Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport
- National Cancer Institute, National Institutes of Health, United States
- Hiroshima University, Japan
- RIKEN Center for Biosystems Dynamics Research, Japan
Figures
Figure 1
Disrupting localization of APC-dependent RNAs, through competition, does not alter their translation.
(A) Outline of experimental procedure. Sucrose gradients are divided into four fractions based on UV absorbance, an equal amount of spike RNA is added to each, and RNA presence is quantitatively …
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Figure 1—source data 1
File containing values used for generation of the heatmaps and statistics of Figure 1D.
- https://doi.org/10.7554/eLife.44752.003
Figure 2
Disrupting localization of APC-dependent RNAs, through perturbation of detyrosinated microtubules, does not alter their translation.
(A) Schematic on the left indicates experimental procedure used for isolation of protrusions. Migration of cells through microporous filters was induced by addition of LPA and protrusion (Ps) and …
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Figure 2—source data 1
File containing values used for generation of the heatmaps and statistics of Figure 2B.
- https://doi.org/10.7554/eLife.44752.005
Figure 3 with 1 supplement
Validation of single-molecule translation reporter assay.
(A) Schematic of translation reporter constructs for labeling of RNA and nascent protein chains. (B) Live cell imaging snapshot of a cell expressing the control translation reporter. The mCherry …
Figure 3—figure supplement 1
Translation signal of localized reporters reflects active translation.
Cells expressing the indicated translation reporters were imaged just prior to (t = 0 min), or 15 min (t = 15 min) after, addition of the translation inhibitors cycloheximide, harringtonine or …
Figure 4 with 3 supplements
RNAs targeted to protrusions are similarly translated in both internal and peripheral locations.
(A) Live imaging snapshots of cells expressing the indicated translation reporters. GFP/mCherry intensity of individual spots (indicating translation efficiency) was plotted as a function of …
Figure 4—figure supplement 1
Expression levels of translation reporters and comparison with live-cell imaging.
(A) In situ hybridization of cells expressing the indicated translation reporters in the absence or presence of doxycycline. Scale bars: 5 μm. Red line:cell outline. (B) Comparison of the number of …
Figure 4—figure supplement 2
Intensity histograms of translation reporter particles.
Frequency distribution plots of mCherry intensities exhibited by particles of the indicated translation reporters. The existence of a single major peak suggests that the majority of observed …
Figure 4—figure supplement 3
Examples of directionally persistent particles.
(A, B) Snapshots of time lapse imaging of cells expressing the indicated localized translation reporters. The GFP channel was recorded showing translated RNAs. Arrowheads point to particles that …
Figure 5 with 3 supplements
APC-dependent RNAs associate with heterogeneous clusters at the tips of protrusions.
(A-C) The indicated endogenous RNAs were detected by in situ hybridization. Signal intensities of observed spots are shown in the associated surface plot profiles, which also indicate the size of …
Figure 5—figure supplement 1
Intensity histograms of endogenous APC-dependent or control RNAs.
(A) Whole cell masks of cells processed for FISH were used to derive a 2 μm-wide peripheral edge mask. (B) FISH images of the indicated endogenous APC-dependent RNAs or non-localized RNAs (Arpc3 and …
Figure 5—figure supplement 2
Amount of APC-dependent RNAs per cell.
Graph shows the average number of the indicated RNAs detected per cell, based on FISH images such as those shown in Figure 5—figure supplement 1. n > 25 cells; error bars: standard error.
Figure 5—figure supplement 3
Peripheral cluster formation by MS2-reporter RNAs.
Localized reporter RNAs, which carry the 3’UTR of Rab13 or Net1 RNAs, as well as 24 copies of MS2-binding sites, were expressed in cells expressing GFP-tagged MS2 coat protein. Snapshots of live …
Figure 6
RNA clusters at the tips of protrusions are translationally silent.
