Sequence-Independent Self-Assembly of Germ Granule mRNAs into Homotypic Clusters

Abstract

mRNAs enriched in membraneless condensates provide functional compartmentalization within cells. The mechanisms that recruit transcripts to condensates are under intense study; however, how mRNAs organize once they reach a granule remains poorly understood. Here, we report on a self-sorting mechanism by which multiple mRNAs derived from the same gene assemble into discrete homotypic clusters. We demonstrate that in vivo mRNA localization to granules and self-assembly within granules are governed by different mRNA features: localization is encoded by specific RNA regions, whereas self-assembly involves the entire mRNA, does not involve sequence-specific, ordered intermolecular RNA:RNA interactions, and is thus RNA sequence independent. We propose that the ability of mRNAs to self-sort into homotypic assemblies is an inherent property of an messenger ribonucleoprotein (mRNP) that is augmented under conditions that increase RNA concentration, such as upon enrichment in RNA-protein granules, a process that appears conserved in diverse cellular contexts and organisms.

Keywords: RNA clusters; RNA granules; RNA phase separation; RNA self-assembly; RNA sorting; RNA:RNA interactions; germ granules; homotypic RNA assembly; nanos; oskar.

FIGURE 1:. mRNAs self-sort into homotypic clusters.

See also Figure S1,2 and Table S1. A Embryo with maternally expressed Vasa:GFP transgene. B,C smFISH reveals the spatial distribution of nos (C) in germ granules (B). D Spatial distribution of nos clusters at the posterior pole. E,F Correlation of nos cluster abundance with the Osk:GFP protein (D) and Vasa:GFP protein (D) abundance quantified by the Pearson correlation coefficient (r). Each dot represents one granule. % = coefficient of determination. ≥3483 granules / pair were analyzed. G Position and abundance of CycB (empty blue circles), nos (empty magenta circles) and gcl (empty yellow circles) clusters in over- and under-expression experiments and in WT condition (full blue, magenta and yellow circles, respectively) in Vasa:GFP granules. 33.6 nanometer (nm) on the Y axis indicates co-localization of a doubly-labeled pgc with itself and 408.5 nm shows co-localization between Vasa:GFP and non-granule-enriched ccr4 (Trcek et al., 2015). PCC(Costes) in over- and under-expression assays: mean±SEM of 3 embryos/gene. Cluster abundance: 337 to 3178 clusters /gene/condition. H Co-localization of nos (magenta) with Vasa:GFP granules (green) in WT embryos and embryos treated with RNAi against nos. I Co-localization of gcl (magenta) with Vasa:GFP granules (green) in WT embryos and embryos over-expressing gcl. Scale bar: C,D = 2 μm, B = 10 μm, A = 50 μm.

FIGURE 2:. Germ granule proteins are more mobile than germ granule mRNAs.

See also Figure S3 and Table S2. A,B Fluorescence recovery of Vasa:GFP (A) and nos MS2-MCP:GFP (B) after photobleaching an ROI (magenta square) within germ plasm. C Labeling of nos with MS2-MCP:GFP. D Recovery of Vasa:GFP (mean±STDEV, gray bars) of 5 normalized recovery curves. Magenta line: fit to the data. E Kinetics of fluorescence recovery of core granule proteins and nos. The half-time to recovery (t_1/2 (s)) and the percent mobile fraction of the population that exchanges between granule and the intergranular space is shown. Mean±STDEV of 5 Osk:GFP, 5 Vasa:GFP, 12 Tud:GFP, 9 Aub:GFP and 5 nos MS2-MCP:GFP recovery curves. Scale bar = 10 μm.

FIGURE 3:. Self-assembly into homotypic clusters is RNA sequence independent.

See also Table S3,4. Ai-iii Endogenous nos hybridized with spectrally-distinct smFISH probes (i), displaying highly co-localization (ii) with a PCC(Costes): 0.84±0.01 (mean±SEM of 5 embryos) (iii). Bi-iii Endogenous nos and the nos 3′UTR chimera hybridized with distinct probes (i); poorly co-localized (ii) with a PCC(Costes): 0.55±0.03 (mean±SEM of 6 embryos) (iii). Ci-iii Two full-length nos chimeras hybridized with distinct probes (i) poorly co-localized (ii) with a PCC(Costes): 0.47±0.02 (mean±SEM of 6 embryos) (iii). Scale bar: 1 μm.

FIGURE 4:. mRNA organization within homotypic clusters is disordered.

See also Figure S4 and Table S5,6. A,B Detection scheme of sequence-specific, ordered trans RNA:RNA interactions in using smFISH (magenta circle) and STORM where mRNAs base-pair with their 3′UTRs and become spatially constrained (right panel) compared to the 5′UTR that do not base-pair (left panel). Magenta dot: smFISH probes. Magenta circles: cluster radius recorded by STORM. B In the absence of sequence-specific, ordered trans RNA:RNA interaction, the same radius of the mRNA clusters is recorded regardless of the position of the smFISH probe hybridization. For simplicity, mRNAs are drawn as linear polymers in A, B. C Radii of osk clusters. Statistical significance P: two tailed t test. Magenta and green circles: Alexa647, Alexa568 probes, respectively. Per probe, mean±STDEV of 11 to 30 ROIs is plotted, with localization uncertainty of 15.5±6.2 to 26.6±2.6 nm. D Radii of nos clusters. Magenta and green circles: Alexa647 or Alexa568 probes, respectively. Per probe, mean±STDEV of 17 to 40 ROIs is plotted with localization uncertainty of 12.4±4.3 to 20.7±2.2 nm. E osk mRNA transport particle coupled to a dynein motor (i) (Sanghavi et al., 2013) and homodimerized dimerized via a palindromic sequence (ii) (Jambor et al., 2011). F Germ granule mRNAs distinguish among distinct mRNAs near near cognate mRNAs.