A Membraneless Organelle Associated with the Endoplasmic Reticulum Enables 3'UTR-Mediated Protein-Protein Interactions

Abstract

Approximately half of human genes generate mRNAs with alternative 3' untranslated regions (3'UTRs). Through 3'UTR-mediated protein-protein interactions, alternative 3'UTRs enable multi-functionality of proteins with identical amino acid sequence. While studying how information on protein features is transferred from 3'UTRs to proteins, we discovered that the broadly expressed RNA-binding protein TIS11B forms a membraneless organelle, called TIS granule, that enriches membrane protein-encoding mRNAs with multiple AU-rich elements. TIS granules form a reticular meshwork intertwined with the endoplasmic reticulum (ER). The association between TIS granules and the ER creates a subcellular compartment-the TIGER domain-with a biophysically and biochemically distinct environment from the cytoplasm. This compartment promotes 3'UTR-mediated interaction of SET with membrane proteins, thus allowing increased surface expression and functional diversity of proteins, including CD47 and PD-L1. The TIGER domain is a subcellular compartment that enables formation of specific and functionally relevant protein-protein interactions that cannot be established outside.

Keywords: AU-rich elements; RNA granule; RNA-binding proteins; ZFP36L1; alternative 3′UTRs; collective properties; compartmentalization; membraneless organelle; subcellular organization; subdomain of the endoplasmic reticulum.

Figure 1.. TIS11B assemblies have a reticular pattern and are intertwined with the ER.

(A) The interaction between SET and CD47-LU protein is 3′UTR-dependent, as CD47-SU does not interact with SET. It is unknown how SET is transferred from the 3′UTR to the protein. (B) Fluorescent confocal microscopy of endogenous TIS11B protein in HeLa cells. Transfected GFP-SEC61B visualizes the ER and DAPI stains the nucleus. Two representative cells are shown. Right panel shows higher magnification of demarcated region. (C) Confocal live cell imaging (Airyscan) of HeLa cells after transfection of mCherry (mC)-TIS11B and of GFP-SEC61B to visualize the ER. Shown are three different magnifications. The arrow indicates the plane used for line profile generation. (D) 3D-reconstruction of confocal images, shown as in (C). See also Figure S1.

Figure 2.. TIS granules enrich membrane protein-encoding mRNAs with AREs in a 3′UTR-dependent manner.

(A) GFP-tagged constructs used for RNA-FISH are drawn to scale. Red stars indicate AREs. S, signal peptide. (B) Representative images obtained from RNA-FISH (green) against GFP after transfection of GFP-CD47-LU in HeLa cells. BFP-TIS11B (red) was co-transfected. The white dotted line demarcates the nucleus. Bottom panel shows higher magnification of indicated region. Right panels show line profiles (generated as in Figure 1C) of fluorescence intensities including Pearson′s correlation coefficients (r). (C) As in (B), but after transfection of GFP-CD47-SU. (D) Pearson′s correlation coefficients of line profiles of TIS11B and the indicated mRNAs. N, number of line profiles. Mann Whitney test, ***, P < E-14, **, P = E-6, NS, not significant. (E) As in (B), but after transfection of GFP-CD274-UTR or GFP-CD274-NU. (F) As in (B), but after transfection of GFP-BCL2-LU or GFP-BCL2-NU. See also Figure S2.

Figure 3.. TIS granules enrich specific proteins.

(A) Confocal live cell imaging (Airyscan) of HeLa cells after transfection of mC-TIS11B (red) and GFP-HuR (green). The white dotted line demarcates the nucleus. Line profile as in Figure 2B. (B) As in (A) but after transfection of GFP-TIS11B (red) and mC-HSPA8 (green). (C) Pearson′s correlation coefficients of line profiles of TIS11B and the indicated proteins. N, number of line profiles. See also Figure S3A.

Figure 4.. TIS11B is required for 3′UTR-mediated cell surface localization of CD47 and interaction of SET with CD47-LU.

(A) Co-IP of endogenous SET using GFP-Trap after transfection of GFP-CD47-LU into HeLa cells stably expressing the indicated shRNAs. 2.5 % of input was loaded. TUBULIN was used as additional loading control. shRNA Ctrl, control shRNA. (B) FACS analysis of endogenous CD47 in HeLa cells stably expressing the indicated shRNAs. Mean fluorescent intensity (MFI) values are shown in parentheses. Shown is a representative experiment. Right panel shows mean ± SD of five biological replicates. t-test, ***, P < 0.001. (C) Sequence of TNFα ARE. (D) RNA pulldown of indicated biotinylated RNA oligonucleotides and western blot analysis of endogenous proteins in HeLa cells. 2.5 % of input was loaded. Mock, no RNA was transfected. ARE-1 and ARE-2 differ in the restriction sites used for cloning. HNRNPA1 specifically binds to SU1. TUBULIN served as loading control. (E) FACS analysis of GFP after transfection of the indicated constructs into HeLa cells, shown as in (B). Bottom panel shows surface and total GFP expression as mean ± SD of five biological replicates. t-test, ***, P < 0.001. (F) Co-IP of endogenous SET using GFP-Trap after transfection of the indicated constructs into HeLa cells. 2.5 % of input was loaded. (G) Western blot of the indicated endogenous proteins in HeLa cells stably expressing TIS11B shRNA1. See also Figure S3B–I.