(A, B) Imaging of cells expressing localized translation reporters carrying either the Pkp4 (A) or Rab13 (B) UTRs. White arrowheads point to single RNA molecules. Yellow arrows point to clustered …
Figure 7 with 1 supplement
Endogenous Rab13 RNA is translated in both internal and peripheral locations, and is silenced at the periphery.
(A) Schematic depicting nascent Rab13 protein detection through puro-PLA. Puromycylation leads to detection of both Rab13 released from ribosomes as well as nascent Rab13 at translation sites. …
Figure 7—figure supplement 1
Rab13 protein levels.
Western blot to assess expression and degree of knockdown of Rab13 in lysates of primary human dermal fibroblasts.
Figure 8
Peripheral Rab13 RNA is silenced at retracting protrusions.
(A) Snapshots of time lapse imaging of MDA-MB-231 cells expressing Lifeact-GFP. Arrow points to protrusion that retracts within a few minutes. The full-length movie of this sample is presented in Vid…
Figure 9
Silenced Rab13 RNA at retracting protrusions can be found in heterogeneous clusters.
(A) Outlines of ‘lamellipodia’ or ‘retracting protrusion’ regions used for quantitations Scale bar:10 μm. (B, C) Frequency distribution histograms of signal intensities (in arbitrary units) of Rab13 …
Figure 10 with 1 supplement
Formation of RNA clusters at protrusions is promoted by translational inhibition and requires microtubules.
PolyA RNA was detected in NIH/3T3 cells with the indicated treatments. Boxed regions are enlarged to show the presence (arrows) or absence of polyA RNA granules at the tips of protrusions. Graph …
Figure 10—figure supplement 1
PolyA RNA staining is a more reliable identifier of peripheral clusters in 3T3 cells.
Enlarged views of individual protrusions from cells processed by in situ hybridization to detect the Pkp4 RNA or polyadenylated RNA. Single Pkp4 RNAs can be observed at protrusions in association …
Author response image 1
MDA-MB-231 cells were treated, or not, with harringtonine for 15min.
Fractions of polysome gradients were isolated and analyzed by RT-ddPCR to detect two localized, APC-dependent RNAs (Rab13 and Net1) and a non-localized control RNA (GAPDH).
Videos
Video 1
NIH/3T3 cell expressing scFv-GFP (green), 3x-mCherry-PCP (red) and the control translation reporter.
Frames were acquired sequentially and with no time delay, for the duration of the movie (45 s). A merged image of the two channels is shown. Overlapping red and green spots indicate translation …
Video 2
NIH/3T3 cell expressing scFv-GFP (green), 3x-mCherry-PCP (red) and the control translation reporter.
Frames were acquired sequentially and with no time delay, for the duration of the movie (13 s). A merged image of the two channels is shown. Blue line: cell outline. Scale bar: 5 μm. Single frames …
Video 3
NIH/3T3 cell expressing scFv-GFP (green), 3x-mCherry-PCP (red) and the localized translation reporter carrying the Pkp4 UTR.
Frames were acquired sequentially and with no time delay, for the duration of the movie (36 s). A merged image of the two channels is shown. Blue line: cell outline. Scale bar: 5 μm. Single frames …
Video 4
NIH/3T3 cell expressing scFv-GFP (green), 3x-mCherry-PCP (red) and the localized translation reporter carrying the Rab13 UTR.
Frames were acquired sequentially and with no time delay, for the duration of the movie (63 s). A merged image of the two channels is shown. Blue line: cell outline. Cyan line: nucleus outline. …
Video 5
NIH/3T3 cell expressing scFv-GFP (green), 3x-mCherry-PCP and the localized translation reporter carrying the Rab13 UTR.
Images of the GFP channel were acquired sequentially and with no time delay, for the duration of the movie (13 s). Blue line: cell outline. Scale bar: 3 μm. Single frames of this movie are shown in F…
Video 6
NIH/3T3 cell expressing scFv-GFP (green), 3x-mCherry-PCP and the localized translation reporter carrying the Pkp4 UTR.
