Slo2 potassium channel function depends on a SCYL1 protein

Slo2 potassium channels play important roles in neuronal function, and their mutations in humans cause epilepsies and cognitive defects. However, little is known how Slo2 function is regulated by other proteins. Here we found that the function of C. elegans Slo2 (SLO-2) depends on adr-1, a gene important to RNA editing. However, slo-2 transcripts have no detectable RNA editing events and exhibit similar expression levels in wild type and adr-1 mutants. In contrast, mRNA level of scyl-1, which encodes an orthologue of mammalian SCYL1, is greatly reduced in adr-1 mutants due to deficient RNA editing at a single adenosine in its 3’-UTR. SCYL-1 physically interacts with SLO-2 in neurons. Single-channel open probability of SLO-2 in neurons is reduced by ∼50% in scyl-1 knockout whereas that of human Slo2.2/Slack is doubled by SCYL1 in a heterologous expression system. These results suggest that SCYL-1/SCYL1 is an evolutionarily conserved regulator of Slo2 channels.


Introduction 25
Slo2 channels are large-conductance potassium channels existing in mammals as well as invertebrates 26 (Kaczmarek, 2013;Yuan et al., 2000). They are the primary conductor of delayed outward currents in many 27 neurons examined (Budelli et al., 2009;Liu et al., 2014). Human and mouse each has two Slo2 channels 28 (Slo2.1/Slick and Slo2.2/Slack) (Kaczmarek, 2013), whereas the nematode C. elegans has only one (SLO-29 2). These channels are abundantly expressed in the nervous system (Bhattacharjee et al., 2002;  In a genetic screen for suppressors of a sluggish phenotype caused by expressing a hyperactive SLO-2 57 in worms, we isolated mutants of several genes, including adr-1, which encodes one of two ADARs in C. 58 elegans (ADR-1 and ADR-2). While ADR-2 has deaminase activity and plays an indispensable role in the 59 A-to-I conversion, ADR-1 is catalytically inactive but can regulate RNA editing by binding to selected In adr-1(lf) mutants, a lack of A-to-I conversion at a specific site in scyl-1 3'-UTR causes reduced scyl-1 64 expression. Knockout of scyl-1 severely reduces SLO-2 current in worms while coexpression of SCYL1 65 with human Slack in Xenopus oocytes greatly augments channel activity. These results suggest that SCYL-66 1/SCYL1 likely plays an evolutionarily conserved role in physiological functions of Slo2 channels. 67 Mutations or knockout mammalian SCYL1 may cause neural degeneration, intellectual disabilities, and adr-1 mutants suppress sluggish phenotype of slo-2(gf) 74 dramatically smaller and VA5 resting membrane potential was much less hyperpolarized in slo-2(lf) than 125 wild type. While adr-1(lf) also caused significantly decreased outward currents and less hyperpolarized 126 resting membrane potential in VA5, it did not produce additive effects when combined with slo-2(lf) (Fig.  127 4 A-C). These results suggest that adr-1(lf) affects motor neuron outward current and resting membrane 128 potential through SLO-2. 129 We next determined whether adr-1(lf) also alters PSC bursts. We found that adr-1(lf) caused an increase 130 in the duration and mean charge transfer rate of PSC bursts without altering the burst frequency compared 131 with wild type (Fig. 4 D and E). These phenotypes of adr-1(lf) were similar to those of slo-2(lf) and did not 132 become more severe in the double mutants ( Fig. 4 D and E), suggesting that ADR-1 modulates 133 neurotransmitter release through SLO-2. The similar effects of adr-1(lf) and slo-2(lf) on PSC bursts are in 134 contrast to their differential effects on VA5 outward currents and resting membrane potential. This 135 discrepancy suggests that there might be a threshold level of SLO-2 deficiency to cause a similar change in 136 PSC bursts. 137

