Dechorionated embryos were fixed at the interface of a 1:1 solution of 10% formaldehyde:100% heptane for 20 min, followed by 100% methanol devitellinization. After permeabilization in ethanol, embryos were washed 4 times for 15 min in PBT and then once for 20 min in Wash Buffer (10% 20× SCC, 10% formamide). They were then incubated overnight at 37 °C in Hybridization Buffer (10% formamide, 10% 20× SSC, 400 µg/mL tRNA, 5% Dextran sulfate, 1% VRC (Vanadyl Ribonucleoside Complexes, Sigma)) with anti-nos probes (Supplementary information, Table S2 ) coupled to CAL Fluor Red 590 (Stellaris). Embryos were washed in Wash Buffer at 37 °C and then in 2× SCC, 0.1% Tween at room temperature before mounting (Pro-Long Gold antifade reagent, Invitrogen). Microscopy was performed using a Leica SP8 confocal scanning microscope. Data were processed and analyzed using the ImageJ software.
Ribonucleoside vanadyl complexes
Manufactured by Merck Group
Sourced in United States
Ribonucleoside vanadyl complexes are a type of lab equipment used for various research applications. They are compounds formed by the complexation of vanadium ions with ribonucleosides. The core function of these complexes is to serve as tool compounds for investigating the interactions and properties of ribonucleosides in different experimental settings.
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Lab products found in correlation
Sw41ti rotor, by Beckman Coulter (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Sp8 scanning confocal microscope, by Leica (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Mammalian protease inhibitor cocktail, by Merck Group (1 mentions)
Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions)
Rnase free dnase, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Rnase a, by Qiagen (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
Anti c myc agarose conjugated beads, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Ultracentrifugation system, by Beckman Coulter (1 mentions)
Density fractionation system, by Brandel Inc (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
α actinin antibody, by Merck Group (1 mentions)
μ slide 8 well chamber, by Ibidi (1 mentions)
13 protocols using ribonucleoside vanadyl complexes
1
In Situ Hybridization of nos mRNA
2
Affinity Purification and RNase Treatment for Protein Interaction Analysis
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For MS sequence determination, HEK 293T cells in T75 flasks were transfected using FuGENE HD (Promega) with 15 μg of FL-SMCR8, C9orf72-FL, or pcDNA6/myc-His B empty vector and expanded for approximately 45 h, followed by whole cell lysate preparation by sonication using a Diagenode Bioruptor. IP and sample recovery were as previously described [75 (link), 76 ]. Treatment of samples with 25 μg/ml DNase-free RNase (Roche) and 25 μg/ml RNaseA (Qiagen) was conducted in the absence of RNase inhibitors.
For other protein extracts, tissues or cells were lysed in RIPA buffer (Sigma) with Mammalian Protease Inhibitor Cocktail and phenylmethanesulfonyl fluoride (Sigma) and homogenized by Diagenode Bioruptor. For tissues, 2 mm ziconium silicate beads (Next Advance, Inc.) were added to the tubes. Supernatants were recovered by centrifugation at 11K rpm at 4 °C for 15 min and resuspended in 3X SDS loading buffer.
For each interaction co-IP (Fig.2 ), extracts from approximately 5 × 106 293T cells in T75 flasks transfected with tagged SMCR8 and test protein constructs were prepared in 700 μl of lysis buffer (160 mM NaCl, 50 mM Tris, 1 mM EDTA, 0.25% NP40) containing protease and phosphatase inhibitors (Sigma), and RNasin (Qiagen) and vanadyl ribonucleoside complexes (Sigma) as required, and immunoprecipitated as previously described [75 (link), 76 ].
For other protein extracts, tissues or cells were lysed in RIPA buffer (Sigma) with Mammalian Protease Inhibitor Cocktail and phenylmethanesulfonyl fluoride (Sigma) and homogenized by Diagenode Bioruptor. For tissues, 2 mm ziconium silicate beads (Next Advance, Inc.) were added to the tubes. Supernatants were recovered by centrifugation at 11K rpm at 4 °C for 15 min and resuspended in 3X SDS loading buffer.
For each interaction co-IP (Fig.
3
Cell Lysis and mRNP Extraction
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After 48 h from transfection, cells were collected by centrifugation (1000× g) at 4 °C for 10 min and washed several times with 10 mL of ice-cold PBS. Cell pellets were lysated with an equal volume of polysomal lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.0, 0.5% NP-40) with protease inhibitor cocktail and freshly added 1 mM DTT, 100 U/mL RNase inhibitor (RNaseOut, Invitrogen, Waltham, MA, USA) and 400 mM Vanadyl ribonucleoside complexes (Sigma-Aldrich). mRNP lysate was incubated on ice for 5 min and stored at −80 °C. mRNP lysates were thawed on ice and centrifuged at 15,000× g for 15 min to clear lysate of large particles. Cleared supernatants were transferred to new tubes and stored on ice.
