The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis
The complexity of post-transcriptional gene regulation has exponentially expanded in recent years as the repertoire of RNA binding proteins ( RBPs ) in an organism as well as their binding sites have been uncovered ( Castello et al . , 2012; Wessels et al . , 2016; Beckmann et al . , 2015; Brannan et al . , 2016 ) . However , connecting this information to how different RBPs alter mRNA splicing , stability , as well as translation is only beginning to be elucidated , and likely varies in different cell- and tissue-types as well as during development . Adenosine deaminases that act on RNA ( ADAR ) are RBPs best known for converting adenosine ( A ) to inosine ( I ) within double-stranded RNA ( dsRNA ) , including double-stranded regions of transcripts and small RNA precursors ( Nishikura , 2010; Goodman et al . , 2012 ) . Transcriptome-wide identification of A-to-I editing sites from a number of species has indicated that RNA editing is prevalent in animal transcriptomes both in coding and non-coding regions of the genome , but the genomic distribution of these editing sites vary in different organisms ( Savva et al . , 2016 ) . As inosine is a biological mimic of guanosine , A-to-I editing has the potential to affect gene expression by altering coding potential , splice site selection , and/or small RNA binding ( Deffit and Hundley , 2016; Nishikura , 2016; Tajaddod et al . , 2016 ) . In addition , recent studies have shown mammalian ADARs play editing-independent roles through binding to mRNAs and influencing the association of other RBPs on the same target RNA ( Anantharaman et al . , 2017; Wang et al . , 2013; Bahn et al . , 2015 ) . One of the primary biological functions of ADARs is to promote proper neuronal function ( Li and Church , 2013; Behm and Öhman , 2016 ) . The mammalian brain contains the highest level of inosine , and A-to-I editing within coding regions of specific human transcripts , such as ion channels and receptors , alters the physiological properties of the encoded proteins , a requisite for proper neuronal function ( Paul and Bass , 1998; Tariq and Jantsch , 2012; Rosenthal and Seeburg , 2012 ) . Consistent with this important role of ADARs in the mammalian nervous system , alterations in ADAR protein levels and editing activity have been observed in human neuropathological diseases , including Alzheimer’s disease , amyotrophic lateral sclerosis and many cancers , including brain tumors ( Mannion et al . , 2015; Bajad et al . , 2017; Tomaselli et al . , 2014 ) . Studies in model organisms provide additional evidence for a critical role for ADARs within the nervous system . Mice lacking ADAR2 die of epileptic seizures by postnatal day 20 due to under-editing of one adenosine within the coding region of the glutamate receptor , which results in excessive calcium influx and neuronal excitoxicity ( Higuchi et al . , 2000 ) . Loss of Drosophila melanogaster ADAR ( dADAR ) results in severe behavioral abnormalities , including extreme uncoordination , tremors , and a lack of courtship , as well as age-dependent neurodegeneration ( Palladino et al . , 2000 ) . Similar to the mouse and fly model organisms , loss of ADARs in C . elegans results in abnormal neuronal function as evidenced by defective chemotaxis to a number of volatile chemicals sensed by the AWA and AWC neurons ( Tonkin et al . , 2002 ) . Loss of C . elegans ADARs likely affects the ability of the neurons to sense these chemicals as the chemotaxis defect is less severe with increasing doses of these volatiles ( Tonkin et al . , 2002 ) . To date , the targets responsible for the chemosensory defects of C . elegans ADARs remain unknown . In the present study , we sought to understand the neurobiological effects of RNA editing in worms by dissecting the neural gene regulatory role of ADR-2 , the only A-to-I editing enzyme in C . elegans . Though multiple studies have used high-throughput sequencing to assess the editome and the role of ADR-2 , these studies have been limited to analyzing RNA isolated from whole worms ( Whipple et al . , 2015; Zhao et al . , 2015; Goldstein et al . , 2017 ) . However , a recently developed method utilized chemomechanical disruption of worms followed by fluorescent activated cell sorting ( FACS ) to obtain cells of interest from whole worms ( Spencer et al . , 2014; Kaletsky et al . , 2016 ) . By expressing a fluorescent marker in neural cells , we used this method to isolate and sequence the transcriptome of neural cells from wild-type and adr-2 deficient worms . We performed the first unbiased tissue-specific assessment of RNA editing in C . elegans . High-throughput sequencing combined with detection of A-to-I editing events using our newly developed SAILOR software revealed over 7300 editing sites in the neural editome . Additionally , a differential expression analysis identified 169 genes whose expression was changed upon loss of adr-2 . To identify potential ADR-2 targets responsible for defects in chemotaxis , our study further focused on genes known to regulate this biological process . Here , clec-41 , a gene previously found to be important for proper worm locomotion ( Simmer et al . , 2003 ) , was found to be expressed and edited within the 3’ untranslated region ( UTR ) in neural cells . In addition , clec-41 mRNA transcripts were differentially expressed in neural cells lacking adr-2 . Strikingly , transgenic expression of clec-41 in neural cells of adr-2 deficient worms was sufficient to rescue the aberrant chemotaxis of these animals . Furthermore , expression of a mutant ADR-2 protein incapable of deamination was not sufficient for proper clec-41 expression or chemotaxis , indicating deamination is required for both gene regulation and chemotaxis . In sum , this is the first study to link noncoding A-to-I editing and altered expression of a specific transcript with a neurological consequence resulting from loss of ADARs .
