Differential expression of the three independent CaM genes coding for an identical protein: Potential relevance of distinct mRNA stability by different codon usage

Cell Calcium 107 (2022) 102656 Available online 8 October 2022 0143-4160/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Review Differential expression of the three independent CaM genes coding for an identical protein: Potential relevance of distinct mRNA stability by different codon usage Mads Munk a , Eduardo Villalobo b , Antonio Villalobo c , * , Martin W. Berchtold a , * a Department of Biology, University of Copenhagen, 13 Universitetsparken, Copenhagen Ø DK-2100, Denmark b Facultad de Biología, Departamento de Microbiología, Universidad de Sevilla, Avenida Reina Mercedes 6, Sevilla E-41012, Spain c Cancer and Human Molecular Genetics Area – Oto-Neurosurgery Research Group, University Hospital La Paz Research Institute (IdiPAZ), Paseo de la Castellana 261, Madrid E-28046, Spain ARTICLE INFO Keywords: Calmodulin genes Codon usage Epigenetic control miRNA control Mutation-mediated diseases Phylogeny Pseudogenes ABSTRACT The Ca 2+ -sensor protein calmodulin (CaM) is a major regulator of multiple cell functions. A unique and puzzling feature of human, and all so far investigated mammals, is the presence of three distinct CaM genes on different chromosomes, which code for identical proteins. How this case of apparent genetic redundancy evolved and why it could be to the advantage of the mammalian organisms is not well established. With a main focus on humans, this article aims to review existing literature addressing how the genes nonetheless differ in function. Clearly, the three CaM genes are differentially expressed in different tissues, during development, in response to different stimuli, and other factors including environmental conditions. As shown in hippocampal neurons, different mRNAs from the CAM genes may even localize differently within the same cell. Regulation of CaM gene expression is achieved by a variety of regulatory elements present in the three genes, including different promotor/insulator elements and 3′- and 5′-noncoding regions differing in length and sequence, as well as regulation by epigenetic factors and miRNAs. Here, we hypothesize that predicted differences in mRNA stability and translational efficiency due to divergent codon usage could play an additional regulatory role as the three genes differ markedly in their use of synonymous codons. CALM3, predicted to produce a relatively stable mRNA may be important where the transcription level is low or transiently absent, e.g. during spermatogenesis. In contrast, CALM2 with a predicted much shorter mRNA half-life, may provide better temporal control of CaM levels. Deciphering the underlying mechanisms responsible for all this complexity may help to understand why this unique multigenic arrangement may be an advantage for the optimal spatio-temporal expression of CaM in higher eukaryotes. Finally, we discuss the expression of the CaM genes in selected human pathologies, and how mutations in these genes are responsible for the appearance of serious congenital syndromes, mainly affecting the heart, and although less known, possibly also affecting the functionality of the central nervous system and other organs. Abbreviations 3′/5′-UTR, 3′- and 5′-untranslated regions; ALS, amyotrophic lateral sclerosis; AP1, activator protein 1; AP2, activator protein 2; bp, base pairs; CALM1,2,3/Calm1,2,3, calmodulin-1,2,3 genes; CaM, calmodulin; CaML, calmodulin-like protein; CaMK-II, calmodulin-dependent protein kinase-II; Ca v 1.2, L-type voltage-gated Ca 2+ channel 1.2; CCR4-NOT, carbon catabolite repression negative on TATA-less; cDNA, copy DNA; CDS, coding sequence; CPEB, cytoplasmic polyadenylation element binding protein; CPVT, catecholaminergic polymorphic ventricular tachycardia; CRE, cAMP-response element; * Corresponding authors. E-mail