4
Immunoprecipitation of C-Myc Complexes
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Anti-C-Myc-agarose conjugated beads (Sigma-Aldrich) were pre-incubated in NT2 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) supplemented with 5% BSA for at least 1 h before use. After several washes, beads were resuspended in 850 μL of ice-cold NT2 buffer with 200 units of RNase inhibitor (RNase Out, Invitrogen, Waltham, MA, USA), 400 mM of Vanadyl ribonucleoside complexes (Sigma-Aldrich), 10 μL of 100 mM DTT and EDTA to 20mM.
5
Polysome Fractionation and RNA Extraction
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For polysome fractionation, cycloheximide (Sigma, 100μg/ml; Interchim, Montluçon, France) was added to the medium for 5 and/or 15 min, prior to harvesting. The medium was then removed and the cells washed with ice-cold PBS containing 100 μg/mL cycloheximide. The cells were then scraped, centrifuged at 800g for 5 min at 4°C and cytoplasmic RNA was obtained by lysis (20 strokes of a p1000 pipet) of the cell pellet in 1 mL of polysome buffer containing 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40, and 40 mM vanadyl ribonucleoside complexes (VRC), 100 μg/mL cycloheximide (CHX), 20 mM dithiothreitol (DTT), and 1 mM phenylmethanesulfonyl fluoride (PMSF), all reagents were supplied by Sigma. Mitochondria and membrane debris were removed by centrifugation (10,000g, 5 min, 4°C). The post-mitochondrial supernatants were overlaid onto a 15-40% sucrose gradient and spun at 38,000rpm for 2 h at 4°C in a SW41Ti rotor (Beckman Coulter, Fullerton, CA, USA). Fractions were collected from the top of each gradient using a 240 nm UV reader-coupled fraction collector (Brandel, Glasgow, UK). Free mRNPs, monosomes and polysomes were located through the interpretation of UV gradient traces. RNA extraction was done using the acid phenol approach detailed previously [8] (link).
6
Polysome Profiling of Caenorhabditis
7
Immunocytochemical Staining of NRCM
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NRCM was cultured in a μ-slide 8-well chamber (IBIDI GmbH, Germany) and fixed with freshly prepared 4% paraformaldehyde at room temperature for 15 minutes. Then, NRCM was permeabilized with 0.5% Triton X-100 and 2 mM Ribonucleoside Vanadyl Complexes (Sigma) on ice for 10 min followed by washing twice with 2x SSC for 10 min each time. The probe (10 ng/mL) was denatured in the hybridization buffer at 90°C for 10 minutes and then immediately chilled on ice for 5 min. NRCM was hybridized and incubated with primary antibodies: α-actinin antibody (1 : 200, Sigma) in a hybridization oven at 37°C overnight. After hybridization, the chamber was washed in 2× SSC, 50% formamide for 3 × 5 mins at 42°C, 2× SSC for 3 × 5 mins at 42°C, 1× SSC for 3 × 5 mins at 42°C, 4× SSC for 2 × 10 mins at room temperature, and 2× SSC for 1 hr at 65°C; Cy3-labeled streptavidin and secondary antibodies were added to the NRCM for 1 hr at RT. After washing three times with PBS, Hoechst (1 : 1000, Keygen) was used to label the nucleus for 30 min, and images were captured by a confocal microscope (Carl Zeiss, Thuringia, Germany).
8
Oligo-conjugated WGA Tissue Staining
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The primary probe staining procedure was similar to those in previous reports27 (link)–29 (link). Tissue sections were stained with oligo-conjugated WGA in 1× hank’s balanced salt solution (HBSS) buffer for 20 min at 37 °C. Samples were post-fixed in 4% PFA for 10 min. Tissue sections were permeabilized using 0.5% Triton in DPBS for 20 min at room temperature before primary probe staining. Tissue samples were incubated for 5 min in pre-hybridization buffer composed of 50% (vol/vol) formamide and 2 mM Ribonucleoside vanadyl complexes (Sigma-Aldrich, R3380) in 2× saline sodium citrate (2× SSC) (Invitrogen, AM9765). Tissue samples were then stained with primary probes in primary hybridization buffer, containing 24-28 μM primary probes, 50% (vol/vol) formamide, 0.1% yeast tRNA (Invitrogen, 1885325), 1% (vol/vol) murine RNase inhibitor (NEB, M0314S), 10% (wt/vol) dextran sulfate (Millipore, S4030), and 2 μM anchor probe (a 15-nt sequence of alternating dT and thymidine-locked nucleic acid with a 5′ -acrydite modification) in 2× SSC, in a humidity chamber at 37 °C for 24 h. After staining, samples were washed for 15 min with 2× SSCT (2× SSC, 0.1% (vol/vol) Tween 20) twice at 60 °C and then once for 15 min at room temperature.