To determine the role of ADR-2 in neural cells , the neural transcriptome of C . elegans was isolated from wild-type and adr-2 deficient worms . To accomplish this , larvae at the first stage of development ( L1 larvae ) were subjected to chemomechanical disruption followed by FACS ( Spencer et al . , 2014 ) . Both wild-type and adr-2 ( - ) strains express green fluorescent protein ( GFP ) driven by the pan-neural promoter , rab-3 , that allows for isolation of neural cells . This technique is robust in dissociating the larvae to single cells , while also resulting in mostly live cells ( Figure 1A ) . The FACS profile indicated that 22% of live cells were GFP positive , consistent with the proportion of neural cells in L1 worms ( 222/558 ) and similar to previously published studies using this technique ( Figure 1B ) ( Spencer et al . , 2014 ) . Consistent with neural enrichment in the GFP+ cells , qRT-PCR of a known neural gene , syntaxin ( unc-64 ) ( Saifee et al . , 1998 ) indicated an enrichment in the isolated neural cells compared to non-neural cells ( Figure 1C ) , whereas expression of the muscle gene , myo-3 ( Ardizzi and Epstein , 1987 ) , was depleted in the isolated neural cells compared to the non-neural cells ( Figure 1C ) . To understand the role of ADR-2 in the neural transcriptome , RNA-sequencing ( RNA-seq ) of poly-A+ selected RNA isolated from wild-type ( N2 ) and adr-2 ( - ) neural cells was performed . To identify and estimate A-to-I editing events from the neural transcriptome of C . elegans , we developed the SAILOR ( Software for Accurately Identifying Locations Of RNA editing ) software , a publicly available improvement in both speed and accuracy of the algorithm described in Washburn et al . ( 2014 ) . Briefly , strand-specific RNA-seq reads were aligned to the C . elegans genome ( ce11 ) to identify single nucleotide changes between RNA and DNA ( Figure 2A ) . To eliminate possible genomic mutations that mimic A-to-I editing events , known single nucleotide polymorphisms ( SNPs ) and single nucleotide variants ( SNVs ) contained within Wormbase ( WS254 ) annotations were eliminated . To measure the accuracy of editing events called , a confidence score was assigned using a beta distribution that considers both read depth and editing site percentage at each site . Only sites with a confidence score greater than 99% were considered for downstream analyses . This approach identified only 28 editing events in the adr-2 ( - ) neural transcriptome , whereas 7377 events were identified in the wild-type neural transcriptome . After subtracting the sites that were identified in the adr-2 ( - ) neural transcriptome , 7361 A-to-I editing sites were predicted for the wild-type neural transcriptome ( Supplementary file 1 ) . The 7361 predicted editing sites mapped to 549 genes , 104 of which are novel edited targets ( Supplementary file 1 ) ( Goldstein et al . , 2017 ) . To independently validate the predicted editing sites , Sanger sequencing assays were performed for 7 genes that had several predicted editing events either in introns or 3’ untranslated regions ( UTRs ) . As low level editing events are difficult to discern in Sanger sequencing assays , the validation focused on sites that were identified as having greater than 10% editing in the RNA-seq data . Importantly , 86 of 86 predicted editing sites ( excluding 4 sites in which Sanger sequencing chromatogram had too much noise to accurately determine the presence of a true editing event ) were confirmed ( Figure 2B and Supplementary file 1 ) . This data indicates the false discovery rate ( FDR ) of SAILOR is less than 1% , which is a significant improvement over the 6% FDR reported in our previously described algorithm ( Washburn et al . , 2014 ) . Furthermore , Sanger sequencing identified 87 additional editing sites in these 7 genes , demonstrating SAILOR can identify A-to-I editing sites on highly edited transcripts ( Supplementary file 1 ) . The initial assessment of the distribution of A-to-I editing events in the neural transcriptome indicated that nearly one-third of the 7361 editing sites were in intergenic regions . As the complete untranslated region ( UTR ) sequences are not annotated for all C . elegans genes and previous studies have indicated that many editing sites fall 1 , 000–2 , 000 base-pairs from the annotated UTRs ( Zhao et al . , 2015; Whipple et al . , 2015 ) , the genomic distribution of editing sites was re-analyzed including annotations of 2000 base-pairs upstream and 2000 base-pairs downstream of a genic region ( Supplementary file 1 ) . This analysis indicated that 92 . 4% of the 7361 editing sites were within genic regions , with the vast majority of these events occurring in the noncoding regions of protein-coding genes , including introns and UTRs ( Figure 2C ) . Importantly , analysis of the unique genes identified as editing targets revealed the same 549 genes using both genic assessments , consistent with the idea that the 2000 base-pair regions upstream and downstream of genic regions are likely unannotated 5’ and 3’ UTRs . It is interesting to note that in contrast to the identification of A-to-I editing events in coding regions of neuronally important mRNAs in squid , octopus , fly , and mammalian brains ( Alon et al . , 2015; St Laurent et al . , 2013; Ramaswami et al . , 2013; Hwang et al . , 2016; Graveley et al . , 2011; Liscovitch-Brauer et al . , 2017 ) , the worm neural RNA-seq data identified very few A-to-I editing events within coding regions . Of the over 7300 events identified , only 83 editing sites ( approximately 1% ) were in coding regions . These data are consistent with the number of coding region editing sites previously identified from RNA isolated from whole-worms , and we identified nearly all of the same edited transcripts , including the heavily edited coding regions of histone genes ( Wu et al . , 2011 ) . Together , this data indicates that re-coding editing events are not enriched in the C . elegans nervous system and demonstrates that the role of C . elegans ADARs in the nervous system is not to generate proteomic diversity , but rather to fine-tune gene expression . To