Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport - PubMed (original) (raw)
Translational regulation of protrusion-localized RNAs involves silencing and clustering after transport
Konstadinos Moissoglu et al. Elife. 2019.
Abstract
Localization of RNAs to various subcellular destinations is a widely used mechanism that regulates a large proportion of transcripts in polarized cells. In many cases, such localized transcripts mediate spatial control of gene expression by being translationally silent while in transit and locally activated at their destination. Here, we investigate the translation of RNAs localized at dynamic cellular protrusions of human and mouse, migrating, mesenchymal cells. In contrast to the model described above, we find that protrusion-localized RNAs are not locally activated solely at protrusions, but can be translated with similar efficiency in both internal and peripheral locations. Interestingly, protrusion-localized RNAs are translated at extending protrusions, they become translationally silenced in retracting protrusions and this silencing is accompanied by coalescence of single RNAs into larger heterogeneous RNA clusters. This work describes a distinct mode of translational regulation of localized RNAs, which we propose is used to regulate protein activities during dynamic cellular responses.
Keywords: RNA granule; RNA transport; cell biology; cell migration; chromosomes; gene expression; human; local translation; mouse; RAB13; NET1; APC-dependent RNAs; puro-PLA; cell protrusion; Suntag; translation reporter; detyrosinated microtubule; RNA localization.
Conflict of interest statement
KM, KY, TW, GC, SM No competing interests declared
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 assessed with nanoString analysis. (B, C) Representative absorbance profiles of polysome gradients of control, puromycin treated (B) or Pkp4-cUTR-expressing cells (C). Inset in (B) shows an enlargement of the polysome region. (D) Heat maps showing RNA presence in polysome gradient fractions, based on nanoString analysis, under the indicated conditions. Gene names are shown on the left. Values indicate averages of 3 independent experiments. Statistically significant differences compared to the corresponding control fractions are indicated by asterisks (2-way ANOVA with Dunnett’s multiple comparisons test).
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 cell body (CB) samples were isolated from control or parthenolide (PTL) treated cells. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n = 3; error bars: standard error). *: p-value<0.04 by two way ANOVA with Bonferroni’s multiple comparisons test against the corresponding control. Parthenolide treatment specifically reduces the enrichment of APC-dependent RNAs at protrusions. (B) Representative absorbance profiles of polysome gradients of control and PTL-treated cells, and heat maps showing RNA presence in polysome gradient fractions, based on nanoString analysis. Gene names are shown on the left. Values indicate averages of 3 independent experiments. No statistically significant differences were detected by 2-way ANOVA with Dunnett’s multiple comparisons test against the corresponding control fractions.
Figure 3.. 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 channel detects the 3x-mCherry-PCP protein. Bright spots correspond to RNA molecules. Diffuse signal results from free 3x-mCherry-PCP. The GFP channel detects the scFv-GFP antibody. Bright spots overlap with RNA spots (merge image) and correspond to nascent protein at translation sites. Diffuse signal results from free scFv-GFP or scFv-GFP bound to the reporter protein released after translation. (C) Cells expressing the control translation reporter, containing PP7 repeats (+PP7), or a reporter without PP7 repeats (-PP7), were imaged live. mCherry intensity overlapping with translation sites (GFP spots) was measured and normalized to the intensity observed in nearby cytoplasmic regions with diffuse signal. Value of 1 indicates that there is no mCherry concentration at translation sites. (D) The same cytoplasmic areas, of cells expressing the control translation reporter, were imaged before and after puromycin addition. GFP/mCherry intensity of individual spots was calculated as a measure of translational efficiency. n > 100 (C) and n > 500 (D) spots from multiple cells observed in three independent experiments; error bars: standard error; ****: p-value<0.0001 by Student’s t-test. Scale bars: 5 μm.
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 lactimidomycin. Graphs indicate the relative change in number of detectable translation sites (one indicating no change). n = 13–17 cells per condition. error bars: standard error; ****: p-value<0.0001 by one way ANOVA with Tukey’s multiple comparisons test. Scale bars: 10 μm.
Figure 4.. 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 distance from the cell edge. More than 200 particles were analyzed from approximately 20 cells. Best fit curves with 95% confidence intervals are overlaid on the graphs. Scale bars: 5 μm. (B) Cumulative frequency distribution plot of translation reporter particles (from panel A) with increasing distance from the cell edge.
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 translation reporter particles detected per cell, after doxycycline addition, by in situ hybridization or live-cell imaging. Expression levels, assessed by FISH, are not significantly higher than endogenous APC-dependent RNAs (compare to values presented in Figure 5—figure supplement 2). Fewer particles are detected by live-cell imaging likely indicating that fast-moving particles cannot be resolved with our current acquisition methods.
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 particles are single molecules.
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 move in a directionally persistent manner during the time of observation. Scale bars: 3 μm. Full length movies of these samples are presented in Video 5 (A) and Video 6 (B).
Figure 5.. 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 each image in microns. In internal regions all detected RNAs exist as single molecules. At the tips of protrusions, they exist in clusters of multiple RNAs. (D) In situ hybridization images and surface plot profiles of endogenous Ddr2 and Net1 RNAs detected in the same cell. Peripheral clusters can contain distinct RNA species. (E) In situ hybridization images and surface plot profiles of endogenous Pkp4 RNA and polyA RNA detected in the same cell. Peripheral clusters are characterized by a visible accumulation of polyA RNA. (Note that only enlarged views of individual protrusions are shown in panels A-E). (F) Whole cell masks of cells processed for FISH were used to derive a 2 μm-wide peripheral edge mask. (G) Whole-cell FISH images of the indicated endogenous RNAs (for additional examples see Figure 5—figure supplement 1). Scale bars: 15 μm. (H) For each RNA, signal intensity histograms of all detected particles found within the 2μm-wide peripheral edge area, were used to group particles into single RNAs or RNA clusters (see Figure 5—figure supplement 1 ). Table lists number of particles in each category for the indicated RNAs. p-values based on Fisher’s exact test against Arpc3 RNA. (I) Percent of overlap of the indicated RNA clusters with polyA clusters. n = number of particles observed in ca. 25 cells.