9
FISH Procedure for RNA Localization
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FISH was performed as described previously (Rouquette et al., 2005 (link)) using a 5’ Cy3-labeled 5’ITS1 probe (5’-CCTCGCCCTCCGGGCTCCGTTAATGATC -3’; Microsynth).
Cells grown on coverslips were fixed in 4% PFA/PBS for 30 min at room temperature. After two rinses with PBS, cells were permeabilized by overnight incubation with 70% ethanol. Cells were rehydrated by two incubations for 5 min in 10% formamide in 2X SSC (30 mM tri-sodium citrate dihydrate pH 7.0, 300 mM NaCl). Hybridization was performed for 4 hr at 37°C in 10% formamide in 2X SSC supplemented with 10% dextran sulfate, 10 mM ribonucleoside vanadyl complexes (Sigma-Aldrich, cat#R-3380), 50 μg/ml BSA (Sigma-Aldrich, cat#B-2518), 0.5 μg/μl tRNA (Sigma-Aldrich, cat#R-1753), and 0.5 ng/μl Cy3-5’ITS1 probe. Subsequently, cells were washed twice for 30 min with 10% formamide in 2X SSC before washing for 5 min with PBS and mounting of the coverslips using Vectashield (Vector Laboratories). After imaging by confocal microscopy, the cytoplasmic signal was measured with CellProfiler version 4.2.0 (McQuin et al., 2018 (link)) and quantified with the GraphPad Prism software.
Cells grown on coverslips were fixed in 4% PFA/PBS for 30 min at room temperature. After two rinses with PBS, cells were permeabilized by overnight incubation with 70% ethanol. Cells were rehydrated by two incubations for 5 min in 10% formamide in 2X SSC (30 mM tri-sodium citrate dihydrate pH 7.0, 300 mM NaCl). Hybridization was performed for 4 hr at 37°C in 10% formamide in 2X SSC supplemented with 10% dextran sulfate, 10 mM ribonucleoside vanadyl complexes (Sigma-Aldrich, cat#R-3380), 50 μg/ml BSA (Sigma-Aldrich, cat#B-2518), 0.5 μg/μl tRNA (Sigma-Aldrich, cat#R-1753), and 0.5 ng/μl Cy3-5’ITS1 probe. Subsequently, cells were washed twice for 30 min with 10% formamide in 2X SSC before washing for 5 min with PBS and mounting of the coverslips using Vectashield (Vector Laboratories). After imaging by confocal microscopy, the cytoplasmic signal was measured with CellProfiler version 4.2.0 (McQuin et al., 2018 (link)) and quantified with the GraphPad Prism software.
10
Visualization of RNA-Protein Interactions
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After fixing with 4% PFA in PBS, the cells were permeabilized with 0.3% Triton in PBS at RT for 10 min. The cells were washed twice with 5 mM MgCl2 (Sigma-Aldrich) in PBS and incubated with in situ hybridization buffer at 37 °C for 90 min. The in situ hybridization buffer is composed of 100 nM Cy3-labeled 2’OMe (CAG)7 (Gene Design, Osaka, Japan), 2 × SSC (Nacalai), 10% dextran sulfate (Merck, Darmstadt, Germany), 40% formamide (Sigma-Aldrich), 0.2% BSA, 0.1 mg/ml herring sperm DNA (Promega), 0.1 mg/ml baker's yeast transfer RNA (Sigma-Aldrich) and 4 mM ribonucleoside vanadyl complexes (Sigma-Aldrich). Then, the cells were washed with PBS-T at RT for 5 min and at 45 °C for 30 min. Thereafter, immunocytochemistry for MBNL1 and nuclear counterstain were performed as described above, except that anti-MBNL1 antibody was incubated at RT for one hour. After fixing with acetone-MeOH, immunocytochemistry for MBNL1 was performed using antibodies diluted in 2 × SSC, as described in a previous report25 (link). The labeled cells were treated with 4% PFA + 5 mM MgCl2 in PBS at RT for one minute and 40% formamide in 2 × SSC for one minute. Then, in situ hybridization and nuclear counterstain were performed as described above. The samples were visualized and photographed under a BZ-X700 (× 100 objective).
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