Regulator of CAlcineuriN 1 (RCAN1) is a highly conserved protein, first identified in fungi and yeasts as an inhibitory regulator to calcineurin, a calcium/calmodulin-dependent phosphatase (Gorlach et al., 2000; Kingsbury and Cunningham, 2000). RCAN1 is highly expressed in the striated muscle, the nervous system and induced, during pathological conditions such as neurodegeneration (Crawford et al., 1997; Fuentes et al., 2000; Wiese et al., 1995; Yang et al., 2000). RCAN1 suppresses dephosphorylation of a multi-functional transcription factor NFAT, which is a well-known substrate for calcineurin, and enhanced calcineurin activity induces RCAN1 expression via negative feedback (Fuentes et al., 2000; Yang et al., 2000). Therefore, RCAN1 overexpression due to trisomy 21 in Down syndrome plays a critical role in the pathology, because of RCAN1’s inhibitory interaction with calcineurin (Ermak et al., 2001; Fuentes et al., 1995; 1997). However, there is evidence that RCAN1 is also required for optimal calcineurin activity. Low calcineurin activity was observed in RCAN1 null mutant yeast, accompanied by phenotypes similar to RCAN1-overexpressing cells (Kingsbury and Cunningham, 2000). In addition, relatively low RCAN1 expression in physiological conditions activates the calcineurin pathway, when RCAN1 is functionally phosphorylated at highly conserved serine sites (Hilioti et al., 2004; Kingsbury and Cunningham, 2000; Li et al., 2015). Therefore, RCAN1’s regulatory modes on calcineurin is complicated, depending on the cellular and developmental contexts of organisms, and the biochemical and molecular properties of RCAN1.

Down syndrome model transgenic mice overexpressing RCAN1 exhibit a variety of phenotypic features that mimic Down syndrome such as cranial structure malformation, learning and memory deficits, disorganized neuronal differentiation and tumorigenesis suppression (Dierssen et al., 2011; Kurabayashi and Sanada, 2013; Martin et al., 2012; Reynolds et al., 2010). Interestingly, RCAN1 knock-out mice also show phenotypes similar to transgenic mice, such as impaired spatial learning and memory, and abnormal long-term potentiation (Hoeffer et al., 2007). In addition, both RCAN1 knock-out mice and RCAN1-1S TG mice displayed reduced exocytosis levels (Keating et al., 2008). Neuronal differentiation is also defective in transgenic flies overexpressing RCAN1 and loss-of-function mutant flies, presumably due to disturbed axonal extension associated with actin dynamics dysregulation (Chang and Min, 2009; Chang et al., 2003; Wang et al., 2012; 2016). In C. elegans, RCAN-1 overexpression resulted in calcineurin loss-offunction-like phenotypes, such as small body size, cuticle defects, small brood size, slow growth and resistance to serotonin-mediated egg-laying, while RCAN-1 deletion mutants showed cryophilic thermotaxis behavior, which is similar to calcineurin gain-of-function mutants (Lee et al., 2003; Li et al., 2015). Therefore, finely regulated RCAN-1 expression level is critical for normal organism physiology, in which the conserved calcineurin/RCAN1 pathways play a pivotal role in various biological processes.

RCAN1 binds to calcineurin through specified binding motifs. The PxIxIT motif is required for binding to calcineurin, which is also present in many calcineurin substrates such as a transcripttion factor NFAT (nuclear factor of activated T-cells) (Aramburu et al., 1998; Li et al., 2015; Mehta et al., 2009). RCAN1 also harbors SP repeats containing conserved serines whose phosphorylation is required for calcineurin stimulatory regulation in yeasts (Hilioti et al., 2004; Kingsbury and Cunningham, 2000). We previously reported the highly conserved PxIxIT motif, not the less conserved PKIIQT motif, is critical for full RCAN1 inhibitory regulation on TAX-6/calcineurin in C.elegans. Interestingly, C.elegans RCAN1 SP repeat phosphorylation is critical in RCAN1 TAX-6/calcineurin inhibitory regulation.

In this study, we investigated two available C.elegans rcan-1 deletion mutants, rcan-1(tm1925) and rcan-1(tm2021), in which RCAN was not detected by western blot analysis (Li et al., 2015). Both mutants showed normal brood size, serotonin-stimulated egg-laying, and male tail structure. However, rcan-1(tm1925) mutants were smaller than wild type animals, while rcan-1(tm2021) mutants were comparable. Also, the deletion mutation in rcan-1(tm1925) made tax-6(lf) animals even smaller, whereas that in rcan-1(tm2021) made tax-6(gf) bigger. Also, hyperactive egg-laying in tax-6(gf) was suppressed in both rcan-1(tm1925) and rcan-1(tm2021) mutant background. We present evidence that rcan-1(tm2021) may produce truncated proteins containing the PxIxIT motif, but are missing SP repeats at low expression levels, whereas rcan-1(tm1925) is a true null mutant. This study suggests that rcan-1 might be either an inhibitory or a stimulatory calcineurin regulator in different genetic and biological contexts in C. elegans.

