Slo2 potassium channel function depends on RNA editing-regulated expression of a SCYL1 protein
- Department of Neuroscience, University of Connecticut Health Center, United States
Figures
Figure 1
Loss-of-function mutations of adr-1 suppress phenotypes caused by a hyperactive SLO-2.
(A) Diagram of ADR-1 domain structures and locations of the non-sense mutations in the adr-1 mutants. ADR-1 has two double-stranded RNA-binding motifs (dsRBM) and a pseudodeaminase domain. (B) Mutations of adr-1 mitigated an inhibitory effect of hyperactive or gain-of-function (gf) SLO-2 on locomotion through acting in neurons. adr-1 rescue was achieved by expressing GFP-tagged wild-type ADR-1 in neurons under the control of Prab-3 (same in C and D). Sample sizes were 10–12 in each group. (C) adr-1(zw96) reduced an augmenting effect of slo-2(gf) on motor neuron whole-cell outward currents. Pipette solution I and bath solution I were used. Sample sizes were 7 wild type, 8 slo-2(gf), 9 slo-2(gf);adr-1(zw96), and 8 slo-2(gf);adr-1(zw96) rescue. (D) adr-1(zw96) mitigated an inhibitory effect of slo-2(gf) on postsynaptic current (PSC) bursts at the neuromuscular junction. The vertical dotted lines over the sample traces mark PSC bursts, which are defined as an apparent increase in PSC frequency accompanied by a sustained current (downward baseline shift) lasting >3 s. Pipette solution II and bath solution I were used. Sample sizes were 12 wild type, and 7 in each of the remaining groups. All values are shown as mean ± SE. The asterisks indicate statistically significant differences between indicated groups (*p<0.05, ***p<0.001) based on either two-way (C) or one-way (D) ANOVA with Tukey's post hoc tests.
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Figure 1—source data 1
Raw data and numerical values for data plotted in Figure 1.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig1-data1-v2.xlsx
Figure 2
Loss-of-function mutation of adr-2 suppressed the effects of gain-of-function (gf) slo-2 on locomotion and motor neuron whole-cell currents.
(A) adr-2(gv42) alleviated an inhibitory effect of slo-2(gf) on locomotion speed. The sample size was 10–12 in each group. (B) adr-2(gv42) largely reversed an augmenting effect of slo-2(gf) on whole-cell currents in VA5 motor neuron. Sample sizes were 11 wild type, 8 slo-2(gf), 11 slo-2(gf);adr-2(gv42), and 10 adr-2(gv42). All data are shown as mean ± SE. Pipette solution I and bath solution I were used. The asterisks indicate statistically significant differences (*p<0.05; ***p<0.001) whereas ‘ns’ stands for ‘no significant difference’ between the indicated groups based on either one-way (A) or two-way (B) ANOVA with Tukey's post hoc tests.
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Figure 2—source data 1
Raw data and numerical values for data plotted in Figure 2.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig2-data1-v2.xlsx
Figure 3
ADR-1 is coexpressed with SLO-2 in many neurons and localized in the nucleus.
(A) Expression of an adr-1 promoter (Padr-1)::GFP transcriptional fusion in worms resulted in strong GFP signal in many neurons (NR, nerve ring; VNC, ventral nerve cord; TG, tail ganglion) and weak GFP signal in body-wall muscles (BWM) and intestine (Int). (B) GFP-tagged ADR-1 (ADR-1::GFP) colocalized with a mStrawberry-tagged HIS-58 nucleus marker, as indicated by fluorescence images of VNC motor neurons. (C) adr-1 and slo-2 are co-expressed in many neurons but show differential expressions in the pharynx (Phx) and Int. Scale bar = 20 µm in in all panels.
Figure 4 with 1 supplement
ADR-1 contributes to motor neuron whole-cell currents and regulates postsynaptic current (PSC) bursts through SLO-2.
(A) Representative VA5 whole-cell current traces. (B) Current (I) - voltage relationships of the whole-cell currents. Sample sizes were 8 wild type, 7 slo-2(lf), 9 adr-1(zw96), 7 slo-2(lf);adr-1(zw96), and 9 adr-1(zw96) rescue. (C) Resting membrane potentials of VA5. Sample sizes were 6 wild type, and 7 in each of the remaining groups. (D) Representative traces of spontaneous PSCs with PSC bursts marked by vertical dotted lines. (E) Comparisons of PSC burst properties. Sample sizes were 8 slo-2(lf);adr-1(zw96), 6 adr-1(zw96) rescue, and 12 in each of the remaining groups. All values are shown as mean ± SE. The asterisks indicate statistically significant differences (*p<0.05, ***p<0.001) compared with wild type whereas ‘ns’ stands for no significant difference between the indicated groups based on either two-way (B) or one-way (C and E) ANOVA with Tukey's post hoc tests. Pipette solution I and bath solution I were used in (A) and (C). Pipette solution II and bath solution I were used in (D).