ADR-1 regulates SLO-2 function through SCYL-1 138
Given that our results suggest that RNA editing is important to SLO-2 function, we determined whether 139 adr-1(lf) causes deficient editing or decreased expression of slo-2 mRNA by comparing RNA-seq data 140 between adr-1(lf) and wild type. The adr-1(zw96) allele was chosen for these analyses to minimize potential 141 complications by mutations of other genes introduced in adr-1 mutants isolated from the genetic screen. 142 Unexpectedly, no RNA editing event was detected in slo-2 transcripts, and slo-2 mRNA level was similar 143 between wild type and the adr-1 mutant (Fig. 4-figure supplement 1). These results suggest that ADR-1 144 might regulate SLO-2 function through RNA editing of another gene. 145 A previous study identified 270 high-confidence editing sites in transcripts of 51 genes expressed in C. 146 elegans neurons (Washburn et al., 2014). We suspected that the putative molecule mediating the effect of 147 ADR-1 on SLO-2 might be encoded by one of these genes, and the mRNA level of this gene may have 148 reduced expression in adr-1(lf). Therefore, we compared transcript expression levels of these genes 149 (excluding those encoding transposons) quantified from our RNA-Seq data between wild type and adr-150 1(zw96). The transcripts of most genes showed either no decrease or only a small decrease, but two of these 151 genes, rncs-1 and scyl-1, were reduced greatly in adr-1(lf) compared with wild type (Fig. 5). rncs-1 is not 152 a conceivable candidate for the putative SLO-2 regulator because it is a non-coding gene expressed in the 153 hypodermis and vulva (Hellwig and Bass, 2008). On the other hand, scyl-1 is a promising candidate because 154 it encodes an orthologue of mammalian SCYL1 important to neuronal function and survival (Pelletier, 155 2016). We therefore focused our analyses on scyl-1. Like its mammalian homologs, SCYL-1 has an amino-156 terminal kinase domain that lacks residues critical to kinase activity, and a central domain containing five 157 HEAT repeats (HEAT for Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1) 158 (Pelletier, 2016). SCYL-1 shares 38% identity and 60% similarity with human SCYL1. Notably, amino 159 acid sequence in the HEAT domain, which is often highly degenerative (Pelletier, 2016), shows a very high 160 level of sequence homology (53% identity and 76% similarity) between these two proteins ( Fig. 5-figure  161 supplement 1). 162 We first examined the expression pattern of scyl-1 by expressing GFP reporter under the control of scyl-163 1 promoter (Pscyl-1). An in vivo homologous recombination approach was used in this experiment to 164 include a large fragment of genomic DNA sequence upstream of the scyl-1 initiation site. Specifically, a 165 0.5-kb genomic fragment upstream of the scyl-1 initiation site was cloned by PCR and fused to GFP. The 166 resultant plasmid was co-injected with a fosmid covering part of the scyl-1 coding region and 32 kb 167 sequence upstream of the initiation site into wild type worms. In vivo homologous recombination between 168 the plasmid and the fosmid is expected to result in a Pscyl-1::GFP transcriptional fusion that includes all 169 the upstream sequence in the fosmid. After successful creation of a transgenic strain expressing the Pscyl-170 1::GFP transcriptional fusion, we crossed the transgene into the Pslo-2::mStrawberry strain, and examined 171 the expression patterns of GFP and mStrawberry. We observed co-expression of scyl-1 and slo-2 in many 172 ventral cord motor neurons (Fig. 6). However, most other neurons expressing slo-2 (e. g. head and tail 173 neurons) did not appear to express scyl-1. In addition, scyl-1 expression was detected in some cells that did 174 not express slo-2, including the excretory cell, spermatheca, vulval muscle cells, and intestinal cells (Fig.  175   6). 176 We next determined whether SCYL-1 is related to SLO-2 function. To this end, we created a mutant, 177 scyl-1(zw99), by introducing a stop codon after isoleucine 152 using the CRISPR/Cas9 approach, and 178 examined the effect of this mutation on VA5 delayed outward currents. scyl-1(zw99) showed a substantial 179 decrease in VA5 outward currents compared with wild type; and this phenotype was non-additive with that 180 of slo-2(lf) and could be rescued by expressing wild type SCYL-1 in neurons (Fig. 7A). These results 181 suggest that SCYL-1 contributes to SLO-2-dependent outward currents. 182 The decrease of delayed outward currents in scyl-1(lf) could have resulted from decreased expression or 183 function of SLO-2. We first determined whether scyl-1(lf) alters SLO-2 expression by crossing a stable 184 (near 100% penetrance) Prab-3::SLO-2::GFP transgene from an existing transgenic strain of wild-type 185 genetic background (Liu et al., 2018) into scyl-1(zw99), and comparing GFP signal between the two strains. 186 We found that GFP signal in the ventral nerve cord was similar between wild type and the scyl-1 mutant 187 ( Fig. 7B), suggesting that SCYL-1 does not regulate SLO-2 expression. We then determined whether 188 SCYL-1 regulates SLO-2 function by obtaining inside-out patches from VA5 and analyzing SLO-2 single-189 channel properties. SLO-2 showed >50% decrease in open probability (Po) without a change of single-190 channel conductance in scyl-1(zw99) compared with wild type, and this mutant phenotype was completely 191 rescued by neuronal expression of wild-type SCYL-1 (Fig. 8A). Analyses of single-channel open and 192 closed events revealed that SLO-2 has two open states and three closed states, and that the decreased Po of 193 SLO-2 in scyl-1(lf) mainly resulted from decreased events of long openings (Fig. 8B) and increased events 194 of long closures (Fig. 8C). 195 The observed effects of scyl-1(lf) on SLO-2 single-channel properties suggest that SCYL-1 may 196 physically interacts with SLO-2. We performed bimolecular fluorescence complementation (BiFC) assays 197 (Hu et al., 2002) to test this possibility. In these assays, full-length SCYL-1 tagged with the carboxyl 198 terminal portion of YFP (YFPc) was coexpressed in neurons with either full-length, amino terminal portion, 199 or carboxyl terminal portion of SLO-2 tagged with the amino terminal portion of YFP (YFPa) (Fig. 9A). 200 YFP fluorescence was observed in ventral cord motor neurons when either the full-length or the C-terminal 201 portion of SLO-2 was used but not when the N-terminal protein was used in the assays (Fig. 9B). These 202 results suggest that SCYL-1 physically interacts with SLO-2, and this interaction depends on SLO-2 203 carboxyl terminal portion. 204