addresses: [email protected] (A. Villalobo), [email protected] (M.W. Berchtold). Contents lists available at ScienceDirect Cell Calcium journal homepage: www.elsevier.com/locate/ceca https://doi.org/10.1016/j.ceca.2022.102656 Received 1 July 2022; Received in revised form 1 September 2022; Accepted 25 September 2022 Cell Calcium 107 (2022) 102656 2 CREB, cAMP-response element binding protein; CSC, codon stabilization coefficient; CTCF, CCCTC-binding protein; DACT3, disheveled binding antagonist of β-catenin 3 gene; DACT3-AS1, DACT antisense RNA 1; EPCAM-DT, epithelial cell adhesion molecule divergent transcript lncRNA; GAPDHP38, glyceraldehyde 3-phosphate dehydrogenase pseudogene 38; GNG8/Gng8, G protein subunit γ8 gene; HEK, human embryonic kidney; IP 3 , inositol-1,4,5-trisphosphate; IVF, idiopathic ventricular tachycardia; LINC00642, lncRNA-642; LINC02317, lncRNA-2317; lncRNA, long non-coding RNA; LQTS, long-QT syndrome; Mef2a, myocytes enhancer factor 2a; Mir-3558, microRNA-3558; MLCK, myosin light-chain kinase; NFAT, nuclear factor of activated T cells; NMDA, N-methyl-D-aspartate; NRDE2/Nrde2, nuclear RNA interference defective-2 gene; Pnma8a/b, paraneoplastic Ma antigen protein 8a/8b gene; Pnmal2, paraneoplastic Ma antigen-like protein 2 gene; PPP5D1P, Ser/Thr protein phosphatase tetratricopeptide repeat domain containing 1 pseudogene; PSMC1/Psmc1, 26S proteasome subunit - ATPase 1 gene; PTGIR/Ptgir, prostaglandin I2 receptor gene; qPCR, quantitative polymerase chain reaction; RPL21P11, ribosomal protein L21 pseudogene 11; Rps6ka5, ribosomal protein S6 kinase a5 gene; RyR2, ryanodine receptor 2; siRNA, small interfering RNA; SNP, single nucleotide polymorphism; SP1, specificity protein 1; STPG4/Stpg4, sperm-tail PG-rich repeat containing 4 gene; TPM, transcripts per million; TTC7A/Ttc7b, tetratricopeptide repeat domain 7A/7b gene. nTPM, normalized transcripts per million 1. Introduction The evolutionary selection of Ca 2+ ions as a key signaling element, based on its charge, ionic radius, coordinating and chemical properties, was accompanied by the appearance of proteins holding distinct Ca 2+ - binding motifs. This made it possible to transduce changes in the concentration of free Ca 2+ , first in the interior of protocells and early prokaryotes, and thereafter in the cytosol and lumen of the different intracellular compartments of more complex eukaryotic cells (reviewed in [1]). Among the proteins containing Ca 2+ -binding EF-hand motifs [2] in eukaryotes, calmodulin (CaM) stands out by its ubiquity, high phylogenetic conservation, a high number of targeted proteins under its control and the versatility of cellular functions, in which this protein is involved (reviewed in [3–7]). CaM has two pairs of EF-hands located at each of the N- and C-lobes connected by a flexible linker (reviewed in [8]). Furthermore, due to the ability of each lobe to act independently, CaM has the capacity to bridge different or identical target proteins, leading to dimerization, or interact with distinct regions of the same protein forming new functional domains [9] (reviewed in [8]). Upon Ca 2+ binding to CaM, a series of hydrophobic patches containing methionine residues are exposed and its 3D-structure undergoes modifications allowing interaction with target proteins, thereby regulating their functions. Nevertheless, CaM is also able to bind to and control some proteins in its Ca 2+ -free form (reviewed in [10,11]). CaM gene(s) are present in all so far examined eukaryotes, from human to single cell organisms such as yeast. CaM is not only a ubiquitous protein among eukaryotes, but is also extraordinary well conserved among them [12]. This is exemplified by the fact that the protein is identical in all studied vertebrates, even though the genes show remarkable diversity at the nucleotide level [12]. An alignment of CaM sequences from different invertebrates shows only few