further assess the role of ADR-2 in regulating the neural transcriptome , the RNA-seq datasets from three independent biological replicates of wild-type and adr-2 ( - ) neural cells were assessed for differential gene expression using DESeq ( Love et al . , 2014 ) ( Supplementary file 2 ) . This analysis identified 76 genes that were ≥2 fold upregulated in neural cells lacking adr-2 as compared to wild-type neural cells and 93 genes that were ≥2 fold downregulated . Of these 169 differentially regulated genes , only four genes were identified as ADR-2 editing targets in neural cells , suggesting ADR-2 can also regulate expression of many genes via a mechanism that is independent of direct editing of the target mRNA . To validate the differential expression values obtained from the RNA-seq dataset , qRT-PCR was performed for two genes from each category ( Figure 3A and B ) . Expression of these genes reflected the fold-change determined by the RNA-seq analysis . Interestingly , qRT-PCR analysis of the expression of these genes in RNA isolated from whole L1 larvae did not follow the same trend ( Figure 3C ) . This reveals that loss of adr-2 regulates expression of genes at a tissue-specific level in C . elegans and suggests that these changes may not be observed in whole worm analysis . Previous studies have shown that worms lacking adr-2 ( - ) exhibit defects in chemotaxis ( Tonkin et al . , 2002 ) , yet the underlying mechanism has yet to be determined . To attempt to elucidate ADR-2 target mRNAs that are important for proper neural function , the neural editome identified above was queried for genes involved in chemotaxis or locomotion . Editing sites were predicted in 82 genes that are associated with locomotion or chemotaxis GO terms ( Supplementary file 1 ) . To narrow down this list of potential neurobiologically important targets , we focused on previous phenotypic data which demonstrated that , similar to the loss of adr-2 , loss of adr-1 results in worms that are defective in sensing organic compounds ( Tonkin et al . , 2002 ) . ADR-1 shares similar sequence and domain structure to ADR-2 , though it lacks critical amino acids necessary for deamination ( Tonkin et al . , 2002 ) . As ADR-1 promotes editing by ADR-2 at specific adenosines ( Washburn et al . , 2014 ) , the similar phenotypes of adr-1 ( - ) and adr-2 ( - ) worms suggests that ADR-1 promotes editing by ADR-2 at sites important for proper chemotaxis . Using the assumption that ADR-1 regulates neurobiological transcripts of interest , the 82 edited genes involved in locomotion and chemotaxis were queried against a list of transcripts identified in a transcriptome-wide RNA immunoprecipitation of ADR-1 bound targets ( unpublished data ) . Of the 82 edited genes that are involved in proper worm locomotion/chemotaxis , 28 were identified as bound by ADR-1 ( Supplementary file 1 ) . Of these genes , only one , clec-41 , was also identified as differentially expressed in neural cells lacking adr-2 ( Supplementary file 2 ) . Focusing on clec-41 , the role of ADR-1 in regulating clec-41 expression and editing in neural cells was further explored . First , ADR-1 binding to clec-41 was examined using a RNA immunoprecipitation assay for ADR-1 ( Figure 4A ) . Analysis of two independent biological replicates indicated a >20 fold enrichment of clec-41 mRNA in immunoprecipitations from wild-type worms compared to those lacking adr-1 . Thus , consistent with the transcriptome-wide RIP-Seq , clec-41 is a mRNA target of ADR-1 . To test whether editing of clec-41 was regulated by adr-1 , RNA from adr-1 ( - ) neural cells was isolated and compared to RNA from wild-type and adr-2 ( - ) neural cells . RT-PCR and Sanger sequencing of the clec-41 3’ UTR was performed for all three strains . Consistent with clec-41 transcripts being bona fide targets of adenosine deamination , the wild-type neural cells exhibited A-to-I editing at 40 individual adenosines within the clec-41 3’ UTR and this editing was absent in neural cells lacking adr-2 ( Figure 4B and Figure 4—figure supplement 1A ) . Compared to RNA from wild-type neural cells , editing of clec-41 is significantly altered at 12 sites in RNA isolated from adr-1 ( - ) neural cells ( Figure 4B and Figure 4—figure supplement 1A ) . ADR-1 regulation of editing at these adenosines was not observed in RNA isolated from L1 worms ( Figure 4B and Figure 4—figure supplement 1B ) . In sum , only 2 of the 12 ADR-1 regulated editing sites exhibited similar editing levels between the L1 and neural cell RNA , suggesting neural-specific regulation of editing at these sites by ADR-1 . To examine the role of adr-1 in regulating clec-41 expression , qRT-PCR of RNA from both isolated neural cells and L1 worms was performed . Loss of adr-1 resulted in a three-fold reduction of clec-41 expression in neural cells ( Figure 4C ) . Consistent with the differential expression data , loss of adr-2 resulted in a similar three-fold downregulation and the regulatory effect of either adr-1 or adr-2 cannot be observed in RNA isolated from L1 worms ( Figure 4C and D ) . This suggests that both ADR-1 and ADR-2 are required for proper clec-41 expression and editing in neural cells . This is consistent with previous data that indicated a similar chemotaxis defect for worms lacking either adr-1 or adr-2 and worms lacking both adr-1 and adr-2 ( Tonkin et al . , 2002 ) . Despite the fact that the similarity in chemotaxis phenotypes has been known for over a decade , this study is the first to identify an editing target whose expression is regulated by both C . elegans ADARs . Furthermore , this study brings into light the need to assess editing and gene expression at a tissue-specific level in C . elegans , as this molecular phenotype cannot be observed in RNA isolated from L1 worms . As our gene regulatory data indicates that both C . elegans ADARs are important for proper clec-41 expression and a previous genome-wide screen suggested clec-41 was important for proper locomotion/chemotaxis , we sought to directly assess the role of clec-41 in regulating the