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 P4hb). For each RNA, the associated graphs present a frequency distribution histogram of the signal intensities (in arbitrary units) of all detected particles found within the 2μm-wide peripheral edge area, as shown in (A). Intensities > 400 were all grouped in one bin. Number of analyzed particles: 150–1500, depending on the RNA, imaged in >25 cells. The existence of one major peak indicates that the majority of analyzed particles reflect single RNA molecules. Particles with higher intensities (>400) reflect homotypic RNA clusters. (See Figure 5H). Note that peripheral clusters of APC-dependent RNAs are not observed in all cells. The amount of each RNA in clusters is a small proportion of the total RNA amount.
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 cell images show that the RNAs exist as single particles in internal regions and as clusters at the tips of protrusions. Scale bars: 5 μm.
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 RNAs at the tips of protrusions. mCherry intensity, distance from the edge and GFP/mCherry intensity are plotted for either single RNAs or RNA clusters observed in the same protrusions. error bars: standard error; n = 10 for RNA clusters, n > 25 for single RNAs, from 4 or eight different cells; p-value: *<0.02, ***<0.001 by Student’s t-test. Scale bars: 5 μm.
Figure 7.. 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. Pre-treatment with cycloheximide (CHX) prevents release of nascent protein. (B) Rab13-puro-PLA signal in primary human dermal fibroblasts transfected with control siRNAs, or siRNAs against Rab13, or pre-treated for 15 min with anisomycin (Aniso), cycloheximide (CHX) or harringtonine (Harr). Representative images from some of the conditions are shown on the left and quantitations in the graph. (C) In situ hybridization of Rab13 and polyadenylated (polyA) RNA in primary dermal fibroblasts. Graph shows the average number of Rab13 RNA particles detected per cell. (D, E) Images as those shown in (C) and (B) respectively were used to quantify a peripheral distribution index (PDI) at different times after plating on fibronectin. (F) Cell area of dermal fibroblasts at various timepoints after plating on fibronectin. Error bars: standard error. Number of cells analyzed in 2–4 independent experiments are shown within each bar. For (F) > 145 cells were analyzed for each timepoint. p-value: **<0.01, ***<0.001 by one-way ANOVA with Dunnett’s multiple comparisons test, compared to control or indicated samples. Scale bars: 15 μm.
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 Video 11. (B) In situ hybridization of Rab13 and polyadenylated (polyA) RNA in MDA-MB-231 cells and PDI quantitations. Arrows point to Rab13 RNA in retracting protrusions. (C) Quantitation of Rab13-puro-PLA signal in MDA-MB-231 cells under the indicated conditions. (D) Representative images of Rab13-puro-PLA and phalloidin staining in MDA-MB-231 cells exhibiting retracting protrusions. Note that Rab13-puro-PLA signal is absent in retracting protrusions (arrows). (E) Rab13-puro-PLA intensity in lamellipodia or retracting protrusions. (See Figure 9A for representative outlines). (F) Percent of retracting protrusions positive for Rab13 RNA or puro-PLA signal based on images such as those shown in (B) and (D). Error bars: standard error. Number of cells analyzed in 2–3 independent experiments are shown within each bar. p-value: **<0.01, ***<0.001, ****<0.0001 by Student’s t-test (B, E) or one-way ANOVA with Dunnett’s multiple comparisons test, compared to control (C). Scale bars: 10 μm.
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 RNA particles within lamellipodia or retracting protrusions of MDA-MB-231 cells, as shown in (A). Intensities > 400 were grouped in one bin and indicate RNA clusters. Table lists numbers of single RNAs or RNA clusters observed in 32 cells. p-value by Fisher’s exact test. Essentially identical results were obtained in three independent experiments. (D) Retracting MDA-MB-231 protrusions (outlined in blue) stained for Rab13 and polyA RNAs. Based on the staining pattern, protrusions were grouped into three categories: Rab13-/polyA- do not exhibit visible Rab13 clusters or obvious local accumulations of polyA RNA; Rab13+/polyA- exhibit clusters of Rab13 RNA (arrows) but no obvious polyA clusters; Rab13+/polyA +exhibit Rab13 clusters which coincide with obvious corresponding polyA clusters (arrows). Values indicate average fraction of protrusions in each category ± standard error. n = 60 from two independent experiments. Scale bars: 10 μm.
Figure 10.. 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 shows scoring of protrusions for the presence of polyA RNA granules. Values are mean and standard error of at least three independent experiments. For each experiment approximately 300 protrusions from more than 25 cells were observed. p-value: *<0.02, **<0.01, ***<0.001 by one-way ANOVA with Dunnett’s multiple comparisons test, compared to control. Scale bars: 10 μm.
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 with accumulated polyA RNA signal indicative of peripheral clusters (upper panels; white arrows). We also observe peripheral polyA RNA clusters that do not contain any detectable Pkp4 RNAs (lower panels; yellow arrowhead). Scale bars: 5 μm (upper panels), 10 μm (lower panels).
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).
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