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Figure 4—source data 1
Raw data and numerical values for data plotted in Figure 4.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Comparison of slo-2 transcript level between wild type and adr-1 mutant.
Shown are mean ± SE of three RNA-seq experiments.
Figure 5 with 1 supplement
Normalized transcript expression levels of selected genes in adr-1(zw96) mutant.
The genes were selected based on the detection of ADR-1-dependent RNA editing events in their transcripts reported in an earlier study (Washburn et al., 2014). Transcript expression level of each gene in the mutant is normalized by that in the wild type. Shown are mean ± SE from three biological replicates of RNA-seq experiments.
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Figure 5—source data 1
Raw data and numerical values for data plotted in Figure 5.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Alignment of amino acid sequences between C. elegans SCYL-1 (W07G4.3, www.wormbase.org) and human SCYL1 (hSCYL1, GenBank: NP_065731.3).
Identical residues are highlighted in black, while similar ones (in size or polarity) in blue. The three residues that are essential for kinase activity in eukaryotic protein kinases are shown in red above the alignment at corresponding locations. Both proteins contain five HEAT repeats (marked by horizontal green lines) in the central portion. The scyl-1 mutant allele zw99 was made by introducing a stop codon after the residue I152 (indicated by an arrow) using the CRISPR/Cas9 approach.
Figure 6
scyl-1 and slo-2 are coexpressed in ventral cord motor neurons but differentially expressed in other cells.
In transgenic worms coexpressing Pscyl-1::GFP and Pslo-2::mStrawberry transcriptional fusions, GFP signal was observed in ventral nerve cord (VNC) motor neurons, the large H-shaped excretory (EXC) cell, uterine ventral (UV) cells, and spermatheca (Spe) while mStrawberry signal was detected in VNC motor neurons, body-wall muscles (BMW), and many other neurons. Scale bar = 20 µm.
Figure 7
SCYL-1 contributes to motor neuron outward currents through SLO-2.
(A) Sample whole-cell current traces of VA5 motor neurons and the current-voltage relationships. Sample sizes were 9 wild type, 7 slo-2(lf), 10 scyl-1(zw99), 7 slo-2(lf);scyl-1(zw99), 7 adr-1(zw96);scyl-1(zw99), and 7 scyl-1(zw99) rescue. The rescue strain was created by expressing wild-type scyl-1 under the control of Prab-3. All values are shown as mean ± SE. The asterisks (***) and pound signs (###) indicate statistically significant differences (p<0.001) between the indicated groups and from wild type, respectively, whereas ‘ns’ stands for no significant difference between the indicated groups (two-way ANOVA with Tukey's post hoc tests). (B) GFP signal in ventral cord motor neurons was indistinguishable between wild type and scyl-1(zw99) worms expressing GFP-tagged full-length SLO-2 under the control of Prab-3. Scale bar = 20 µm.
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Figure 7—source data 1
Raw data and numerical values for data plotted in Figure 7.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig7-data1-v2.xlsx
Figure 8
Single-channel open probability (Po) of SLO-2 is decreased in scyl-1 mutant.
(A) Representative SLO-2 single-channel currents from inside-out patches of the VA5 motor neuron, and comparisons of Po and single-channel amplitude between wild type (n = 14), scyl-1(zw99) (n = 15), and scyl-1(zw99) rescued by expressing wild-type scyl-1 in neurons under the control of Prab-3 (n = 11). (B and C) Fitting of open and closed dwell time histogram to exponentials, and comparisons of τ values and relative areas (A) of the fitted components. All the open and closed events of each group were pooled together to plot the dwell time histograms. Statistical comparisons shown below were based on the mean τ values of individual recordings with each dot representing the mean value of one recording. Pipette solution III and bath solution II were used. All values are shown as mean ± SE. The asterisks indicate a significant difference between the indicated groups (*p<0.05, ***p<0.001, one-way ANOVA with Tukey's post hoc tests).
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Figure 8—source data 1
Raw data and numerical values for data plotted in Figure 8.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig8-data1-v2.xlsx
Figure 9
SCYL-1 physically interacts with SLO-2 in neurons.