scyl-1 expression depends on RNA editing at a specific 3'-UTR site 205
Our RNA-Seq data revealed eight high-frequency (>15%) adenosine-to-guanosine editing sites in scyl-1 206 transcripts of wild type (Fig. 10A). All these editing sites are located within a predicted 746 bp hair-pin 207 structure in the 3' end of scyl-1 pre-mRNA, which contains an inverted repeat with >98% complementary 208 base pairing (Fig. 10B). Interestingly, RNA editing at only one of the eight sites was significantly 209 undermined (by 74%) in adr-1(zw96) compared with wild type (Fig. 10A). Sanger sequencing of scyl-1 210 mRNA and the corresponding genomic DNA from wild type, adr-1(zw96), and adr-2(gv42) confirmed that 211 RNA editing at this specific site was deficient in both the adr-1 and adr-2 mutants whereas editing at an 212 adjacent site was deficient only in the adr-2 mutant (Fig. 10C), suggesting that RNA editing at the site 213 impaired by adr-1(lf) might be important to scyl-1 expression. To test this possibility, we fused GFP coding 214 sequence in-frame to a genomic DNA fragment covering part of the last exon of scyl-1 and 5 kb downstream 215 sequence, and expressed it in neurons under the control of Prab-3 (Fig. 10D). We also made a modified 216 plasmid construct in which adenosine (A) was changed to guanosine (G) at the specific ADR-1-dependent 217 editing site to mimic the editing (Fig. 10D). In transgenic worms harboring the original genomic sequence, 218 no GFP signal was detected in neurons (Fig. 10E). In contrast, strong GFP signal was observed in neurons 219 of transgenic worms expressing the A-to-G mutated genomic sequence (Fig. 10E). Taken together, the 220 results suggest that ADR-1 plays a key role in scyl-1 expression by promoting RNA editing at a specific 221 site in its 3'-UTR. 222

