EGFP insertional mutagenesis reveals multiple FXR2P fibrillar states with differing ribosome association in neurons

Abstract

RNA-binding proteins (RBPs) function in higher-order assemblages such as RNA granules to regulate RNA localization and translation. The Fragile X homolog FXR2P is an RBP essential for formation of neuronal Fragile X granules that associate with axonal mRNA and ribosomes in the intact brain. However, the FXR2P domains important for assemblage formation in a cellular system are unknown. Here we used an EGFP insertional mutagenesis approach to probe for FXR2P intrinsic features that influence its structural states. We tested 18 different in-frame FXR2P^EGFP fusions in neurons and found that the majority did not impact assemblage formation. However, EGFP insertion within a 23 amino acid region of the low complexity (LC) domain induced FXR2P^EGFP assembly into two distinct fibril states that were observed in isolation or in highly-ordered bundles. FXR2P^EGFP fibrils exhibited different developmental timelines, ultrastructures and ribosome associations. Formation of both fibril types was dependent on an intact RNA-binding domain. These results suggest that restricted regions of the LC domain, together with the RNA-binding domain, may be important for FXR2P structural state organization in neurons.

Keywords: Fragile X syndrome; Local protein synthesis; Low complexity; RNA granule; RNA-binding protein.

Fig. 1.

Unbiased insertional mutagenesis screen identifies a discrete region of the FXR2P LC domain as a regulator of assemblage formation in neurons. (A) Schematic of EGFP insertional mutagenesis approach used to generate a set of 18 unique, full-length EGFP-transposed FXR2P constructs (FXR2P^EGFP; see Materials and Methods). Each green tag marks one specific EGFP insertion site within a FXR2P^EGFP construct. EGFP insertion at a given residue is denoted as FXR2P^[#] where ‘#’ is the amino acid insertion position in FXR2P. (B–D) Cultured cortical neuron co-transfected with FXR2P^[217] (green) and diffusible cell fill TdTomato (red). FXR2P^[217] is present diffusely in the soma (C) and in granules in dendrites (D). (E–G) FXR2P^[416] is present in Type A fibril bundles in both soma (F) and dendritic processes (G). (H–J) FXR2P^[435] is present in dendritic granules (arrows) as well as Type B fibril bundles within soma (I) and dendritic processes (J). (K–M) FXR2P^[456] is present in dendritic granules; no fibrils were observed in either soma (L) or processes (M). All cultures are DIV14. Representative images from n=3 experiments. Scale bars: 20 µm. Tud, Tudor domain; NLS, nuclear localization signal; KH1, RNA-binding KH1 domain; KH2, RNA-binding KH2 domain; NES, nuclear export sequence; NOS, nucleolar targeting signal.

Fig. 2.

Time course of Type A and B fibril bundle expression in cultured neurons. (A) DIV6 neuron co-transfected with FXR2P^[416] (green) and TdTomato (red). Type A fibril bundles are detected in the cell body and extending into dendritic processes. Arrows mark localization of fibrils in dendrites. Dendritic FXR2P granules are not observed. (B) Type A fibril bundles observed in soma and dendrites of a DIV28 neuron. Arrows mark FXR2P fibrils in a dendrite. (C) Dendrite of a DIV6 neuron co-expressing FXR2P^[439] (green) and TdTomato (red). FXR2P^[439] is present in dendrites only in granules at this time (arrows). (D) In DIV14 neurons, FXR2P^[435] localizes to discrete, spherical, nest-like structures with a fibrillar substructure (arrowhead). These nest-like structures are closely associated to newly forming Type B fibril bundles. (E) FXR2P^[435] forms Type B fibril bundles in a DIV28 neuron that extend from the cell body into dendritic processes. (F) Western blot analysis of protein lysates from DIV6 neuronal cultures expressing FXR2P^[217], FXR2P^[416] or FXR2P^[435] probed with antibodies to FXR2P and γ-actin (loading control; ∼43kD). Note that all the FXR2P^[EGFP] fusions are expressed at similar levels. Equivalent results were observed in two independent experiments. The anti-FXR2P detected both the FXR2P^[EGFP] fusions (upper band ∼122kD; double asterisk) as well as endogenous FXR2P (lower band ∼95kD; single asterisk). Representative images from n=3 experiments for DIV6 and DIV14; n=2 for DIV28. Scale bars: 20 µm.

Fig. 3.

FXR2P contains a C-terminal LC domain that is intrinsically disordered. (A) PONDR-FIT predicts that a low complexity region in the C-terminus of FXR2P (residues 388–673) is intrinsically disordered. PONDR-FIT scores > 0.5 indicate a probability towards intrinsic disorder. (B) FoldIndex predicts that the LC region of FXR2P is intrinsically unfolded (gray shading shows less than zero). However, no region within FXR2P was predicted to be prion-like by either the PLAAC algorithm (red, log-likelihood ratio score below zero) or the PAPA algorithm (green, log-odds ratio score below dashed green line). (C) ZipperDB predicts that regions around residues 415 and 500 have increased fibrillization propensity. [Note that the apparent increased fibril-forming propensity of regions N-terminal to the low complexity domain (light shading) are due to the folded nature of these domains and are not relevant to this analysis.] (D) Phase separation prediction based on the per-residue pi-contact propensity indicates that the low complexity region of FXR2P has increased propensity to form pi-contacts compared to its folded domains. Dotted lines represent PScore thresholds for enrichment of pi-contacts (PScore ≥ 4) or depletion of pi-contacts (PScore ≤ −2). (E) ANCHOR predicts multiple disordered binding regions within the LC domain of FXR2P (blue, score > 0.5; darker blue signifies higher ANCHOR score). IUPRED predicts the LC domain as intrinsically disordered segment (red, score >  0.5 are predicted as disordered). Red lines below the residue position denote the location of the 217, 416, 435/439 and 456 EGFP insertion sites.

