In vivo reconstitution finds multivalent RNA–RNA interactions as drivers of mesh-like condensates
- Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, United States
- Structural Biology Program, Memorial Sloan Kettering Cancer Center, United States
Figures
Figure 1 with 2 supplements
RNA determines the morphology of dynamic mesh-like RNA granules in cells.
(A) Confocal live-cell imaging of HeLa cells after the transfection of mCherry-tagged TIS11B. GFP-SEC61B was co-transfected to visualize the endoplasmic reticulum. The white dotted line demarcates …
Figure 1—figure supplement 1
Mutation of the TIS11B RNA-binding domain generates sphere-like granules in cells.
(A) Fluorescence recovery after photobleaching of TIS11B 5 hr after transfection of mGFP-TIS11B into HeLa cells. Scale bar, 1 µm. (B) Shown is the amino acid sequence of the wild-type (WT) TIS11B …
Figure 1—figure supplement 2
In the context of various multivalent domains, the TIS11B RNA-binding domain generates mesh-like condensates in vivo.
(A) Confocal live-cell imaging of HeLa cells after the transfection of mCherry-tagged TIS11B RNA-binding domain (RBD). GFP-SEC61B was co-transfected to visualize the endoplasmic reticulum (ER). The …
Figure 2 with 2 supplements
Specific RNAs induce formation of dynamic mesh-like condensates in vitro.
(A) Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 16 hr of …
Figure 2—figure supplement 1
Specific RNAs induce mesh-like condensates in vitro.
(A) Schematic of the FUS-TIS chimeric protein. (B) SDS-PAGE of the purified FUS-TIS protein. FUS-TIS was tagged with His-MBP-mGFP at the N-terminus and with Strep-tag II at the C-terminus and …
Figure 2—figure supplement 2
Specific RNAs induce mesh-like condensates in vitro.
(A–D) Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 2 hr of …
Figure 3 with 5 supplements
RNAs predicted to have large disordered regions have a high propensity to induce network formation in vitro.
(A) Distribution of length of sphere- and network-forming RNAs. Mann–Whitney test, Z = −2.76, p=0.004. See also Figure 3—source data 1. (B) Number of AU-rich elements in sphere- and network-forming …
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Figure 3—source data 1
Length, number of AU-rich elements, GC-content, and normalized ensemble diversity values of the 47 experimentally tested 3′UTRs.
- https://cdn.elifesciences.org/articles/64252/elife-64252-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Specific RNAs with various lengths induce mesh-like condensates in vitro.
Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 16 hr of incubation. …
Figure 3—figure supplement 2
RNA alone does not induce phase separation in vitro.
Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the absence or presence of the indicated in vitro transcribed RNAs after 16 hr of incubation. …
Figure 3—figure supplement 3
Predicted RNA secondary structures and their corresponding normalized ensemble diversity values for examples of sphere-forming, network-forming, and highly structured RNAs.
(A, B) Centroid RNA secondary structure of two sphere-forming 3′UTRs predicted by RNAfold. The color code represents base-pairing probability. See also the URLs in Figure 3—source data 1. (C, D) As …
Figure 3—figure supplement 4
The normalized ensemble diversity (NED) value of RNAs is highly predictive for their ability to form sphere- or mesh-like condensates.
(A) Totally 10 out of 11 RNAs with a high NED value were predicted correctly to induce network formation. Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS …
Figure 3—figure supplement 5
In a size-restricted dataset, the number of AU-rich elements does not predict mesh-like condensate formation.
(A) Number of AU-rich elements in RNAs with high normalized ensemble diversity (NED) or low NED values. See also Figure 3—source data 1. Mann–Whitney test, NS, not significant. (B) Nucleotide …
Figure 4
RNAs work additively to induce formation of granule networks in vitro.
(A) RNAs co-localize in mesh-like condensates. Representative confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of two different in vitro …
Figure 5 with 1 supplement
A multivalent RNA matrix is responsible for mesh-like condensate formation in vitro.
(A) Schematic of RNAs with strong local secondary structures that are predicted to induce spherical condensates. (B) Schematic of RNAs with large disordered regions that form extensive …
Figure 5—figure supplement 1
Extensive intermolecular RNA–RNA interactions are responsible for formation of mesh-like condensates in vitro.
(A) The TLR8 3′UTR (gray) and the MYC 3′UTR (green) are predicted by RNAfold to dimerize. The secondary structure prediction shows extensive base-pairing between the two RNAs, indicated by the black …
Figure 6 with 1 supplement
Formation of an extensive mRNA network is sufficient for the reconstitution of mesh-like condensates in vivo.