changes (2 or 3 amino acids), mostly located in the C-terminus, with respect to vertebrate CaM (Fig. 1a). Of interest, the presence, in most cases, of a threonine at position 144 and serine at position 148 in CaM of invertebrates, in contrast to vertebrates, having a glutamine and an alanine, respectively, at these positions, may suggest that these positions in invertebrate CaM could serve as specific phosphorylation targets for Ser/Thr-protein kinases, as it occurs in different Ser/Thr residues of CaM in many organisms (reviewed in [13,14]). A single CaM gene is present in most non-metazoan eukaryotes, including yeast/fungi [15–20], protozoans [21,22], and green algae [23]. However, there are some exceptions, as for example in Trypanosoma cruzi and T. brucei, which have eight and three tandemly repeated CaM genes, respectively, either in two different loci or in a single one [24,25]. Fig. 1b shows an alignment of the sequences of CaM from different protists and yeast/fungi, as compared with vertebrate CaM. Interestingly, the highest number of substitutions is found in the C-ter- minal helix of EF-hand IV. Even though Sacharomyces cerevisiae shows the lowest identity (59% with vertebrate CaM), it can functionally be replaced by vertebrate calmodulin [26]. In plants, several CaM isoforms and multiple CaM-like proteins (CaML) encoded by distinct genes are expressed. For example, in rice 5 CaM genes and 32 CaML genes and in Arabidopsis 7 CaM genes and 50 CaML genes are present with differential structural features and specific target recognition properties. We are not considering protist, invertebrate or plant CaMs and CaMLs in this review, as extensive information is available on the properties, functions and evolution of both protein families, particularly in plants (reviewed in [27–29]). It is well known that many organisms harbor multiple and distinct CaM genes giving rise to identical proteins [30], as for example the three CaM genes present in all so far investigated mammals [31]. The coding sequences for the three human CaM genes (CALM1,2,3) share only about 80% identity at the nucleotide level [12]. Comparing nucleotide coding sequences of CALM1, CALM2 and CALM3 of rat and human reveals similarities of 88, 90 and 90%, respectively. In chicken, 2 CaM genes (CALM1,2) are present with an identity of about 85%, and identities of about 92% with their human counterparts [32,33]. This redundancy of identical proteins being expressed by multiple independent genes is puzzling. Seemingly, this phenomenon may represent, at least in part, a safeguard for cell function and viability in case of mutational mishaps. However, something more is perhaps hidden behind this phenomenon. Evidence is mounting that there is differential expression of the three CaM genes in a given tissue, as for example in the white matter of the rat spinal cord [34], among many other tissues. Furthermore, it has been shown that natural occurring monogenic CaM mutations [35] provoke organ-specific dysfunctions, as shown for the heart. This underscores either that the expression of the single affected CaM gene is the only one expressed in specific regions of the myocardium (see Section 4.1), or alternatively, that the expression of the other CaM genes is not apt to rescue the dysfunction. This may occur either because: (i) the three CaM genes are not expressed in sufficient quantities; (ii) they present extemporal expression timing; (iii) the other expressed wild-type CaM is targeted to a different subcellular location; (iv) and/or probably most important, because the arrhythmogenic mutant CaM proteins may work in a dominant negative fashion on target proteins (e.g. RyR2, CaMK-II, SK2 channel, Ca v 1.2 channel) and therefore their function cannot be compensated by wild-type CaM [36–38]. M. Munk et al. Cell Calcium 107 (2022) 102656 3 2. From a single EF-hand Ca 2þ -binding primordial protein to CaM with four