Human Slo2.2/Slack is regulated by SCYL1 223
The HEAT domain of SCYL proteins play important roles in protein-protein interactions but generally 224 varies considerably in amino acid sequence for interactions with different partners (Yoshimura and Hirano, 225 2016). The high level of sequence homology of the HEAT domain between mammalian SCYL1 and worm 226 SCYL-1 (Fig. 5-figure supplement 1) promoted us to test whether mammalian Slo2.2/Slack is also 227 regulated by SCYL1. We expressed human Slack either alone or together with mouse SCYL1 in Xenopus 228 oocytes, and analyzed Slack single-channel properties. Coexpression of SCYL1 caused ~130% increase in 229 Slack Po (Fig. 11A). The channel has at least two open states and two closed states. SCYL1 increased the 230 duration and proportion of the long open state; and decreased the proportion but increased the duration of 231 the long closed state (Fig. 11 B and C). These effects of SCYL1 on Slack are similar to those of SCYL-1 232 on SLO-2 single-channel properties (Fig. 8), suggesting that regulation of Slo2 channel function is likely a 233 conserved physiological function of SCYL-1/SCYL1 proteins. 234 235

Discussion 236
This study shows that both ADR-1 and SCYL-1 are critical to SLO-2 physiological function in neurons. 237 While ADR-1 enhances SLO-2 function indirectly through regulating the expression level of SCYL-1, the 238 latter do so directly. These conclusions are supported by multiple lines of evidence, including the isolation 239 of adr-1(lf) mutants as suppressors of SLO-2(gf), the inhibition of SLO-2 activities by either adr-1(lf) or 240 scyl-1(lf), the reduction of scyl-1 transcript expression in adr-1(lf) and correlation between scyl-1 RNA 241 editing and gene expression, the SLO-2 carboxyl terminal-dependent reconstitution of YFP fluorophore in 242 BiFC assays with SCYL-1, and the inhibitory effects of scyl-1(lf) on SLO-2 single-channel activities. 243 Importantly, we found that the human Slack is also regulated by SCYL1. 244 The biological significance of RNA editing at non-coding regions is only beginning to be appreciated. 245 A recent study with C. elegans identified many neural-specific A-to-I editing sites in the 3'-UTR of clec-246 41, and found that adr-2(lf) causes both an elimination of these editing events and a chemotaxis defect 247 (Deffit et al., 2017). Although it is unclear how clec-41 expression is controlled by these editing events, 248 and a direct link between the chemotaxis defect of adr-2(lf) mutant and the decreased clec-41 expression 249 remains to be established, these results suggest that RNA editing at non-coding regions might have 250 important biological functions. In the present study, we demonstrate that A-to-I RNA editing at the 3'UTR 251 of scyl-1 controls its expression, and that SCYL-1 contributes to neuronal whole-cell currents through a 252 direct effect on the SLO-2 channel. The results of these two studies have provided a glimpse of the 253 biological roles of 3'-UTR RNA editing in gene expression and neuronal function. 254 Our results demonstrate that RNA editing at a single site in the 3'-UTR could have a profound effect in 255 promoting gene expression. The A-to-I conversion at the specific editing site of scyl-1 increases base 256 pairing in the putative double-stranded structure of the 3'-UTR (Fig. 10B). Increased base paring in a 257 double-stranded RNA generally facilitates RNA degradation. It is therefore intriguing how such an 258 increased paring in the 3'-UTR may cause increased gene expression. One possibility is that editing at this 259 site helps recruit a specific RNA-binding protein to the 3'-UTR to prevent scyl-1 mRNA from degradation. 260 Although the exact mechanism remains to be determined, it is a remarkable first example that a specific 261 RNA editing site at the 3'UTR plays a crucial role in gene expression. In summary, this study demonstrates that ADAR-mediated RNA editing controls the expression of 293 SCYL-1, which interacts with SLO-2 to allow SLO-2 perform its physiological functions. Moreover, this 294 study shows that this regulatory mechanism is conserved with mammalian SCYL1 and Slo2. Our findings 295 reveal a new molecular mechanism of Slo2 channel regulation, and provide the bases for investigating how 296