(A) Representative confocal image of in vivo reconstitution of mesh-like condensates using the MS2 system. mCherry-SUMO-SIM fused to the MS2 coat protein was transfected into HeLa cells. Constructs …
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Figure 6—source data 1
Transcriptome-wide analysis on normalized ensemble diversity values of 3′UTRs.
- https://cdn.elifesciences.org/articles/64252/elife-64252-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
mRNAs with large disordered regions are enriched in AU-rich elements.
(A) The number of AU-rich elements in 3′UTRs correlates with 3′UTR length. The dataset contains all mRNAs expressed in HeLa cells. 3′UTRs without AU-rich elements, N = 2504, 3′UTRs with 1–3 AU-rich …
Figure 7 with 1 supplement
An RNA matrix prevents full fusion of spherical condensates, thus promoting arrangement into filamentous structures in vitro.
(A) Confocal 3D time-lapse imaging of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of the TLR8 3′UTR (200 nM) after 30 min of incubation. Scale bar, 2 µm. …
Figure 7—figure supplement 1
Extensive intermolecular RNA–RNA interactions are responsible for formation of mesh-like condensates in vitro.
(A) Representative high-resolution confocal images of phase separation experiments using purified mGFP-FUS-TIS (10 µM) in the presence of Cy5-labeled TLR8 3′UTR RNA after 16 hr of incubation. Scale …
Tables
Table 1
Chimeric proteins investigated for mesh-like condensate formation.
RBDmut, RNA-binding domain mutant.
| Multivalent domain | RNA-binding domain | RNA | Diffusive pattern | Sphere-like condensate | Mesh-like condensate |
|---|---|---|---|---|---|
| TIS11B | √ | ||||
| HuR | √ | ||||
| FUS IDR | √ | ||||
| SUMO-SIM | √ | ||||
| TIS11B-N/C | TIS11B RBDmut | √ | |||
| TIS11B-N/C | TIS11B | √ | |||
| TIS11B-N/C | HuR | √ | |||
| FUS IDR | TIS11B | √ | |||
| FUS IDR | HuR | √ | |||
| SUMO-SIM | TIS11B | √ | |||
| SUMO-SIM | MS2 | √ | |||
| SUMO-SIM | MS2 | Singlevalent RNA | √ | ||
| SUMO-SIM | MS2 | Multivalent RNA | √ |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HeLa | Jonathan S. Weissman | N/A | A human cervical cancer cell line (female origin). |
| Strain, strain background (Escherichia coli) | BL21(DE3) | NEB | C2527H | Chemically competent E. coli cells. |
| Antibody | Anti-α-tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T9026, RRID:AB_477593 | WB (1:5000). |
| Antibody | Anti-mCherry (mouse monoclonal) | Abcam | Cat# ab125096, RRID:AB_11133266 | WB (1:5000). |
| Antibody | Anti-HuR (rabbit polyclonal) | Millipore | Cat# 07-1735, RRID:AB_1977173 | WB (1:2000). |
| Antibody | IRDye 680RD anti-rabbit IgG secondary antibody (donkey polyclonal) | LI-COR Biosciences | Cat# 926-68073, RRID:AB_10954442 | WB (1:10,000). |
| Antibody | IRDye 800CW anti-mouse IgG secondary antibody (donkey polyclonal) | LI-COR Biosciences | Cat# 926–32212, RRID:AB_621847 | WB (1:10,000). |
| Transfected construct (human) | pcDNA-SP-GFP-CD47-LU | Berkovits and Mayr, 2015 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-GFP-ELAVL1-LU | Ma and Mayr, 2018 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-SP-GFP-CD274-UTR | Ma and Mayr, 2018 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-SP-GFP-FUS-UTR | Ma and Mayr, 2018 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-GFP-SEC61B | Ma and Mayr, 2018 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-SEC61B | Ma and Mayr, 2018 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-TIS11B | Ma and Mayr, 2018 | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-TIS11B CC | This paper | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-TIS11B FF | This paper | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-TIS11B KK | This paper | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-TIS11B RK | This paper | N/A | See Materials and methods. |
| Transfected construct (human) | pcDNA-mCherry-TIS-HuR RBD | This paper | N/A | See Materials and methods. |
| Transfected construct (human) | pmCherry-SUMO10-SIM5 | Liam J. Holt (NYU) | N/A | See Materials and methods. |
| Transfected construct (human) | pmCherry-SUMO10-SIM5-TIS | This paper | N/A | See Materials and methods |
| Transfected construct (human) | pcDNA-mGFP-FUS-TIS | This paper | N/A | See Materials and methods. |
| Recombinant DNA reagent | pET28a | Dirk Remus (MSKCC) | N/A | Bacterial expression vector. |
| Recombinant DNA reagent | pDZ2087 | Addgene | Cat# 92414 | Bacterial expression of TEV protease. |
| Recombinant DNA reagent | pET28a-6xHis-MBP-mGFP-FUS-TIS-Strep-Tag II | This paper | N/A | Bacterial expression of 6xHis-MBP-mGFP-FUS-TIS-Strep Tag II. See Materials and methods. |