chemotaxis defects associated with loss of ADARs . To test this , a worm line expressing clec-41 under the control of the pan-neuronal rab-3 promoter ( rab3p ) was generated . To visually follow the clec-41 transgene , the worms were co-injected with a rab3p::GFP reporter . As the chemotactic effects of restoring clec-41 expression to adr-2 ( - ) worms were of interest , the transgenic wild-type worm generated by microinjection was crossed to C . elegans adr-1 ( tm668 ) ; adr-2 ( ok735 ) mutant male worms . After identifying progeny that were wild-type for the adr-1 locus , worms homozygous for the adr-2 ( ok735 ) mutation and carrying the rab3p::clec-41 transgene as well as wild-type worms carrying the transgenes were isolated from the F2 progeny . Neural expression of clec-41 from the transgene in both the wild-type and adr-2 ( - ) worms was confirmed by performing qRT-PCR on neural cells isolated from the transgenic worms . Compared to wild-type worms , both transgenic lines express higher levels of clec-41 in neural cells ( Figure 5A ) . As previous chemotaxis studies indicated that adr-2 ( - ) worms are defective in sensing volatile organic chemicals that are sensed by AWA and AWC neurons ( Tonkin et al . , 2002 ) , we tested whether transgenic expression of clec-41 in neural cells altered chemotaxis to benzaldehyde ( sensed by AWC ) or trimethylthiazole ( sensed by both AWA and AWC ) ( Bargmann et al . , 1993 ) . Here , plates containing normal worm growth media were marked as shown in Figure 5B . Immediately prior to addition of worms , the paralysis agent sodium azide as well as a control ( Ethanol ) or the chemoattractant were spotted on opposite sides of the plate . Worms were placed in the center and allowed to move for one hour , after which the worms were counted to determine a chemotaxis index . Similar to previous results obtained using a different deletion allele of adr-2 , worms lacking adr-2 ( - ) ( adr-2 ( ok735 ) ) exhibited reduced chemotaxis indices to both benzaldehyde and trimethylthiazole compared to wild-type worms ( Figure 5C and D ) . Strikingly , expression of clec-41 within the neural cells of adr-2 ( - ) worms led to a significant increase in the chemotaxis indices ( Figure 5C and D ) . Importantly , the increased expression of clec-41 within neural cells did not significantly increase the chemotaxis index over wild-type worms . In addition , expression of the clec-41 3’ UTR in neural cells was not sufficient to restore chemotaxis to adr-2 ( - ) worms nor was expression of clec-41 in nonneural tissue ( Figure 5—figure supplement 1A and B ) . This data suggests restoring CLEC-41 expression within neural cells is sufficient to prevent disruptions in chemotaxis specifically associated with loss of adr-2 . As ADAR family members are known to perform both editing-dependent and independent mechanisms of gene regulation , we sought to directly assess the role of A-to-I RNA editing in clec-41 mRNA expression and chemotaxis . In this regards , we queried worm strains from The Million Mutation Project ( Thompson et al . , 2013 ) for missense mutations in conserved residues of the adr-2 deaminase domain . The adr-2 ( gk777511 ) strain has a single nucleotide mutation that results in the incorporation of an arginine instead of a conserved glycine at amino acid 184 ( G184R ) and will hereafter be referred to as the adr-2 ( G184R ) worms ( Figure 6A ) . To determine whether the G184R mutation affects deaminase activity , RT-PCR and Sanger sequencing of the clec-41 3’ UTR was performed on RNA isolated from wild-type , adr-2 ( - ) and adr-2 ( G184R ) worms . At all 40 adenosines edited in wild-type worms , editing was absent in adr-2 ( G184R ) worms , similar to worms lacking adr-2 ( Figure 6B , data not shown ) . To examine whether the reduced editing of the ADR-2 G184R mutant was a result of loss of RNA binding , an ADR-2 RNA immunoprecipitation assay was performed . Using a custom antibody that is specific for ADR-2 ( Figure 6—figure supplement 1 ) , similar levels of ADR-2 protein were immunoprecipitated from both wild-type and adr-2 ( G184R ) worms , whereas no ADR-2 protein was immunoprecipitated from the adr-2 ( - ) worms ( Figure 6C ) . Analysis of two independent biological replicates indicated a >20 fold enrichment of clec-41 mRNA in immunoprecipitations from both wild-type and adr-2 ( G184R ) worms compared to those lacking adr-2 , indicating that the ADR-2 G184R mutant binds clec-41 similar to wild-type ADR-2 ( Figure 6C ) . To directly assess the impact of loss of RNA editing on clec-41 neural gene expression , qRT-PCR of RNA from neural cells isolated from adr-2 ( G184R ) worms , as well as wild-type and adr-2 ( - ) worms , was performed . Neural mRNA expression of adr-2 was similar between the wild-type and adr-2 ( G184R ) neural cells ( Figure 6D ) . However , clec-41 expression was reduced three-fold in neural cells expressing the ADR-2 G184R mutant compared to wild-type neural cells , mirroring the adr-2 ( - ) neural cells ( Figure 6E ) and suggesting deamination is required for proper neural expression of clec-41 . Similarly , chemotaxis in the adr-2 ( G184R ) mutant worms was impaired compared to wildtype , further implicating A-to-I editing and clec-41 expression in regulating this biological function ( Figure 6F ) . This is the first study to link a phenotype associated with loss of C . elegans ADAR enzymes to editing-dependent regulation of a specific gene .