EF-hands EF-hand motifs, first described in the Ca 2+ buffering protein parvalbumin by Kretsinger and Nockolds in 1973 [39], are structural elements found in a large number of proteins of roughly 30 amino acids, containing a loop of 12 residues, providing the Ca 2+ coordinating amino residues, between two perpendicularly arranged alpha helices. Most EF-hands are able to bind Ca 2+ , though with greatly variable affinities. Calcium is bound in a pentagonal bipyramid configuration [40] with oxygen atoms of acidic amino acids and water. As EF-hand proteins have been found in prokaryotes (reviewed in [41]) it is possible that EF-hands have arisen from a single non-eukaryotic organism that gave rise to eukaryotes or that EF-hands have been evolved in early eukaryotes and then were transmitted to prokaryotes [42–45]. Alternatively, it cannot be excluded that EF-hands may have evolved independently in prokaryotes and eukaryotes. In most cases, two EF-hands, generated by duplication, are placed together in a characteristic way providing enhanced stability and leading to increased Ca 2+ binding affinity. A single protein may have several mostly paired EF-hands. Calmodulin, as well as some other proteins including troponin C and myosin light chains, contains 2 pairs of EF- hands distributed in 2 lobes. The fact that domains 1 and 3, as well as 2 and 4, are most similar points to duplication of a single domain with 2 EF-hands, which themselves have evolved from a single one EF-hand domain. Interestingly, the 4 EF-hands in CaM have different structural and functional properties, which is believed to contribute to the huge flexibility of CaM to interact with and regulate a large number (>300) of target proteins. 3. The three mammalian CaM genes Mammalian CaM is special in that it is encoded by 3 different nonallelic genes, placed on three different chromosomes, all producing an identical protein sequence [30]. In human, for instance, extensive use of synonymous codons means only about 80% identity at the nucleotide level [12]. On the other hand, the individual gene orthologs among different mammals have higher identities. The three CaM genes have the same intron-exon boundaries, which are also conserved in some non-mammalian species. Chicken and frog lack the CALM3 gene, and only have two CALM genes, which are orthologs to mammalian CALM1 and CALM2, indicating that CALM3 was either developed at a later evolutionary step, or that it was present but lost again in these species. Having three different promotors and 5′/3′-noncoding RNA sequences, the three CaM genes of mammals are differentially regulated during development (reviewed in [46]), and their mRNAs were shown to be Fig. 1. a. Comparison of CaM sequences in invertebrates. Multisequence alignment of CaM from the indicated invertebrates, as compared with vertebrate CaM (highlighted in red), using the CLUSTALW multi-alignment tool (https://www.genome.jp/tools-bin/clustalw). Identical residues are marked (*). Residues differing from the ones in vertebrate CaM are highlighted in red (non-conserved substitutions) and blue (conserved substitutions). b. Comparison of CaM sequences in lower eukaryotes. Multisequence alignment of CaM from the indicated lower eukaryotes species, as compared with vertebrate CaM (highlighted in red), using the CLUSTALW tool (https://www.genome.jp/tools-bin/clustalw). Identical residues are marked (*). Residues differing from the ones in vertebrate CaM are highlighted in red (non-conserved substitutions) and blue (conserved substitutions). M. Munk et al. Cell Calcium 107 (2022) 102656 4 differentially localized in neuronal cells. 