Images of the GFP channel were acquired sequentially and with no time delay, for the duration of the movie (19 s). Blue line: cell outline. Scale bar: 3 μm. Single frames of this movie are shown in F…
Video 7
NIH/3T3 cell expressing tdMCP-GFP (green) and a MS2-reporter RNA carrying the Rab13 UTR.
Images of the GFP channel were acquired sequentially and with no time delay, for the duration of the movie (29 s). A single frame of this movie is shown in Figure 5—figure supplement 3. Fainter …
Video 8
NIH/3T3 cell expressing tdMCP-GFP (green) and a MS2-reporter RNA carrying the Net1 UTR.
Images of the GFP channel were acquired sequentially and with no time delay, for the duration of the movie (22 s). A single frame of this movie is shown in Figure 5—figure supplement 3. Fainter …
Video 9
Primary human dermal fibroblast expressing Lifeact-GFP.
Images were acquired every 6 min for a total of 3 hr. Note that each protrusion is very dynamic undergoing retracting and extending phases within minutes.
Video 10
Primary human dermal fibroblast expressing Lifeact-GFP.
Images were acquired every 6 min for a total of 3 hr. Note that each protrusion is very dynamic undergoing retracting and extending phases within minutes.
Video 11
MDA-MB-231 cell expressing Lifeact-GFP.
Images were acquired every 3 min for a total of 51 min. Arrow points to protrusion that retracts over a period of few minutes. Note that lamellipodial regions undergo constant dynamic retracting and …
Video 12
MDA-MB-231 cell expressing Lifeact-GFP.
Images were acquired every 3 min for a total of 51 min. Arrow points to protrusion that retracts over a period of few minutes. Note that lamellipodial regions undergo constant dynamic retracting and …
Video 13
NIH/3T3 fibroblast expressing Lifeact-GFP.
Images were acquired every 6 min for a period of 1 hr. Note that, in comparison to MDA-MB-231 cells, NIH/3T3 cells exhibit much slower dynamics with protrusions persisting relatively unchanged for …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | MDA-MB-231 | ATCC | ATCC Cat# HTB-26, RRID: CVCL_0062 | |
| Cell line (Mus musculus) | NIH/3T3 | ATCC | RRID:CVCL_0594 | |
| Cell line (Homo sapiens) | Primary dermal fibroblasts | provided by Dr. Ramiro Iglesias-Bartolome (NCI, NIH) | ||
| Cell line (Mus musculus) | NIH/3T3-PCFvGdelta1-PP7 (control translation reporter) | This study | NIH/3T3 cells expressing SunTag-based control translation reporter. | |
| Cell line (Mus musculus) | NIH/3T3-PCFvGdelta1-deltaPP7 (control translation reporter) | This study | NIH/3T3 cells expressing SunTag-based control translation reporter with deletion of the PP7 repeats. | |
| Cell line (Mus musculus) | NIH/3T3-PCFvGdelta1-Rab13U (translation reporter/Rab13 UTR) | This study | NIH/3T3 cells expressing SunTag-based translation reporter with the mouse Rab13 UTR. | |
| Cell line (Mus musculus) | NIH/3T3-PCFvGdelta1-Pkp4U (translation reporter/Pkp4 UTR) | This study | NIH/3T3 cells expressing SunTag-based translation reporter with the mouse Pkp4 UTR. | |
| Cell line (Mus musculus) | NIH/3T3-tdMCP-GFP_pIND20-b24bs/Net1 (MS2 reporter/Net1 UTR) | This study | NIH/3T3 cells expressing MS2 reporter with the mouse Net1 UTR. | |