| Recombinant DNA reagent | T7-CD47 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-ELAVL1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-CD274 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-FUS 3′UTR | This paper | N/A | T7 RNA polymerase- based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-CD44 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-VSIG10 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-IL10 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-TNFSF11 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-GPR39 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-TLR8 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-GPR34 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-TNFAIP6 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-MYC 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-PLA2G4A 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-HEATR5B 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-PPP1R3F 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-DRD1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-FAM72B 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-MCOLN2 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-TSPAN13 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-LHFPL6 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-FAM174A 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-VPS29 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-ADPGK 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-ASPN 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-CASP8 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-CLCA2 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-EOMES 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-ESCO1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-GLYATL3 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-HNRNPH3 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-HOGA1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-LPAR4 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-LRBA 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-LYPLAL1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-ODF2 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-PRKDC 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-RHOA 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-SHQ1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-SLC39A6 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-SLC5A9 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-SMIM3 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-SNTN 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-SOSTDC1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-STBD1 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-TP53TG3 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Recombinant DNA reagent | T7-TTC17 3′UTR | This paper | N/A | T7 RNA polymerase-based in vitro transcription. See Materials and methods. |
| Sequence-based reagent | Biotinylated RNA oligo, TNFα ARE-1 | Ma and Mayr, 2018 | RNA oligonucleotides | 5′-CACUUGUGAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUUA −3′ |
| Peptide, recombinant protein | FUS-TIS | This paper | N/A | Recombinant 6xHis-MBP-mGFP-FUS-TIS-Strep Tag II protein purified from bacteria. See Materials and methods. |
| Peptide, recombinant protein | Bovine serum albumin (BSA) | New England Biolab | Cat# B9000S | |
| Commercial assay or kit | Streptavidin C1 beads | Invitrogen | Cat# 65002 | Streptavidin pulldown assay. |
| Commercial assay or kit | QuikChange Lightning Multi Site-Directed Mutagenesis Kit | Agilent Technologies | Cat# 210513 | Site-directed mutagenesis. |
| Commercial assay or kit | MEGAscript T7 Transcription Kit | Invitrogen | Cat# AMB13345 | In vitro T7 transcription. |
| Commercial assay or kit | Quick Star Bradford Protein Assay Kit | Bio-Rad | Cat# 5000202 | Bradford assay –protein quantitation. |
| Chemical compound, drug | Lipofectamine 2000 | Invitrogen | Cat# 11668019 | |
| Chemical compound, drug | Dextran T500 | PHARMACOSMOS | Cat# 40030 | |
| Chemical compound, drug | Desthiobiotin | Sigma-Aldrich | Cat# D1411-1G | |
| Chemical compound, drug | Zinc chloride | Sigma-Aldrich | Cat# 793523-100G | |
| Chemical compound, drug | Imidazole | Sigma-Aldrich | Cat# I2399-100G | |
| Chemical compound, drug | IPTG | Gold Biotechnology | Cat# I2481-EZ10 | |
| Chemical compound, drug | PMSF | Sigma-Aldrich | Cat# 11359061001 | |
| Chemical compound, drug | DTT | Sigma-Aldrich | Cat# 10708984001 | |
| Software, algorithm | FIJI | NIH | https://fiji.sc/ | |
| Software, algorithm | ZEN | ZEISS | https://www.zeiss.com/microscopy/int/downloads/zen.html | |
| Software, algorithm | GraphPad Prism 7 | GraphPad Software | https://www.graphpad.com/scientific-software/prism | |
| Software, algorithm | Odyssey | LI-COR Biosciences | https://www.licor.com/bio/products/imaging_systems/odyssey/ | |
| Other | Ni-NTA Agarose | Qiagen | Cat# 30230 | His tag purification. |
| Other | StrepTrap column | GE Healthcare | Cat# 28907547 | Strep tag II purification. |
| Other | Amicon Ultra-centrifugal filters-50K | EMD Millipore | Cat# UFC905024 | Concentrating protein samples. |
| Other | 384-well glass-bottom microplate | Greiner Bio-One | Cat# M4437-16EA | Glass-bottom microplate for confocal imaging. |