3.1. CaM genes and pseudogenes. Comparison of their genomic organization: intron-exon organization, chromosomal localization 3.1.1. CaM genes The chromosomal loci of the three human CaM genes were first reported in 1993 [31] to be on chromosomes 14, 2 and 19 for CALM1, CALM2 and CALM3, respectively (Table 1), as analyzed by in situ hybridization of metaphase spreads of human lymphocytes, and confirmed with the precise physical localization after the human genome was sequenced [47–49]. CALM1 and CALM3 are located on the forward DNA strand while CALM2 is placed on the reverse strand [47–50] (see Fig. 2). The CALM2 gene is the longest of the three CaM genes [51], and all human CALM genes (as well as all known mammalian CaM genes) have 6 exons and 5 introns (see Table 1). Of interest and supporting a common evolutionary history, the intron/exon boundaries with respect to the coding sequences are conserved, not only in human CALM1, CALM2, and CALM3 [52], but also in other mammals, as well as in the two chicken CaM genes. Table 2 summarizes the sequences of the 10 intron/exon boundaries in the CaM genes of human, rat, mouse and chicken, showing that all of them are conserved. The internal exons 2–5, containing only coding sequences, are identical in length, while exons 1 and 6, with mostly noncoding sequences, are different in length (see Table 1). Exon 1 includes the 5′-UTR as well as the ATG start codon at its 3′-end, while exon 6, includes the 10 C-terminal codons as well as the 3′-UTR sequence (see Table 1). The location of the orthologous genes upstream and downstream of the three CaM genes in human, rat and mouse is similar (see Fig. 2). The genes PSMC1/Psmc1 and NRDE2/Nrde2, encoding the 26S proteasome subunit-ATPase 1 and the nuclear exosome regulator NRDE2 (necessary for RNA interference-2), are located upstream of CALM1/Calm1 in human and rat, respectively. The genes STPG4/Stpg4 and Ttc7b, coding for the sperm-tail PG-rich repeat containing 4 protein and the tetratricopeptide repeat domain 7b protein are located downstream of CALM1 in human rat and mouse, respectively. The gene encoding the 7a isoform of the latter protein is located upstream of CALM2/Calm2 in human and mouse, while it is placed downstream in rat. Moreover, the genes PTGIR/Ptgir and GNG8/Gng8, coding the prostaglandin I2 receptor and the G protein subunit g8, respectively, are located downstream of CALM3 in human, and upstream of Calm3 in rat and mouse. The occurrence of synteny, in the chromosome regions where the CaM genes in the three species are located, reinforces the idea of the common evolutionary history, as stated above. Whether regulatory elements in the mentioned genes located in the genomic regions close to the CaM genes have an influence on the transcriptional activities of the 3 CaM genes is unknown. Even though mammalian CaM genes encode identical proteins, as described above, the nucleotide sequence varies quite markedly [53]. As an example, Fig. 3a shows extensive differences in the nucleotide coding sequence of the three CaM genes in human, presenting more than one hundred nucleotide differences, almost exclusively from differences in the third codon position. Similar numbers of variations are also found in rat (Fig. 3a) and mouse (not shown) and mostly in the same positions. In contrast, only less than half of the variations are found when comparing the nucleotide coding sequences of the three CaM genes in human versus Table 1 Human calmodulin genes and mRNAs. Properties CALM1 CALM2 CALM3 Refs. Chromosome locus 14q24-q31 (14q32.11) 2p21.1-p21.3 (2p21) 19q13.2-q13.3 (19q13.32) [31,47–49] Gene length (bp) 11,240 16,885 9453 [47–49] Exons length (bp) Exon 1 (251) Exon 2 (31) Exon 3 (144) Exon 4 (107) Exon 5 (136) Exon 6 (3573) Exon 1 (71) Exon 2 (31) Exon 3 (144) Exon 4 (107) Exon 5 (136) Exon 6 (721) Exon 1 (108) Exon 2 (31) Exon 3 (144) Exon 4 (107) Exon 5 (136) Exon 6 (1658) [50,52] Introns length (bp) Intron 1 (2831) Intron 2 (1163) Intron 3 (2459) Intron 4 (410) Intron 5 (174) Intron 1 (5676) Intron 2 (8071) Intron 3 (126) Intron 4 (427) Intron 5 (918) Intron 1 (4389) Intron 2 (2339) Intron 3 (141) Intron 4 (257) Intron 5 (143) [50,52] Promoter TATA