Strains

Wild type N2 and tax-(ok2065) were obtained from Caenorhabditis Genetics Center (CGC) at the University of Minnesota. rcan-1 mutants, rcan-1(tm1925) and rcan-1(tm2021) were obtained from National Bioresource Project Japan. tax-6(jh107) was isolated by standard TMP/UV mutagenesis (Park et al., 2001a).

Transgenic lines

pPD95.77 and pPD49.26 were generous gifts from Andy Fire. To make a RCAN-1 translational expression construct, a rcan-1 genomic sequence, as well as approximately 3 kb of 5′ up-stream sequence was cloned into pPD95.77. Primers were previously described (Li et al., 2015).

Immunostaining

Immunostaining against RCAN-1 was performed as previously described (Park et al., 2001b).

Serotonin stimulated egg-laying assay

Serotonin stimulated egg-laying was conducted as previously described with some modification (Trent et al., 1983). Worms were placed in 96-well plates containing M9 buffer either with or without 12.5 mM serotonin. After 90 min, the number of eggs laid by each worm was counted.

Body size

Worms were synchronized at L4, and then adult body sizes were measured using a Zeiss microscope and Axio Image software.

rcan-1 expression pattern and mutant information

rcan-1 was expressed in various tissues including male tail and hypodermal seam cells, which also highly expressed tax-6/calcineurin (Bandyopadhyay et al., 2002; Lee et al., 2003). To further explore in which organs rcan-1 may physiologically function, we expressed RCAN-1 GFP fusion protein driven by its own promoter in worms. RCAN-1::GFP is expressed in various head neurons, pharyngeal muscle, ventral nerve cord, and hypodermal seam cells where immunostaining for RCAN-1 was also prominent (Figs. 1A–1C, and 1F–1I). GFP fluorescence was also detected in an excretory canal cell and vulva (Figs. 1D and 1E).

rcan-1 regulates a body size and serotonin-stimulated egg-laying of C. elegans

Worms overexpressing RCAN-1 exhibited pleiotrophic phenotypes similar to tax-6 loss-of-function mutants, suggesting RCAN-1 is inhibitory to tax-6/calcineurin (Lee et al., 2003). To further investigate rcan-1 functions, we examined two available rcan-1 deletion mutant alleles, rcan-1(tm1925) and rcan-1(tm2021). These mutants have a brood size comparable to wild type animals, and a male tail with normal morphology (Supplementary Fig. 1)(Li et al., 2015). Because RCAN-1 is highly expressed in seam cells (Fig. 1I), and tax-6 loss-of-function mutants are small (Kuhara et al., 2002), we measured body size for those two rcan-1 alleles, asking the question whether rcan-1 played a role in growth. tm1925 animals were slightly smaller than wild types, while tm2021 worms exhibited normal body length (Fig. 2). We further investigated tax-6 mutant body size and various rcan-1;tax-6 double mutants. Both tax-6(ok2065) loss-of-function (lf) and tax-6(jh107) gain-of-function (gf) mutant worms were significantly smaller than wild types. Interestingly, tm1925 allele did not affect tax-6(jh107)gf, but shrank tax-6(ok2065)lf smaller. However, tm2021 allele made tax-6(jh107)gf bigger, but did not affect tax-6(ok2065)lf. In addition, those body size regulation effects were not observed in cnb-1(jh103) in which intact tax-6 was present, but no calcineurin activity. To further explore differential phenotype modulation by these two alleles, we also investigated egg-laying stimulation by serotonin which is dependent on tax-6 activity. Egg-laying activity in both tm1925 and tm2021 mutant worms were slightly stimulated by serotonin, though the average number of eggs laid per worm was comparable to wild types (Figs. 3A and 3D). Both alleles suppressed enhanced egg-laying of tax-6(jh107)gf, but only tm1925 reversed tax-6(ok2065)lf reduced egg-laying activity (Fig. 3B and 3D). Again, differential modulation by the two different alleles was not observed in cnb-1(jh103)ls background, suggesting that tax-6 abnormal activity was associated with body length modulation by tm1925 and tm2021 alleles (Fig. 3C and 3D).

tm1925 is a true null mutant whereas tm2021 is expression-competent

The tm1925 allele had a large deletion in the 3^rd intron and part of the 4^th exon, and additions of several nucleotides near the deletion site in the 4^th exon, while the tm2021 allele had a deletion in the 2^nd and 3^rd exons (Fig. 4A). Both alleles were once predicted to be null mutants, because no RCAN-1 protein was detected by western blot analyses (Li et al., 2015). To analyze rcan-1 transcriptional products in each allele mutant, we amplified rcan-1 cDNA sequences isolated from mutant worms, and sequenced the RT-PCR products (Figs. 4A–4C). Among the detected transcripts, a splicing donor site was created at the deletion site in the 3rd intron in the tm1925 transcript A, while the 3^rd intron undeleted starting region was transcribed, linking to the remaining 4^th exon in the tm1925 transcript B. Both transcripts contained a newly created stop codon (Fig. 4B). For the tm2021 transcript, the A transcript, in which splicing between the 1^st exon and the 4^th exon occurred, was a major product, while the B transcript contained a piece of the 1^st intron and the 3^rd exon was minor (Figs. 4D–4E).