Mutant screening and mapping 324
An integrated transgenic strain expressing Pslo-1::SLO-2(gf) and Pmyo-2::YFP (transgenic marker) in the 325 wild-type genetic background was used for mutant screen. L4-stage slo-2(gf) worms were treated with the 326 chemical mutagen ethyl methanesulfonate (50 mM) for 4 hours at room temperature. F2 progeny from the 327 mutagenized worms were screened under stereomicroscope for animals that moved better than the original 328 slo-2(gf) worms. 17 suppressors were isolated in the screen and were subjected to whole-genome 329 sequencing. Analysis of the whole-genome sequencing data showed that 2 mutants have mutations in the 330 adr-1 gene (www.wormbase.com). Identification of adr-1 mutants was confirmed by the recovery of the 331 sluggish phenotype when a wild-type cDNA of adr-1 under the control of Prab-3 was expressed in slo-332 2(gf);adr-1(zw81) double mutants. 333

Analysis of expression pattern and subcellular localization 348
The expression pattern of adr-1 was assessed by expressing GFP under the control of 1.8-kb adr-1 promoter 349 Rockingham, VT, USA). 373

Behavioral assay 374
Locomotion velocity was determined using an automated locomotion tracking system as described 375 previously (Wang and Wang, 2013). Briefly, a single adult hermaphrodite was transferred to an NGM plate 376 without food. After allowing ~30 sec for recovery from the transfer, snapshots of the worm were taken at Pipette solution III contained (in mM) 150 K + gluconate, 1 Mg 2+ gluconate and 10 HEPES (pH 7.2). 418

Data Analyses for Electrophysiology 432
Amplitudes of whole-cell currents in response to voltage steps were determined from the mean current  indicate statistically significant differences (* p < 0.05; *** p < 0.001) whereas "ns" stands for "no 635 significant difference" between the indicated groups based on either one-way (A) or two-way (B) ANOVA 636 with Tukey's post hoc tests. 637 The following source data are available for Figure 2:  properties. Sample sizes were 8 slo-2(nf101);adr-1(zw96), 6 adr-1(zw96) rescue, and 12 in each of the 652 remaining groups. All values are shown as mean ± SE. The asterisks indicate statistically significant 653 differences (*p < 0.05, ***p < 0.001) compared with wild type whereas "ns" stands for no significant 654 difference between the indicated groups based on either two-way (B) or one-way (C and E) ANOVA with 655 Tukey's post hoc tests. Pipette solution I and bath solution I were used in (A) and (C). Pipette solution II 656 and bath solution I were used in (D). 657 The following figure supplement and source data are available for Figure 4: The scyl-1 mutant allele zw99 was made by introducing a stop codon after the residue I 152 (indicated by an 673 arrow) using the CRISPR/Cas9 approach. 674 Source data 1. Raw data and numerical values for data plotted in Figure 5. values are shown as mean ± SE. The asterisks (***) and pound signs ( ### ) indicate statistically significant 684 differences (p < 0.001) between the indicated groups and from wild type, respectively, whereas "ns" stands 685 for no significant difference between the indicated groups (two-way ANOVA with Tukey's post hoc tests). 686 (B) GFP signal in ventral cord motor neurons was indistinguishable between wild type and scyl-1(zw99) 687 worms expressing GFP-tagged full-length SLO-2 under the control of Prab-3. Scale bar = 20 µm. 688 The following source data are available for Figure 7: 689 Source data 1. Raw data and numerical values for data plotted in Figure 7. were used. All values are shown as mean ± SE. The asterisks indicate a significant difference between the 697 indicated groups (* p < 0.05, *** p < 0.001, one-way ANOVA with Tukey's post hoc tests). 698 The following source data are available for Figure 8: The following source data are available for Figure 10: 729 Source data 1. Raw data and numerical values for data plotted in Figure 10. Sample sizes were 13 in both groups. All values are shown as mean ± SE. The asterisks (***) indicate a 736 significant difference compared between the indicated groups (p < 0.001, unpaired t-test). 737 The following source data are available for Figure 11: 738 Source data 1. Raw data and numerical values for data plotted in Figure 11. 739