In this study , we have provided the first analysis of the tissue-specific role of ADR-2 , both in A-to-I editing and in regulating neural gene expression in C . elegans . Combining neural cell isolation with RNA-sequencing and editing site detection with SAILOR , we identified over 7300 A-to-I editing sites in the C . elegans neural transcriptome , including 104 novel edited genes . In addition to identifying neural editing events , the neural RNA-seq data revealed 169 genes to be misregulated in worms lacking editing . The intersection of these two datasets lead to the identification of clec-41 , a gene that was expressed three-fold lower in adr-2 ( - ) neural cells and whose transcripts contained multiple 3’ UTR editing events . Importantly , using a combination of transgenic worms and chemotaxis assays , we demonstrated that transgenic expression of clec-41 in neural cells was sufficient to rescue the aberrant chemotaxis of adr-2 deficient worms and that editing is required for proper clec-41 expression and chemotaxis . Although ADARs play critical roles in editing codons of neuronal transcripts to generate the proteomic diversity needed for proper neuronal function in mammals , our data indicate the aberrant behavioral phenotypes caused by lack of C . elegans adr-2 are a result of altered neural gene expression rather than recoding events . Consistent with this idea , previous studies indicated that loss of critical components of the RNA interference ( RNAi ) pathway , namely rde-1 or rde-4 , can restore proper chemotaxis to adr mutant worms ( Tonkin and Bass , 2003 ) . However , previous attempts to identify genes antagonistically regulated by RNAi and ADARs were unable to pinpoint transcripts that underlie the chemotactic behavior ( Wu et al . , 2011 ) . Interestingly , previous transcriptome-wide studies of gene expression in RNAi mutant worms identified elevated levels of clec-41 in dcr-1 , rde-4 and rde-1 mutant worms ( Welker et al . , 2007 ) . Similarly , data from our lab has indicated that neural cells from rde-4 ( ne299 ) mutant worms have increased levels of clec-41 compared to wildtype worms ( unpublished data ) . Therefore , despite the conserved role of ADARs in regulating proper neurological function and essential roles for A-to-I editing within codons of neurologically important targets in certain organisms , our data indicate studies addressing the role of ADARs in the nervous system need to focus not only on direct editing targets but also on differentially expressed genes . In this regard , our analysis revealed 169 genes to be misregulated in adr-2 ( - ) neural cells; however , A-to-I editing was only detected in the transcripts of four of these genes . At present , it is unclear how ADR-2 is regulating expression of these genes . Several recent studies have identified editing-independent mechanisms by which mammalian ADARs promote gene expression by either promoting or inhibiting binding of other RBPs . Specifically , human ADAR1 was shown to promote binding of the mRNA stabilizing RBP , HuR , while human ADAR2 was shown to inhibit the mRNA degrading RBP , poly ( A ) specific ribonuclease deadenylase ( PARN ) ( Anantharaman et al . , 2017; Wang et al . , 2013 ) . Future work will test whether the mRNAs with altered expression in adr-2 ( - ) neural cells are directly bound by ADR-2 and if this binding alters other RBPs that influence the expression of these targets . Our study identified altered expression of clec-41 as a critical gene regulatory function of ADR-2 . Strikingly , restoring clec-41 expression in neural cells was sufficient to prevent disruptions in chemotaxis in adr-2 ( - ) worms . It is currently unclear how CLEC-41 , a predicted C-type lectin protein , functions to regulate proper chemotaxis . However , previous studies have determined that clec-41 is regulated by the Protein Kinase D pathway , and that the DKF-2 B isoform of Protein Kinase D is expressed in ~20 chemosensory neurons , including the AWA and AWC neurons that are responsible for the aberrant chemotaxis defects observed in adr-2 deficient worms ( Ren et al . , 2009; Fu et al . , 2009 ) . Future work aimed at determining whether clec-41 expression is specifically altered in AWA and AWC neurons lacking adr-2 ( - ) will be critical to understanding how CLEC-41 contributes to proper chemotaxis . In addition , experiments aimed at understanding the role of specific editing events and cellular factors , including the Protein Kinase D pathway , to regulate clec-41 expression in neural cells will broaden our understanding of the gene regulatory functions of ADARs . This study utilized novel methodology to assess A-to-I RNA editing in a specific tissue and developed a robust platform for easy identification of editing . The SAILOR software is publicly available and designed for ease of use to run with one single command , requiring only a BAM-formatted file of the sequence alignments , a FASTA-formatted reference genome sequence ( of any organism or cell-type ) , and a BED-formatted file of known SNPs . Notably , SAILOR allows the user to specify a range of filtering criteria including: Non A-to-I mismatch rate , location of mismatches ( to account for biases at the end of reads ) , and a minimum read coverage required to call variants . Users may relax any of these filtering criteria and/or pursue analysis of A-to-I editing sites with lower confidence scores . However , altering these criteria may result in a higher false discovery rate , therefore it is critical to utilize Sanger sequencing to verify a portion of all predicted sites . Our validation by Sanger sequencing revealed 87 additional editing sites not detected using our stringent SAILOR parameters , but either lowering read coverage requirements or decreasing confidence scores to 90% allowed for recovery of 54 of these sites . The complete environment is defined using Common Workflow Language ( Amstutz et al . , 2016 ) and packaged inside a Singularity ( Kurtzer et al . , 2017 ) container . This allows users the flexibility to run the entire workflow on a wide variety of platforms ( workstation , cloud or HPC clusters ) immediately after downloading the single executable file . In sum , this study is the first of its kind in the RNA editing field to span from developing novel methodology for tissue-specific target identification to organismal behavior , significantly advancing our understanding of ADAR functions in neural cells . Our data indicates the necessity of assessing editing and gene expression at a tissue or cell-type specific level . Identification of the edited target clec-41 as a critical regulator of chemotaxis in ADAR deficient worms would likely not have been possible without the neural-specific identification of gene expression changes as alterations in clec-41 editing and gene expression were not observed in RNA isolated from adr mutant whole worm extracts .