(A) Diagrams of the various fusion proteins used in the BiFC assays (left) and of SLO-2 membrane topology (right). The arrow indicates the split site for SLO-2N and SLO-2C fusions. RCK, regulator of conductance for K+. (B) YFP signal was detected when SCYL-1 was coexpressed with either full-length or the carboxyl terminal portion of SLO-2 but not with the amino terminal portion of SLO-2. Shown are representative fluorescent images of the ventral nerve cord (indicated by arrows) with corresponding DIC images. The bright signals at the top of each fluorescence image was from auto-fluorescence of the intestine. Scale bar = 20 μm. (C) SCYL-1 co-immunoprecipitates with full-length SLO-2 and SLO-2C but not SLO-2N. IP, immunoprecipitation; IB, immunoblot. The molecular masses of the protein standard are indicated on the right. Note that multiple bands are seen in the lanes loaded with GFP fusions. The bands that match predicted molecular masses of SLO-2::GFP, SLO-2N::GFP, and SLO-2C::GFP fusions are indicated with arrows, respectively. The other bands likely resulted from cleaved or partially translated GFP fusion proteins.
Figure 10 with 1 supplement
ADR-1 regulates scyl-1 expression through RNA editing at a specific nucleotide in the 3’-UTR.
(A) RNA editing at one out of eight highly (>15%) edited sites is severely deficient in adr-1(zw96) compared wild type. The percentage of editing was calculated by diving the number of reads containing A-I conversion by the total number of reads at each site. The x-axis indicates the positions of the edited adenosines in chromosome V (NC_003283). Shown are results (mean ± SE) of three RNA-seq experiments. The asterisks (***) indicate a statistically significant difference (p<0.001, unpaired t-test). (B) Diagram showing a predicted hair-pin structure in the 3’ end of scyl-1 pre-mRNA with 746 complementary base pairs. Nucleotide are numbered from the first nucleotide of the 3’-UTR. (C) Chromatograms of scyl-1 mRNA 3’-UTRs of wild type, adr-1(zw96), and adr-2(gv42), and of the corresponding wild type genomic DNA. Two editing sites in wild type mRNA (indicated by arrows) display a mixture of green (adenosine) and black (guanosine) peaks. While both editing events are non-existent in adr-2(gv42), only one of them is inhibited by adr-1(zw96). (D) Diagram of two GFP reporter constructs (wp1923 and wp1924) used to confirm the role of the ADR-1-dependent editing site in gene expression. GFP was placed under the control of Prab-3 and fused to the last exon (blue) of scyl-1 followed by 5 kb downstream genomic sequence. The red bars indicate the inverted repeat sequences that form the double-stranded RNA in the hair-pin structure (B). wp1923 contains the intact genomic sequence of scyl-1 3’-UTR, whereas wp1924 differs from it in an A-to-G conversion mimicking the ADR-1-dependent editing. (E) Effects of the A-to-G conversion on GFP reporter expression. Shown are fluorescent and corresponding DIC images of transgenic worms harboring either wp1923 or wp1924. GFP expression in the head and ventral nerve cord (VNC) was observed only in worms harboring wp1924. The diffused signal below the VNC in fluorescent images was from auto-fluorescence of the intestine (Int). Scale bar = 20 µm. (F) GFP expression from wp1924 was decreased in adr-1(zw96) compared with that in wild type. Scale bar = 20 µm. (G) Statistical comparison of GFP intensity in the VNC between wild type (n = 16) and adr-1(zw96) (n = 19). (***p<0.001, unpaired t-test).
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Figure 10—source data 1
Raw data and numerical values for data plotted in Figure 10.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig10-data1-v2.xlsx
Figure 10—figure supplement 1
Expression of Prab-3::GFP::scyl-1 3’-UTR (A–to–G) was greatly decreased in isolated mutants.
(A) Images from wild type and two representative mutants, zw103 and zw105, expressing Prab-3::GFP::scyl-1 3’-UTR (A–to–G). The GFP signals in the head and the ventral nerve cord (VNC) are almost invisible in the two mutants. The fluorescent signals at the bottom of the right panels were from auto-fluorescence of the intestine (Int). (B) GFP expression levels from Prab-3::GFP::unc-10 3’-UTR are comparable between wild type and the mutants. Scale bars = 20 µm.
Figure 11
Single-channel open probability (Po) of human Slo2.2/Slack is augmented by SCYL1 in Xenopus oocyte expression system.