box CAAT box GC box AGGGA motif CRE bind. site SP1 bind. site AP1 bind. site AP2 bind. site (number) 4 4 1 - - 5 - - 2 4 1 6 2 1 1 2 1 3 - 3 3 8 1 5 [47,51,176] Poly-A signals (number, sequences) (5) 5′-AATAA-3′ 5′-AATAAA-3′ 5′-TTTTAT-3′ (3) 5′-AATAAA-3′ 5′-TTTTAT-3′ (3) 5′-ATAAA-3′ 5′-AATAAA-3′ 5′-AATAAAAT-3′ [94,177] CPEB-binding sites (number, sequence) 1 5′-UUUUUAU-3′ 2 5′-UUUUUAU-3′ None [98] Potential CTCF-binding sites (sequence) 1 5′-CACACCCTGGTGGC-3′ Score 10.9943 5′-TTGCACCTTGTGGA-3′ Score 7.33699 5′-GGAACTGCT-3′ Score 12.137 [178] miRNA Let7a binding to 3′-UTR (sequence) 5′-UACCUC-3′ [103] Staufen2 binding Located in a retained intron within the 3′-UTR [99] 1 The potential CTCF-binding sites shown were determined using the CTCFBSDB 2.0 prediction tool. For each gene, the sequence shown corresponds to that with the highest score in the coding orientation from the output reported. bp, base pair; CPEB, cytoplasmic polyadenylation element binding protein; CTCF, CCCTC-binding protein. M. Munk et al. Cell Calcium 107 (2022) 102656 5 rat, mouse or cow (see Fig. 3b). This shows that there are more differences in the three CaM genes among them in a given species than in a given CaM gene among different vertebrates. 3.1.2. CaM pseudogenes The lack of introns, truncation and mutational disruption of the CDS are the main characteristics of eukaryote pseudogenes (e.g. [52,54,55]), although these characteristics do not always apply. Pseudogenes are widely distributed in the genome of many living organisms, and despite early assumptions of lack of function, a growing number of evidences support the functionality of some of them (reviewed in [56–58]). Some of the mechanisms by which pseudogenes could have a functional role include: (i) they could encode a sequence-altered or truncated functional protein reminiscent of the parental one; (ii) they could be transcribed into siRNAs or antisense RNAs, preventing translation of the parental gene, or yielding lncRNAs, which directly interact and regulate the expression of certain proteins; and (iii) they could favor DNA folding allowing interaction of different chromatin regions, and this could positively or negatively affect the expression of nearby genes, and to allow the conversion of their parental genes into dysfunctional ones by transferring segments via non-allelic recombination (reviewed in [57, 58]). Pseudogenes derived from the CaM genes have been identified in multiple vertebrates, including human [59–61]. Among them, a couple of presumably non-functional intronless pseudogenes, derived from the genuine human CALM1, denoted CALM1P1 and CALM1P2 presenting disruptive mutations in their CDS were first described by Rhyner et al. [52], as well as three retro-pseudogenes derived from the human CALM2 gene [62]. Alu elements, repetitive sequences that are cut by the restriction endonuclease Alu, were identified upstream of the 5′-UTR and downstream of the 3′-UTR in one of these pseudogenes [62]. Alu elements are dispersed by retro-transposition. Transcription of Alu elements, reverse transcription of their RNA and insertion of the resulting cDNA into the targeted genes are the three steps required in the dispersion process, usually leading to multiple effects by modifying functions of other genes [63]. The retro-pseudogenes derived from Fig. 2. Genes located upstream and downstream of the three CaM gene loci in human, rat and mouse. The drawing shows the location of protein-coding genes (blue arrows), pseudogenes (yellow arrows) and some RNA-coding genes (green arrows) upstream and downstream of the three CaM genes (red arrows) in human, rat and mouse. Other predicted genes in the indicated DNA segments are not shown. The length of the genes and inter-gene segments are not drawn to scale. CALM1/ Calm1, calmodulin-1 gene; CALM2/Calm2, calmodulin-2 gene; CALM3/Calm3, calmodulin-3 gene; DACT3, disheveled binding antagonist of