| Cell line (Mus musculus) | NIH/3T3-tdMCP-GFP_pIND20-b24bs/Rab13 (MS2 reporter/Rab13 UTR) | This study | NIH/3T3 cells expressing MS2 reporter with the mouse Rab13 UTR. | |
| Antibody | anti-Rab13 rabbit polyclonal | Novus Biologicals | NBP1-85799 | (1:1,000 WB; 1:200 PLA) |
| Antibody | anti-GAPDH (14C10) rabbit monoclonal | Cell Signaling | 2118 | (1:2,000 WB) |
| Antibody | anti-puromycin (3RH11) mouse monoclonal | Kerafast | EQ0001 | (1:2,000 PLA) |
| Recombinant DNA reagent | pHR-tdPP7-3x-mCherry | Addgene | 74926 | |
| Recombinant DNA reagent | pcDNA4TO-24xGCN4_v4-kif18b-24xPP7 | Addgene | 74928 | |
| Recombinant DNA reagent | pcDNA4TO-24xGCN4_v4-kif18b | Addgene | 74934 | |
| Recombinant DNA reagent | pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE | Addgene | 60906 | |
| Recombinant DNA reagent | pHR-scFv-GCN4-sfGFP-GB1-deltaNLS-dWPRE | This study | Plasmid expressing scFv against GCN4 peptide of the SunTag system, fused to sfGFP, without NLS. Derived from pHR-scFv-GCN4-sfGFP-GB1-NLS-dWPRE after introduction of stop codon before the NLS sequence. | |
| rRcombinant DNA reagent | pInducer 20 | Addgene | 44012 | |
| Recombinant DNA reagent | Phage-ubc-nls-ha-tdMCP-gfp | Addgene | 40649 | |
| Recombinant DNA reagent | pInducer20-24xGCN4_v4-kif18b-24xPP7-Rab13 UTR | This study | Dox-inducible translation reporter carrying the mouse Rab13 3'UTR | |
| Recombinant DNA reagent | pInducer20-24xGCN4_v4-kif18b-24xPP7-Pkp4 UTR | This study | Dox-inducible translation reporter carrying the mouse Pkp4 3'UTR | |
| Recombinant DNA reagent | pInducer20-beta24bs-Rab13 UTR | This study | Dox-inducible MS2 reporter carrying the mouse Rab13 3'UTR | |
| Recombinant DNA reagent | pInducer20-beta24bs-Net1 UTR | This study | Dox-inducible MS2 reporter carrying the mouse Net1 3'UTR | |
| Sequence-based reagent | All Stars Negative control siRNA | Qiagen | 1027281 | |
| Sequence-based reagent | Rab13 siRNA, si-Rab13 #8 | Qiagen | SI02662702 | target sequence: 5’-ATGGTCTTTCTTGGTATTAAA-3’ |
| Sequence-based reagent | FISH probes against mouse Net1 | Thermo Fisher Scientific | VB1-3034209 | |
| Sequence-based reagent | FISH probes against mouse Cyb5r3 | Thermo Fisher Scientific | VB1-18647 | |
| Sequence-based reagent | FISH probes against mouse Cenpb | Thermo Fisher Scientific | VB1-18648 | |
| Sequence-based reagent | FISH probes against mouse Rab13 | Thermo Fisher Scientific | VB1-14374 | |
| Sequence-based reagent | FISH probes against human Rab13 | Thermo Fisher Scientific | VA1-12225 | |
| Sequence-based reagent | FISH probes against mouse Pkp4 | Thermo Fisher Scientific | VB4-600264 | |
| Sequence-based reagent | FISH probes against human Kif18b | Thermo Fisher Scientific | VA6-3170686 | |
| Sequence-based reagent | FISH probes against mouse Ddr2 | Thermo Fisher Scientific | VB1-14375 | |
| Sequence-based reagent | FISH probes against mouse Arpc3 | Thermo Fisher Scientific | VB1-14507 | |
| Sequence-based reagent | FISH probes against mouse P4hb | Thermo Fisher Scientific | VB6-15898 | |
| Sequence-based reagent | Custom-made codeset | NanoString Technologies | Item # 116000002 | |
| Commercial assay or kit | Duolink In Situ Red kit | Sigma Aldrich | DUO92101 | |
| Commercial assay or kit | ViewRNA ISH Cell Assay kit | Thermo Fisher Scientific | QVC0001 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44752.036