(A) Representative traces of single-channel currents from inside-out patches and comparisons of Po and single-channel amplitude between patches with and without mouse SCYL1. (B and C) Dwell time histograms and statistical comparisons of open and closed (≤30 ms in duration) events. The histograms were constructed and the τ values were quantified as described in Figure 8 legend. (D) Dwell time histograms and statistical comparisons of the long closed events. Closed events that were >30 ms in duration of all recordings in each group were pooled together to construct the dwell time histogram. The average duration and frequency of these events were compared between the two groups. Each dot represents the mean value of one recording. Sample sizes were 13 in both groups. All values are shown as mean ± SE. The asterisks indicate a significant difference compared between the indicated groups (*p<0.05, ***p<0.001, unpaired t-test).
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Figure 11—source data 1
Raw data and numerical values for data plotted in Figure 11.
- https://cdn.elifesciences.org/articles/53986/elife-53986-fig11-data1-v2.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background(C. elegans) | N2 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00000001 | Laboratory reference strain (wild type). |
| Strain, strain background (C. elegans) | LY101 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00026423 | Genotype: slo-2(nf101). |
| Strain, strain background (C. elegans) | BB3 | Caenorhabditis Genetics Center | RRID:WB-STRAIN:WBStrain00000435 | Genotype: adr-2(gv42). |
| Strain, strain background (C. elegans) | ZW860 | This paper | Genotype: zwIs139[Pslo-1::slo-2(gf)(wp1311), Pmyo-2::YFP(wp214)]. | |
| Strain, strain background (C. elegans) | ZW876 | This paper | Genotype: zwIs139[Pslo-1::slo-2(gf)(wp1311), Pmyo-2::YFP(wp214)]; adr-1(zw80). | |
| Strain, strain background (C. elegans) | ZW877 | This paper | Genotype: zwIs139[Pslo-1::slo-2(gf)(wp1311), Pmyo-2::YFP(wp214)]; adr-1(zw81). | |
| Strain, strain background (C. elegans) | ZW983 | This paper | Genotype: zwIs139[Pslo-1::slo-2(gf)(wp1311), Pmyo-2::YFP(wp214)]; adr-2(gv42). | |
| Strain, strain background (C. elegans) | ZW1049 | This paper | Genotype: zwEx221[Prab-3::slo-2::GFP]. | |
| Strain, strain background (C. elegans) | ZW1388 | This paper | Genotype: zwEx260[Prab-3::His-58::mStrawberry(p1749), Prab-3::adr-1::GFP(p1374)]. | |
| Strain, strain background (C. elegans) | ZW1394 | This paper | Genotype: adr-1(zw96). | |
| Strain, strain background (C. elegans) | ZW1401 | This paper | Genotype: zwEx261[Padr-1::GFP(wp1872), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1407 | This paper | Genotype: zwIs139[Pslo-1::slo-2(gf)(wp1311), Pmyo-2::YFP(wp214)]; adr-1(zw96). | |
| Strain, strain background (C. elegans) | ZW1408 | This paper | Genotype: zwIs139[Pslo-1::slo-2(gf)(wp1311), Pmyo-2::YFP(wp214)]; zwEx262[Prab-3::adr-1::GFP(p1374);Pmyo-2::mStrawberry (wp1613)]; adr-1(zw96). | |
| Strain, strain background (C. elegans) | ZW1409 | This paper | Genotype: scyl-1(zw99). ZW1410: slo-2(nf101); scyl-1(zw99). | |
| Strain, strain background (C. elegans) | ZW1415 | This paper | Genotype:: zwEx221[Prab-3::slo-2::GFP]; scyl-1(zw99). | |
| Strain, strain background (C. elegans) | ZW1416 | This paper | Genotype: zwEx247[Pslo-2::mStrawberry(wp1776), lin-15(+)]; zwEx263[Pscyl-1::GFP(wp1901+wp1902), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1417 | This paper | Genotype: zwEx264[Prab-3::scyl-1(wp1912), Pmyo-2::mStrawberry (wp1613)]; scyl-1(zw99). | |
| Strain, strain background (C. elegans) | ZW1418 | This paper | Genotype: zwEx247[Pslo-2::mStrawberry(wp1776), lin-15(+)]; zwEx261[Padr-1::GFP(wp1872), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1419 | This paper | Genotype: zwEx265[Prab-3::GFP::scyl-1 3-UTR(wp1923), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1420 | This paper | Genotype: zwEx266[Prab-3::GFP::scyl-1 3’-UTR(A-to-G)(wp1924), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1428 | This paper | Genotype: slo-2(nf101); adr-1(zw96). | |