Worm strains were cultured on NGM plates seeded with E . coli OP50 . The worm strains that express GFP driven by the pan-neural promoter , rab-3: BB76 ( wild-type ) , BB77 ( adr-1 ( tm668 ) ) , and BB78 ( adr-2 ( ok735 ) ) were previously published ( Hundley et al . , 2008 ) . The worms used for the RNA immunoprecipitation were BB19 ( adr-1 ( tm668 ) ) and BB21 ( adr-1 ( tm668 ) +blmEx1[3XFLAG-adr-1 genomic , rab3::gfp::unc-54] ) , both previously published ( Washburn et al . , 2014 ) . The VC40720 strain containing a deaminase mutant of ADR-2 ( gk777511 , chrIII:7232280 C > T ) was obtained from the Caenorhabditis Genetics Center ( CGC ) , but was originally created by the C . elegans Reverse Genetics Core Facility at the University of British Columbia . This strain was backcrossed eight times to generate strain HAH4 ( adr-2 ( gk777511 ) ) . This strain was then crossed to BB78 ( adr-2 ( ok735 ) ) heterozygous males and genotyping was used to identify progeny that were wild-type for the adr-2 locus ( named HAH8 ) or containing either the adr-2 ( ok735 ) mutation ( named HAH9 ) or the adr-2 ( gk777511 ) mutation ( named HAH10 ) . Transgenic worm lines were generated by microinjection into the gonads of young adult N2 worms . The injection mix used for generating transgenic worms contained the following: 10 ng/μl of the transgene of interest , 20 ng/μl of the dominant marker , and 70 ng/μl of 1 kb DNA ladder ( NEB , Ipswich , MA ) . Transgenic strains were maintained by passaging only worms with the dominant marker . The dominant marker used in this study was rab3::gfp::unc-54 , which was previously described ( Hundley et al . , 2008 ) . The transgene expressing the clec-41 genomic locus was generated by amplifying clec-41 from the start codon to 2000 bp downstream of the stop codon and inserting into a modified pBluescript SK plasmid containing the rab3 promoter ( ∼1200 bp ) or the myo-2 promoter ( ~1000 bp ) . The transgene expressing the clec-41 3’UTR downstream of RFP was generated by inserting RFP into a modified pBluescript SK plasmid and the clec-41 3’ UTR downstream of RFP . Transgenic strains expressing these constructs were isolated using standard techniques following microinjection of the preceding plasmids into the gonads of adult hermaphrodites using the Bristol strain N2 and named blm9[rab3::clec-41 , rab3::gfp:unc-54 3' UTR] . The injected rab-3 promoter driven clec-41 transgenic strain was crossed with BB21: adr-1 ( tm668 ) ; adr-2 ( ok735 ) and genotyping was used to identify progeny that were wild-type for the adr-1 locus and contained either the wild-type adr-2 locus ( named HAH1: blm9[rab3::clec-41 , rab3::gfp:unc-54 3' UTR] ) or the mutant adr-2 ( ok735 ) locus ( named HAH2: adr-2 ( ok735 ) +blm9[rab3::clec-41 , rab3::gfp:unc-54 3' UTR] ) . The injected myo-2 driven clec-41 transgenic and rab3p::RFP::clec-41 3’UTR transgenic strains were crossed with BB20 ( adr-2 ( ok735 ) ) and genotyping was used to identify progeny that contained either the wild-type adr-2 locus ( named HAH15 blm12[myo-2::clec-41 , rab3::gfp:unc-54 3' UTR] , HAH11 blm9[rab3::rfp:clec-41 3' UTR , rab3::gfp:unc-54 3' UTR] or the mutant adr-2 ( ok735 ) locus ( named HAH16 adr-2 ( ok735 ) +blm12[myo-2::clec-41 , rab3::gfp:unc-54 3' UTR] , HAH12 adr-2 ( ok735 ) +blm9[rab3::rfp:clec-41 3' UTR , rab3::gfp:unc-54 3' UTR] . Neural cells were isolated from first larval stage ( L1 ) worms using a previously published method ( Spencer et al . , 2014 ) . Eggs were released from gravid adult worms by incubating in 0 . 5 M NaOH in 1 . 2% NaClO for 6 min . Eggs were thoroughly washed with M9 buffer ( 3 g KH2PO4 , 6 g Na2HPO4 , 5 g NaCl , 1 ml 1 M MgSO4 , H2O to 1 L ) and cultured overnight at 20°C to synchronize worms in the first larval stage ( L1 ) . L1 worms were washed with dH2O and digested with freshly thawed SDS-DTT ( 200 mM DTT , 0 . 25% SDS , 20 mM HEPES , pH 8 . 0 3% sucrose , stored at −20°C ) for 2 min on a nutator at room temperature . The digest was thoroughly washed with egg buffer ( 118 mM NaCl , 48 mM KCl , 2 mM CaCl2 , 2 mM MgCl225 mM HEPES , pH 7 . 3 adjusted osmolarity to 340 mOsm with sucrose ) and further digested in freshly made 15 mg/ml Pronase E ( Sigma-Aldrich , St . Louis , MO ) dissolved in egg buffer . During the 20 min Pronase E digestion , the worms were mechanically disrupted using a 200 μl pipette tip . The released cells were washed and resuspended in egg buffer . Cells were stained with the Near IR Live/Dead Fixable dye ( Invitrogen , Carlsbad , CA ) for 30 min prior to FACS sorting to isolate GFP expressing neural cells . Cells were sorted on a BD FACSAria II sorter and analyzed using FACSDiva 6 . 1 . 1 software ( Indiana University Bloomington-Flow Cytometry Core Facility ) . Events were thresholded on FSC-A and SSC-A parameters at a threshold of 600 and 700 respectively . Cells were first gated on FSC-A vs . SSC-A , both analyzed on a log scale , and then doublet discrimination was done using SSC-H vs . SSC-W and FSC-H vs . FSC-W . Single cells were subsequently gated for Near IR Live/Dead Fixable dye negative populations , with final gating set on a GFP-A vs . SSC-A plot with gates for GFP positive and GFP negative cells . 