Fig. 4.

Ultrastructure of FXR2P^[EGFP] Type A and B isolated fibrils and fibril bundles in neurons. Electron microscopy of neurons transfected with non-fibrillar FXR2P^[217] (A,B); Type A fibril-forming FXR2P^[416] (C,D); or Type B fibril-forming FXR2P^[435] constructs (E,F). (A) Cytoplasm of DIV6 neuron expressing FXR2P^[217] is rich in polysomes (arrowheads) and no fibrils are observed. (B) Ribosome (arrowhead)-enriched dendritic granule in a neuron expressing FXR2P^[217]. (C) Isolated Type A fibrils decorated with ribosomes (arrow) from DIV6 neuron transfected with FXR2P^[416]. Note that polyribosomes are not observed in the cytoplasm. (D) Type A fibril bundle (arrow) associated with ribosomes in a neuron expressing FXR2P^[416]. (E) DIV14 neuron transfected with FXR2P^[435] displays ultrastructurally distinct, isolated Type B fibrils decorated with ribosomes (arrows). Polyribosomes are observed in the cytoplasm (arrowheads). (F) Type B fibril bundle (arrow) devoid of ribosomes in a neuron expressing FXR2P^[435]. Note that polysomes are readily observed in the adjacent cytosol (arrowheads). Representative images from n=3 neurons per condition. Scale bars: 250 nm.

Fig. 5.

Disposition and ultrastructural localization of Type A and B FXR2P isolated fibrils and fibril bundles in neurons. (A–C) Electron micrographs of DIV6 cortical neurons transfected with Type A fibril-forming FXR2P^[416]. (A) Low-magnification view of cell soma shows Type A fibril bundles coursing through cytoplasm (arrows). Juxtanuclear amorphous material is also observed (white arrowhead). (B) Isolated Type A fibrils (black arrowhead) and bundles (arrow) in close proximity to granular material (white arrowhead). Note the apposition of the isolated Type A fibril with the granular domain. (C) Type A fibril bundles (arrows) extend from cell soma into dendritic processes. (D–F) Electron micrograph of DIV14 cortical neurons expressing Type B fibril-forming FXR2P^[435]. (D) Low-magnification view of cell shows multiple rigid Type B fibrils extending radially from cell center (arrows). Plane of section is adjacent to the substrate. (E) Isolated Type B ribosome-decorated fibrils (black arrowhead) are restricted to a circular domain in the cytoplasm (white asterisk), which is likely to be a section of the nest-like structures observed by fluorescence microscopy (Fig. 2D). Note that a ribosome-free Type B fibril bundle (white arrow) is juxtaposed to this nest-like structure. (F) Type B fibril bundles (white arrow) are rectilinear and jagged in the cytoplasm as well as devoid of ribosomes. Representative images from n=3 neurons per condition. Scale bars: 2 µm (A,B), 500 nm (B,C,E,F). nuc, nucleus.

Fig. 6.

Ribosomes and mRNA co-localize with Type A but not Type B fibril bundles. (A) A subset of FXR2P^[217] (green) granules colocalize (arrows) with 5S/5.8S rRNA (Y10b antibody; red) in dendrites. Arrowheads denote FXR2P^[217]-only granules. (B–E) Type A fibrils (green) colocalize with 5S/5.8S rRNA (red; B) and polyA+ mRNA (red; C) in dendrites. No signal was observed in fibrils with a polyT control (red; D). Anti-GFP immunostaining (red) of Type A fibrils (green; E). Note the complete co-localization of FXR2P^[416] intrinsic EGFP signals with the anti-GFP immunostain. (F–H) Type B fibrils (green) do not colocalize with either 5S/5.8S rRNA (red; F) or polyA⁺ mRNA (red; G). Intrinsic GFP signal of FXR2P^[435] Type B fibril bundles co-localize with the anti-GFP immunostain (red; H). Representative images from n=2–3 experiments. DIV14 neurons. Scale bars: 10 µm.

Fig. 7.

FXR2P RNA-binding and LC domains collaborate in fibril formation. (A–C) DIV14 neuron co-transfected with (FXR2P^[217ΔLC]; green) and TdTomato (red). FXR2P^[217ΔLC] is diffusely localized throughout the cell and discrete granules are not observed (B and C, respectively). (D) A₂₅₄ traces of sucrose gradients from HEK293T cells expressing either FXR2P^[217] or the RNA-binding mutant FXR2P^[217::I314N]. (E) Western blot for FXR2P in fractions collected from sucrose gradients from either FXR2P^[217] (upper blot) or FXR2P^[217::I314N] (lower blot). FXR2P^[217] predominantly co-sediments in polysome fractions. In contrast, FXR2P^[217::I314N] is enriched in free and monosome fractions and present in low levels in polysome fraction. Equivalent results were observed in two independent experiments. (F–H) FXR2P^[217::I314N] (green) contains a point mutation in the RNA-binding KH2 domain and is diffusely localized in the soma (G) and present in granules in cell processes (H). Compare to Fig. 1B–D. (I–K) FXR2P^[416::I314N] (green) is expressed diffusely in the nucleus and cell processes (J and K, respectively). No granular or fibrillar structures are observed. Compare to Fig. 1E–G. (L–N) FXR2P^[435::I314N] (green) is diffusely distributed in the soma (M) and is present in granules in dendrites (N). No fibrillar structures are observed. Compare to Fig. 1H–J. (O) Summary of Type A and B fibril dynamics and ribosome association in neurons. See text for details. Representative images of n=2–3 experiments. Neurons transfected at DIV3 and analyzed on DIV14. Scale bars: 20 µm.