Figure 5.. Model of TIS granule-mediated protein-protein interactions.

(A) FACS analysis of surface and total GFP in HeLa cells after transfection of GFP-CD47-LU together with mC (grey) or mC-SET (green). Representative plots are shown. (B) As in (A), but after transfection of mC-TIS11B (blue). (C) Surface and total GFP-CD47-LU expression from (A) and (B) shown as mean ± SD of three biological replicates. t-test, ***, P < 0.001. (D) Confocal live cell imaging (Airyscan) of HeLa cells after transfection of GFP-TIS11B (red) and mC-SET (green), shown as in Figure 3A. (E) Pearson′s correlation coefficients of line profiles of TIS11B and SET. N, number of line profiles. (F) Model, see text. The TIGER domain is a new subcellular compartment created by the association of TIS granules and the ER. See also Figure S3J.

Figure 6.. The TIGER domain is necessary and sufficient for the protein-protein interaction between SET and membrane proteins.

(A) RNA-FISH as in Figure 2C, but cells with high TIS11B expression were selected. (B) Co-IP of endogenous SET after transfection of GFP-CD47-SU in HeLa cells. GFP-Trap was performed after expression of mC, mC-HuR or high expression of mC-TIS11B. 2.5 % of input was loaded. TUBULIN is shown as loading control. (C) FACS analysis of surface GFP after transfection of GFP-CD47-SU in HeLa cells, in the presence of mC (grey) or in the presence of mC-TIS11B (blue). Cells with high mC-TIS11B expression were analyzed and shown as in Figure 5B. Right panel shows mean ± SD of three biological replicates. t-test, ***, P < 0.001. (D) Co-IP of endogenous SET using GFP-Trap after transfection of GFP-CD274-UTR or GFP-CD274-NU into HeLa cells. 2.5 % of input was loaded. (E) As in (C), but after transfection of GFP-CD274-UTR (blue) or GFP-CD274-NU (grey) into HeLa cells. Endogenous TIS11B expression was used.

Figure 7.. TIS11B is the scaffold of TIS granules whose assembly is charge pattern-driven.

(A) Net charge distribution of protein sections of wild-type (WT) and mutant TIS11B. RBD, RNA-binding domain, IDR, intrinsically disordered domain, CPM, charge pattern mutant. See also Figure S4B, S4C. (B) Confocal live cell imaging of HeLa cells after transfection of the indicated constructs described in (A) fused to mC together with GFP-SEC61B to visualize the ER. (C) FACS analysis of GFP after transfection of GFP-CD47-LU and the constructs from (A) fused to mC into HeLa cells. Normalized GFP expression (MFI values) shown as mean ± SD of five biological replicates. t-test, ***, P < 0.001. (D) RNA-FISH as in Figure 2B against GFP-CD47-LU mRNA after co-transfection of BFP-labeled WT or CPM2 TIS11B (red) in HeLa cells with stable knock-down of endogenous TIS11B. (E) Pearson′s correlation coefficients of line profiles of TIS11B and the indicated mRNAs from (D) and Figure S5B, shown as in Figure 2D. Mann Whitney test, ***, P < E-21. (F) Confocal microscopy of mC-tagged WT or CPM2 TIS11B and GFP-tagged HSPA8 in HeLa cells with stable knock-down of endogenous TIS11B, shown as in Figure 3B. mC was used as control. (G) Pearson′s correlation coefficients of line profiles of TIS11B and the indicated proteins from (F) and Figures S5C and S5D, shown as in Figure 2D. Mann Whitney test, ***, P < E-12. (H) FRAP of GFP-SET in the TIS granule region (mC-TIS11B) or in the cytoplasm located outside of TIS granules, performed in HeLa cells. Mean fluorescence ± SD from 40 different granule and cytoplasmic regions from 21 cells is shown. Mann Whitney test, P = E-41. (I) Confocal imaging of HeLa cells after transfection of GFP-TIS11B (red) and mC-SET (green). Bottom panel, cells were fixed and permeabilized for 1 hour. The white dotted line demarcates the nucleus. Shown are representative images. See also Figures S4, S5, S6, S7, and Table S1.