200 , 000 cells were sorted into 15 ml conical tubes containing 1 ml Trizol . Both the sample and collected cells were kept at 4°C during and after the sort . Total RNA was isolated from neural cells or from whole worms at the L1 stage using Trizol ( Invitrogen ) followed by treatment with DNase ( Fisher Scientific , Hampton , NH ) and purification using the RNeasy Extraction Kit ( Qiagen , Hilden , Germany ) . qRT-PCR cDNA was synthesized from total RNA using Superscript III ( Invitrogen ) with both random hexomers ( Fisher Scientific ) and oligo dT ( Fisher Scientific ) primers . SybrFast Master Mix ( KAPA , Wilmington , MA ) as well as gene specific primers ( Table 1 ) were used to quantitate gene expression on the Thermofisher Quantstudio 3 . RNA libraries were created from RNA isolated from three independent biological replicates of FACS sorted neural cells from the wild-type ( BB76 ) and adr-2 ( - ) ( BB78 ) worm strains . PolyA +beads ( Invitrogen ) were used to select for mRNA and libraries were generated using the KAPA Strand-Specific RNA Library Kit according to the manufacturer’s instructions . The libraries were sequenced for SE150 cycles on an Illumina NextSeq500 in High Output mode ( Indiana University Center for Genomics and Bioinformatics ) . To accurately call editing sites , we improved upon our previously published bioinformatics algorithm as described in ( Washburn et al . , 2014 ) , updated to accommodate for improved sequencing technology . Briefly , 150 bp single-end , stranded RNA-seq reads were trimmed of sequence adapters , polyA tails , and repetitive elements using cutadapt ( v1 . 9 . 1 ) , and aligned with STAR ( v2 . 4 . 0i ) against RepBase ( v18 ) to remove repetitive elements . Reads were then aligned to ce11 using the following STAR parameters: [outFilterMultimapNmax 10 , outFilterScoreMinOverLread: 0 . 66 , outFiterMatchNminOverLread: 0 . 66 , outFilterMismatchNmax: 10 , outFilterMismatchNoverLmax: 0 . 3] . Sorting and duplicate removal were performed using Samtools 1 . 3 . 1 . Triplicates for each condition ( wild-type and adr2 ( - ) ) were merged into a single bam file prior to site calling to increase coverage to 98 , 161 , 600 reads for wild-type and 94 , 311 , 728 reads for adr-2 ( - ) . SAILOR is publicly available on our github repository with documentation ( https://github . com/yeolab/sailor; copy archived at https://github . com/elifesciences-publications/sailor ) ( Yee et al . , 2017 ) . The following parameters were used to further filter mapped reads prior to site calling: junction overhangs must be 10nt or longer , no insertions or deletions , no mutations within 5nt of the 3’ end , not more than 1 non A-G ( T-C in antisense ) mutation . Samtools ( v1 . 3 . 1 ) mpileup [-d 1000 -E -I -p -o -f -t DP , DV , DPR , INFO/DPR , DP4 , SP] and bcftools ( v1 . 2 . 1 ) call [-O -c -A -i -v] were used to pileup and call variants . Variants were further filtered for read coverage ( minimum 5 reads after BAQ filtering from bcftools ) and ensured that variants are all A-G ( T-C in antisense ) . Variants sharing positions of SNPs contained within Wormbase ( WS254 ) annotations were removed . Candidate editing sites were given a confidence score using a previously described Bayesian model ( Bahn et al . , 2012; Li et al . , 2008 ) based on the number of supporting reads and the percent edited reads . Sites with less than 1% of reads edited and a 99% confidence score were not considered . Sites that saw 100% editing were flagged as possible SNPs . Editing sites called in the adr-2 ( - ) datasets were removed from the final list of called sites . Annotations were gathered from Wormbase WS254 . If two regions overlapped , priority was assigned in the following order: [3'UTR , 5'UTR , CDS , Intron , mRNA , piRNA , ncRNA , tRNA , nc_primary_transcript , miRNA , snoRNA , pre_miRNA , lincRNA , scRNA , antisense_RNA , rRNA , miRNA_primary_transcript , scRNA , 2000 base-pairs downstream from gene , 2000 base-pairs upstream from gene , 2000 base-pairs downstream from ncRNA , 2000 base-pairs upstream from ncRNA , pseudogene , intergenic] . 150 bp single-end , stranded reads trimmed of sequence adapters , polyA tails , and repetitive elements using cutadapt ( v1 . 9 . 1 ) and STAR ( v2 . 4 . 0i ) against RepBase ( v18 ) . Reads were then aligned to ce11 using the following parameters: [outFilterMultimapNmax 10 , outFilterScoreMinOverLread: 0 . 66 , outFiterMatchNminOverLread: 0 . 66 , outFilterMismatchNmax: 10 , outFilterMismatchNoverLmax: 0 . 3] . Mapped reads were sorted using Samtools 1 . 3 . 1 . FeatureCounts was used to count mapped reads to Wormbase ( WS254 ) gene annotations . WT and adr-2 ( - ) triplicates were then normalized and compared for differential expression using DESeq2 . Genes with a greater than 2-fold change in expression were reported . Editing sites were verified using total RNA isolated from neural cells or L1 stage worms . RNA was reverse transcribed using Thermoscript ( Invitrogen ) , and PFX Platinum DNA Polymerase ( Invitrogen ) was used with gene specific primers for PCR amplification ( Table 1 ) . Negative controls without Thermoscript were conducted for each sample to ensure all DNA subjected to sequencing resulted from cDNA amplification . PCR products were gel purified and subjected to Sanger sequencing . To get the list of genes involved in chemotaxis , annotations derived from http://www . geneontology . org/ were intersected with the list of edited genes and filtered for the following GO terms: ( GO:0040011 - locomotion , GO:0043058 - regulation of backward locomotion , GO:0040012 - regulation of locomotion , GO:0006935 - chemotaxis , GO:0040017 - positive regulation of locomotion , GO:0043059 - regulation of forward locomotion , GO:0050919 - negative chemotaxis ) . The ADR-1 RNA immunoprecipitation was performed as previously described ( Washburn et al . , 2014 ) . Briefly , after washing with IP buffer ( 50 mM HEPES [pH 7 . 4]; 70 mM K-Acetate , 5 mM Mg-Acetate , 0 . 05% NP-40 , and 10% glycerol ) , worms were subjected to 3 J/cm2 of UV radiation using the Spectrolinker ( Spectronics , Westbury , NY ) and stored at −80°C . To obtain cell lysates , frozen worms were ground with a mortar and pestle on dry ice . After thawing , the lysate was centrifuged and protein concentration was measured with Bradford reagent ( Sigma-Aldrich ) . Five milligrams of extract was added to anti-FLAG magnetic beads ( Sigma-Aldrich ) that were washed with wash buffer ( WB: 0 . 5 M NaCl , 160 mM Tris-HCl [pH 7 . 5] ) . After incubation for 1 hr at 4°C , the beads were washed with ice-cold WB , resuspended in low-salt WB ( 0 . 11 M NaCl ) , 1 μl RNasin ( Promega , Madison , WI ) , and 0 . 