GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function
- University of Pennsylvania, United States
- University of Massachusetts Medical School, United States
- Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, United States
- Drexel University, United States
Figures
Figure 1 with 2 supplements
The GGGGCC repeat and other microsatellite RNA repeats with high secondary structure content are neuritically localized.
(A–B) GGGGCC repeat RNA is neuritically localized in discrete granules in human iPSNs derived from C9orf72 GGGGCC repeat expansion carriers. (A–C) GGGGCC repeat RNA (red) was detected with a …
Figure 1—figure supplement 1
Neuritic localization of (CAG)100 RNA by in situ hybridization.
(A–B) In situ hybridization of primary rat spinal cord neurons expressing (CAG)100 RNA. The (CAG)100 construct was not MS2-tagged, but contained a leader sequence and translation reporter tags (see F…
Figure 1—figure supplement 2
Microsatellite repeat and control expression constructs.
(A) Constructs used to express control (top three constructs) and microsatellite repeat (bottom nine constructs) RNA in transfected primary rat spinal cord neurons are shown. With the exception of …
Figure 2
Particle formation dependence on CAG and GGGGCC RNA repeat number.
(A–C, E) Rat mixed spinal cord neurons were transfected with NLS-CP-GFP (green; arrowheads) and (A) (CAG)20-MS2, (B) (CAG)40-MS2, (C) (CAG)70-MS2, or (E) (GGGGCC)3-MS2. (CAG)100-MS2 and (GGGGCC)48-MS…
Figure 3 with 1 supplement
(GGGGCC)48 and (CAG)100 RNA assembles into neuritic transport particles and neuritic (GGGGCC)48 RNA correlates with branching defects.
(A–B) Rat spinal cord neurons were transfected with NLS-CP-GFP and (A) (GGGGCC)48-MS2 or (B) (CAG)100-MS2, and the trajectory of motile RNA particles along neuronal processes was captured by …
Figure 3—figure supplement 1
Nuclear (GGGGCC)48-MS2 foci in transfected rat primary spinal cord neurons.
(A) Primary rat spinal cord neurons transfected with NES-CP-GFP (green) and (GGGGCC)48-MS2. (A) Bottom left, a transfected neuron with nuclear (GGGGCC)48-MS2 RNA foci. Top right, a transfected …
Figure 4 with 1 supplement
(GGGGCC)48-induced dendritic arborization defects are modulated by altered levels of transport granule components in Drosophila.
(A–H) Tracings, from confocal Z-series projections, of the cell body and dendritic arbor of class IV da neurons located in the body wall of Drosophila early or late third instar larvae. Expression …
Figure 4—figure supplement 1
Knock down of dFMR1 or orb2 restores (GGGGCC)48-induced branching defects.
(A–G) Tracings from confocal Z-series projections of GAL4477 driven UAS-mCD8::GFP expression (Grueber et al., 2003) in Drosophila class IV da neurons were used for the analyses. (A–E) The GAL4477 …
Figure 5 with 1 supplement
Misregulation of transport granule components in human iPSNs from carriers with a C9orf72 GGGGCC expansion.
(A–C) Neuritic particles consisting of expanded GGGGCC repeat RNA co-label for FMRP. Rat primary spinal cord neurons were transfected with NLS-CP-GFP (green), FMRP-RFP (magenta), and (GGGGCC)48-MS2, …
Figure 5—figure supplement 1
FMRP colocalizes with neuritic (GGGGCC)48-MS2 and (CAG)100-MS2 RNA.
Neuritic particles consisting of expanded GGGGCC or CAG repeat RNA co-label for FMRP. (A–F) Neurons were transfected with NLS-CP-GFP (green), FMRP-RFP (magenta), and (A–C) (GGGGCC)48-MS2, or (D–F) …
Videos
Video 1
Movement of a distal (GGGGCC)48-MS2 particle.
Two identical videos (60 s real-time duration each) are combined vertically; the bottom video displays a tracked particle in green, starting at 22 μm and reaching 40 μm from the cell body. Images …
Video 2
Movement of a distal (CAG)100-MS2 particle.
Two identical videos (40 s real-time duration each) are combined vertically; the bottom video displays the tracked particle and its path in green, starting at 67.0 μm and reaching 111.4 μm from the …
Video 3
Movement of a proximal (CAG)100-MS2 particle.
Two identical videos (97 s real-time duration each) are combined vertically; the bottom video displays the tracked particle and its path in green, starting in the cell body and reaching 6.5 μm into …
Tables
Table 1
Behavior of repeat RNA particles in rat spinal cord neurons.
| Mean average velocity (μm/s) | Average max velocity (μm/s) | Velocity range (μm/s) | Particles tracked in neurites | Particles tracked in cell body | Basal velocity* (μm/s) | |
|---|---|---|---|---|---|---|
| (GGGGCC)48-MS2::GFP n = 11 cells | 1.06 | 1.40 | 0.32–2.67 | 10 | 15 | 0.11 |
| (CAG)100-MS2::GFP n = 2 cells | 1.30 | 1.85 | 0.30–4.73 | 5 | 28 | 0.13 |
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Uninterrupted unidirectional anterograde and retrograde particle runs with an average run distance of 5.3 μm ((GGGGCC)48-MS2), and 6.7 μm ((CAG)100-MS2), were analyzed. (*)The basal velocity is given as a mean average and was estimated by analyzing five particles that underwent corralled movements with an average net displacement of <0.51 μm within 20s. Data are from four (GGGGCC)48-MS2 and two (CAG)100-MS2 independent live imaging sessions. (GGGGCC)48-MS2 and (CAG)100-MS2 were co-expressed with NLS-CP-GFP. CP-GFP, MS2 RNA-binding coat protein fused with green fluorescent protein; NLS, nuclear localization signal.
Table 2
Expression of transport-granule related transcripts in brains of C9orf72 patients.
| Expression in C9orf72 Cortex | Gene subset | Expected percentage | Observed percentage | Chi-squared p-value |
|---|---|---|---|---|
| Upregulated | RNA binding proteins | 7.5 | 15.10 (167/1103) | p<0.0001 |
| mRNA binding proteins | 3.4 | 7.80 (86/1103) | p<0.0001 | |
| FMRP targets | 4.2 | 4.90 (54/1103) | p=0.2568 | |
| STAT5B targets | 3.1 | 6.07 (67/1103) | p<0.0001 | |
| Downregulated | RNA binding proteins | 7.5 | 4.93 (129/2618) | p<0.0001 |
| mRNA binding proteins | 3.4 | 1.72 (45/2618) | p<0.0001 | |
| FMRP targets | 4.2 | 3.40 (89/2618) | p=0.0389 | |
| STAT5B targets | 3.1 | 1.38 (36/2618) | p<0.0001 |
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Comparison of uniquely-identified protein coding genes that were either up- or down-regulated in cortical samples from C9orf72 patients (Donnelly et al., 2013) with transcripts associated with the regulation of RNA. Supplementary file 1 lists the RNA binding proteins upregulated and downregulated, as noted above.
Additional files
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Supplementary file 1
Genes related to RNA granules that are misregulated in brain tissue from C9ORF72 patients.
Lists of the RNA binding proteins upregulated or downregulated in cortical samples from C9orf72 patients (Donnelly et al., 2013). Analysis is presented in Table 2.
- https://doi.org/10.7554/eLife.08881.018
