condensate; block copolymer (BCP); long non-coding RNA (lncRNA); micellization; paraspeckle; phase separation.

FIGURE 1

Biomolecular condensates with RNA scaffolds. Biomolecular condensates with RNA scaffolds in various organisms are illustrated. The scaffold RNAs are described in parentheses if they are identified. Species other than humans are also described in parentheses.

FIGURE 2

RNAs are suitable biomolecules for scaffolds of biomolecular condensates. (A). RNA can induce phase separation in a spatially and temporally regulated manner. Transcription of nuclear architectural RNAs (arcRNAs) (nucleation event) induces nuclear condensates with roles such as reaction crucible, molecular sponge, and chromatin hub. The stress granule is shown as an example of the formation of cytoplasmic condensates with RNA scaffolds. (B). RNA can effectively sequester many proteins into the condensates by liquid-liquid phase separation (LLPS) compared with a stoichiometric decoy mechanism. The NP (NORAD-PUM) body is shown as an example. (C). RNA can create biomolecular condensates with various forms and functions by recruiting a wide variety of RNA-binding proteins (RBPs) (∼1,500 kinds of RBPs in humans).

FIGURE 3

The functional RNA domains of human NEAT1 long non-coding RNA (lncRNA). (A). Schematics show the domains of human NEAT1_2 lncRNA required for the form and function of paraspeckles. These domains include NEAT1_2 stability, isoform switching from NEAT1_1 to NEAT1_2, polyadenylation signal (PAS), UG-repeats that sequester TDP-43 proteins, R-loop formation (Dumelie and Jaffrey, 2017), DNA:RNA triplex formation (Sentürk Cetin et al., 2019), paraspeckle assembly (B block), shell-formation (A and C blocks). The spherical and cylindrical paraspeckles with restricted size (Sx: ∼360 nm in HeLa cells) form through micellization, a type of phase separation. (B) Deletion of the NEAT1_2 middle domain (8–16.6 kb region, B block) causes the formation of smaller paraspeckle foci (magenta). Nuclei are stained with DAPI (blue). (C) Schematics show NEAT1 mutants lacking the 5′ and/or 3′ domains (shell-forming domains) and the paraspeckles constructed by these mutants. Localization of NEAT1_2 within these paraspeckles and their size are shown.

FIGURE 4

ABC triblock copolymer micelle model of the paraspeckle. (A) Schematics of AB amphipathic (di)block copolymers with different block lengths and the micelles they form in water. (B) The 5′ and 3′ domains of NEAT1_2 localize in distinct shell domains of the paraspeckle. The super-resolution images (structured illumination microscopy) with indicated probes are shown. Dotted circles indicate the domains within the paraspeckles where the 5′, 3′, and/or middle domains of NEAT1_2 localize. (C) RNA-binding proteins (RBPs) coat the shell-formation domains and the assembly domain of the NEAT1_2 long non-coding RNA (lncRNA). (D) ABC triblock copolymer micelle model of the paraspeckle. (E) Energetic contributions (1–5) considered in the ABC triblock copolymer micelle model of the paraspeckle are shown in a schematic.

FIGURE 5

Differences of the condensates formed by liquid-liquid phase separation LLPS and micellization. (A) Schematics show condensates formed by LLPS and micellization. Their internal morphologies and behaviors upon transcriptional upregulation are also shown. Different RNA-binding proteins (RBPs) are illustrated as other color circles (blue and yellow). The condensates formed by LLPS and micellization can contain many types of RBPs, although the illustration shows one or two types of RBPs for simplicity. (B) Representative images of the paraspeckles in HAP1 NEAT1 wild type (WT) and Δ5’/Δ3′ cell lines are shown. Insets are magnified images of the paraspeckles in these cell lines. The 5′ domains of NEAT1_2 are shown in green and the middle domains of NEAT1_2 are shown in magenta. Nuclei are shown in blue.

FIGURE 6

Transcription rates influence the shape and internal morphology of the condensates. (A) Free energetic contributions in the sphere-cylinder transition are shown in a table. Free energetically favored states (shape) are shown in magenta when more RNA-protein complexes (RNPs) are incorporated into condensates. Schematics of the transition are also shown. If a large micelle forms, the shape is energetically unfavorable from elastic free energy caused by stretches of RNP polymers in the core. (B) The contribution of the transcription rate in the internal morphology of condensates is shown. Excluded-volume interactions between A (or C) blocks become dominant compared with excluded-volume interactions between A (or C) and B blocks in the core when more RNPs are incorporated into the condensates upon transcriptional upregulation.

FIGURE 7

Potential functional importance of micellization. (A–D) Features of condensates formed through micellization are shown (Left). Potential functional importance related to the features on the left is listed (Right).