| Strain, strain background (C. elegans) | ZW1505 | This paper | Genotype: zwEx273[Prab-3::scyl-1::YFPc(wp1952), Prab-3::slo-2::YFPa(wp1783), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1506 | This paper | Genotype: zwEx274[Prab-3::scyl-1::YFPc(wp1952), Prab-3::slo-2N::YFPa(wp1784), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1507 | This paper | Genotype: zwEx275[Prab-3::scyl-1::YFPc(wp1952), Prab-3::slo-2C::YFPa(wp1785), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1537 | This paper | Genotype: adr-1(zw96);scyl-1(zw99). | |
| Strain, strain background (C. elegans) | ZW1538 | This paper | Genotype: zwEx280[Prab-3::scyl-1::HA(wp1998), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1539 | This paper | Genotype: zwEx281[Prab-3::scyl-1::HA(wp1998), Prab-3::slo-2::GFP(wp1318), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1540 | This paper | Genotype: zwEx282[Prab-3::scyl-1::HA(wp1998), Prab-3::slo-2N::GFP(wp1999), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1541 | This paper | Genotype: zwEx283[Prab-3::scyl-1::HA(wp1998), Prab-3::slo-2C::GFP(wp2000), lin-15(+)]; lin-15(n765). | |
| Strain, strain background (C. elegans) | ZW1544 | This paper | Genotype: zwIs146[Prab-3::GFP::scyl-1 3’-UTR(A-to-G)(wp1924)]. | |
| Strain, strain background (C. elegans) | ZW1545 | This paper | Genotype: zwIs146[Prab-3::GFP::scyl-1 3’-UTR(A-to-G)(wp1924)]; adr-1(zw96). | |
| Strain, strain background (C. elegans) | ZW1549 | This paper | Genotype: zwIs146[Prab-3::GFP::scyl-1 3’-UTR(A-to-G)(wp1924)]; zw103. | |
| Strain, strain background (C. elegans) | ZW1552 | This paper | Genotype: zwIs146[Prab-3::GFP::scyl-1 3’UTR(A-to-G)(wp1924)]; zw105. | |
| Strain, strain background (C. elegans) | ZW1562 | This paper | Genotype: zwEx284[Prab-3::GFP::unc-10 3’-UTR(wp70)]. | |
| Strain, strain background (C. elegans) | ZW1563 | This paper | Genotype: zwEx284[Prab-3::GFP::unc-10 3’-UTR(wp70)]; zw103. | |
| Strain, strain background (C. elegans) | ZW1564 | This paper | Genotype: zwEx284[Prab-3::GFP::unc-10 3’-UTR(wp70)]; zw105. | |
| Antibody | Mouse monoclonal anti-HA | Santa Cruz Biotechnology | Cat# sc-7392, RRID:AB_627809 | WB: 1:500 |
| Antibody | Mouse monoclonal anti-GFP | Santa Cruz Biotechnology | Cat# sc-9996, RRID:AB_627695 | WB: 1:500 |
| Antibody | Donkey anti-Mouse IgG | Thermo Fisher Scientific | Cat# A16011, RRID:AB_2534685 | WB: 1:10000 |
| Antibody | GFP-Trap_MA | ChromoTek | Cat# gtma-20, RRID:AB_2631358 | |
| Commercial assay or kit | ECL Substrate | Bio-Rad | Cat# 1705060 | |
| Commercial assay or kit | mMESSAGE mMACHINE | Ambion | Cat# AM1348 | |
| Software, algorithm | Photoshop CS5 | Adobe | RRID:SCR_014199 | https://www.adobe.com/products/photoshop.html |
| Software, algorithm | Origin | OriginLab | RRID:SCR_014212 | http://www.originlab.com/index.aspx?go=PRODUCTS/Origin |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | https://imagej.nih.gov/ij/ |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | http://www.moleculardevices.com/products/software/pclamp.html |
| Software, algorithm | MATLAB | MathWorks | RRID:SCR_001622 | http://www.mathworks.com/products/matlab/ |
| Software, algorithm | TopHat | PMID:23618408 | RRID:SCR_013035 | http://ccb.jhu.edu/software/tophat/index.shtml |
| Software, algorithm | Trim Galore | Babraham Bioinformatics | RRID:SCR_011847 | http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ |
| Software, algorithm | Track-A-Worm | PMID:23922769 | RRID:SCR_018299 | https://health.uconn.edu/worm-lab/track-a-worm/ |
Additional files
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Source code 1
Track-A-Worm software.
- https://cdn.elifesciences.org/articles/53986/elife-53986-code1-v2.rar
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53986/elife-53986-transrepform-v2.pdf
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Slo2 potassium channel function depends on RNA editing-regulated expression of a SCYL1 protein
eLife 9:e53986.
https://doi.org/10.7554/eLife.53986