The predicted RCAN-1 translational product in tm1925 had the PxIxIT calcineurin binding motif deleted (Figs. 4B and 5A), whereas that in tm2021 retained it, but lost the SP repeat domain including S129 and S133 phosphorylation sites, which are critical for tax-6 activity regulation (Li et al., 2015) (Figs. 4C and 5B). To find out whether the rcan-1 mutant sequences were competent to produce any translational product, we cloned the mutants’ genomic sequences into the GFP expression vector. We found that transgenic worms injected with the tm2021 genomic construct exhibited reasonably detectable green fluorescence at a short exposure, whereas those with the tm1925 showed little fluorescence even after a long exposure (Figs. 5C–5E). To detect any rcan-1:GFP translational products in those transgenic lines, we performed western blot analysis using polyclonal anti-RCAN-1 antibodies (Fig. 5F). 38 kDa of tm2021:GFP fusion was detected, but the predicted tm1925::GFP was not detected due to the stop codons generated by the mutation (Fig. 4B). In addition, no tm1925 predicted products were detected, indicating that tm1925 transcripts were probably not translated, or translational products were subject to fast degradation. Therefore, tm1925 was likely to be a null mutant, and tm2021 might produce a truncated protein retaining the PxIxIT motif, but missing the SP repeats domain, making expression level undetectable by typical Western blot analysis using currently available polyclonal anti-RCAN-1 antibodies.

RCAN1 has been widely shown to be an inhibitory calcineurin regulator, but several lines of studies have shown that phosphorylated RCAN1 is able to stimulate calcineurin at low expression levels (Genesca et al., 2003; Hilioti et al., 2004; Kishi et al., 2007). Also, both knock-out and overexpression lines in various models show similar malfunction in different phenotypes. This study showed that C.elegans rcan-1 may also play a stimulatory role in the calcineurin pathway by investigating a phenotypic variance between two deletion rcan-1 mutants.

Previously considered to be null mutants, both rcan- 1(tm1925) and rcan-1(tm2021) mutants are cryophilic for tax-6 (gf), suggesting that RCAN1 should be an inhibitory calcineurin regulator in thermotaxis behavior (Li et al., 2015). However, these two mutants showed differential phenotypes for body length and serotonin-stimulated egg-laying behavior (Figs. 2 and 3). The tm1925 allele had PxIxIT calcineurin binding motif deletion, and its GFP fusion protein genomic sequence was not detectable, indicating that tm1925 is null (Fig. 4). On the other hand, the tm2021 was predicted to produce a truncated PxIxIT motif. When the tm2021 genomic sequence was cloned in frame into a GFP expression vector, it produced a GFP fusion protein with expected size in transgenic worms that was detected by western blot. The tm1925 mutant animal was shorter than wildtypes, and rcan-1(tm1925);tax-6(ok2065) was even shorter, indicating that RCAN1 may regulate calcineurin-independent growth. The fact that rcan-1(tm2021);tax-6(jh107) were longer than tax-6(jh107)gf also supports RCAN1’s regulatory role in body size. Because tm2021 showed normal body size, the truncated protein seemed to be sufficient to sustain normal growth rate. In the serotonin-mediated egg-laying behavior, those two rcan-1 mutants lay slightly more eggs than wild types, indicating that rcan-1 might be inhibitory for calcineurin in this behavior. However, those rcan-1 mutations significantly suppressed tax-6(jh107)gf, while tm2021 allele was more effective in suppression. These results suggest that rcan-1 may be stimulatory to tax-6 in certain circumstances.

rcan-1 is upregulated in the tax-6(jh107)gf background, presumably due to feedback induction via calcineurin activity (Li et al., 2015). rcan-1 expression level seemed to be very critical in specific tissues, because rcan-1 expression in AFD neurons rescued cryophilic rcan-1 mutant phenotypes better than panneuronal expression (Li et al., 2015). Therefore, the truncated tm2021 allele protein might accumulate more in tax-6(jh107)gf than in the transgenic line expressing GFP fusion. In addition, the elevated truncated protein containing PxIxIT and missing SP repeats may play a role in the potentiated suppression in rcan-1(tm2021); tax-6(jh107). Finally, rcan-1 lack significantly reversed the low tax-6(ok2065)lf egg-laying phenotype. Taken together, these data indicate that rcan-1 is also required for normal egg-laying, and might stimulate calcineurin activity.