5 μl of 20 mg/ml proteinase K ( Sigma-Aldrich ) and incubated at 42°C for 15 min to degrade protein and release bound RNA . Protein samples were subjected to SDS-PAGE and western blotting with a FLAG antibody ( Sigma-Aldrich ) . RNA samples were isolated and qRT-PCR was performed as described above . The ADR-2 RNA immunoprecipitation was performed in a similar manner as described for the ADR-1 RNA immunoprecipitation , except that a custom antibody to ADR-2 was incubated anti-Rabbit IgG magnetic Dynabeads ( Fisher ) before incubating with cell lysates . The ADR-2 antibody was produced by Cocalico Biologicals using partially purified ADR-2 , which was tagged at the N-terminus with maltose binding protein ( MBP ) and purified from Hi5 insect cells using an amylose binding column . The amylose-bound MBP-ADR-2 was injected into rabbits for antibody generation . Adult worms were used to assess chemotaxis behavior using a method previously described ( Kowalski et al . , 2014 ) . 10 μl of 1 M sodium azide was placed at both ends of the plate ( see Figure 5B for location ) and allowed to dry . Once dry , 10 μl of ethanol ( control ) or the chemoattractant diluted in ethanol were placed at either end and allowed to dry . The adult worms were washed 3x in M9 buffer . After the final wash , worms were resuspended in ~200 μl of M9 and 20 μl ( 50–200 worms per plate ) was pipetted onto the center line of the plate . Chemotaxis to benzaldehyde ( 1:1000 dilution in ethanol ) and trimethylthiazole ( 1:10 , 000 dilution ) after 1 hr at room temperature was assessed and the chemotaxis index was determined using the formula below . Three technical replicate plates for each worm strain were used in each of the biological replicates . Chemotaxis index=Number of animals at attractant−Number of animals at controlTotal worms on plate Raw fastq files and their corresponding processed BAM alignments were made available and uploaded to the GEO database . Files generated from wild-type neural cells were labeled as: WT_1 , WT_3 , and WT_6 corresponding to the condition and original sample number . Files generated from adr-2 ( - ) neural cells were labeled as: Adr2-_2 , Adr2-_5 , and Adr2-_7 . A tabbed-separated file containing differential expression between WT and adr-2 ( - ) is also available as diffexp . tsv . Editing sites , approximate fractions , confidence and region annotations made from the editing pipeline are available on GEO accession number GSE98869 as editing_calls . tsv . GEO link for reviewers to access raw data: https://www . ncbi . nlm . nih . gov/geo/query/acc . cgi ? token=khqdcwemjhqbvgp&acc=GSE98869
ADAR proteins alter gene expression both by catalyzing adenosine ( A ) to inosine ( I ) RNA editing and binding to regulatory elements in target RNAs . Loss of ADARs affects neuronal function in all animals studied to date . Caenorhabditis elegans lacking ADARs exhibit reduced chemotaxis , but the targets responsible for this phenotype remain unknown . To identify critical neural ADAR targets in C . elegans , we performed an unbiased assessment of the effects of ADR-2 , the only A-to-I editing enzyme in C . elegans , on the neural transcriptome . Development and implementation of publicly available software , SAILOR , identified 7361 A-to-I editing events across the neural transcriptome . Intersecting the neural editome with adr-2 associated gene expression changes , revealed an edited mRNA , clec-41 , whose neural expression is dependent on deamination . Restoring clec-41 expression in adr-2 deficient neural cells rescued the chemotaxis defect , providing the first evidence that neuronal phenotypes of ADAR mutants can be caused by altered gene expression .
DNA is the blueprint that tells each cell in an organism how it should operate . It encodes the instructions to make proteins and other molecules . To make a protein , a section of DNA known as a gene is used as a template to make molecules known as messenger ribonucleic acids ( or mRNAs for short ) . The message in RNA consists of a series of individual letters , known as nucleotides , that tell the cell how much of a protein should be produced ( referred to as gene expression ) as well as the specific activities of each protein . The letters in mRNAs can be changed in specific cells and at certain points in development through a process known as RNA editing . This process is essential for animals to grow and develop normally and for the brain to work properly . Errors in RNA editing are found in patients suffering from a variety of neuropathological diseases , including Alzheimer’s disease , depression and brain tumors . Humans have millions of editing sites that are predicted to affect gene expression . However , many studies of RNA editing have only focused on the changes that alter protein activity . The ADAR proteins carry out a specific type of RNA editing in animals . In a microscopic worm known as Caenorhabditis elegans the loss of an ADAR protein called ADR-2 reduces the ability of the worm to move in response to chemicals , a process known as chemotaxis . Deffit et al . found that loss of ADR-2 affected the expression of over 150 genes in the nervous system of the worm . To identify which letters in the mRNAs were edited in the nervous system , Deffit et al . developed a new publically available software program called SAILOR ( software for accurately identifying locations of RNA editing ) . This program can be used to detect RNA editing in any cell , tissue or organism . By combining the experimental and computational approaches , Deffit et al . were able to identify a gene that was edited in normal worms and expressed at lower levels in the mutant worms . Increasing the expression of just this one of gene in the mutant worms restored the worms’ ability to move towards a chemical “scent” . Together , these findings suggest that when studying human neuropathological diseases we should consider the effect of RNA editing on the amount of gene expression as well as protein activity . Future work should investigate the importance of RNA editing in controlling gene expression in other diseases including cancers .