β-catenin 3; DACT3-AS1, disheveled binding antagonist of β-catenin 3 antisense RNA 1; EPCAM-DT, epithelial cell adhesion molecule divergent transcript lncRNA; GAPDHP38, glyceraldehyde 3-phosphate dehydrogenase pseudogene 38; GNG8/Gng8, G protein subunit g8; LINC00642, lncRNA-642; LINC02317, lncRNA-2317; Mir- 3558, microRNA-3558; NRDE2/Nrde2, nuclear exosome regulator NRDE2 (necessary for RNA interference-2); Pnma8a/ b, paraneoplastic Ma antigen protein 8a/8b; Pnmal2, paraneoplastic Ma antigen-like protein 2; PPP5D1P, Ser/Thr protein phosphatase tetratricopeptide repeat domain containing 1 pseudogene; PSMC1/Psmc1, 26S proteasome subunit - ATPase 1; PTGIR/Ptgir, prostaglandin I2 receptor; RPL21P11, ribosomal protein L21 pseudogene 11; Rps6ka5, ribosomal protein S6 kinase a5; STPG4/Stpg4, sperm-tail PG-rich repeat containing 4; TTC7A/Ttc7b, tetratricopeptide repeat domain 7A/ 7b. Adapted from [47–50]. M. Munk et al. Cell Calcium 107 (2022) 102656 6 human and murine CALM2 and CaMII/Calm2 genes, respectively, as well as other unrelated genes, were shown to be present in different species, and the evolutionary divergence time between human and other primates, and among different rodent species, was established [64,65]. Four processed CaM pseudogenes of rats, denoted lSC8, lSC9, lSC19, and lSC27 are derived from the genuine CaMI/Calm1 gene (lSC9) or the CaMII/Calm2 gene (lSC8, lSC19 and lSC27), respectively. The lSC9 gene differs from the lSC8 in that it does not contain frameshift mutations in its CDS [66–68]. Transcripts of pseudogenes that do not contain disabling mutations in the CDS could produce CaM-like proteins if they are correctly transcribed and translated and therefore could potentially exert diverse functional roles. Indeed, a functional intronless CaM-like gene in human has been found [52,60]. This pseudogene, identified in the NCBI database (https://www.ncbi.nlm.nih.gov/) as CaM-like protein 3 (CALML3 or CLP) is highly expressed in epithelial cells, and its downregulation during tumor transformation suggests that it may be involved in differentiation processes [60]. The nucleotide coding sequence identity of the human CALML3 transcript with those of CALM1, CALM2 and CALM3 is 70.7%, 69.8% and 80%, respectively. In chicken, an intronless CaM pseudogene denoted cCM1, also identified as CaM-like protein 3 (CALML3), as in human, codes for a 149 residues protein, and is differentially expressed in tissues. Comparing the coding sequence of human and chicken CALML3 shows that they share 79.1% identity. This chicken protein differs in 19 residues from the one encoded by the CaM gene cCL1, 11 of them non-conservative, and it is affected in its Ca 2+ -binding capacity at EF-hands I and IV, but not EF-hands II and III, as critical Ca 2+ coordinating acidic residues are altered in the former EF-hands [55]. The authors of this study suggested that this protein may have a specific role in striated muscle physiology, as this was the only tissue among several others tested, where the authors found expression of this protein. Its subcellular localization and availability may determine its impact on skeletal muscle specific processes. 3.2. Regulation of CaM expression Since CaM is involved in a huge variety of physiological functions, even within a single cell, it is of no surprise that a certain level of CaM expression is not only required in a specific tissue and cell with a given set of CaM targets of different abundance, locations and affinities for CaM, but also in different subcellular compartments. It has been shown in living smooth muscle cells that only 5% of the total CaM concentration is freely diffusible [69] and that, upon increase of cytosolic Ca 2+ , CaM could be translocated to the nucleus. This has been confirmed in cardiomyocytes where soluble free CaM was found in very low concentration (ca. 1% of total) and increasing intracellular Ca 2+ led to translocation of CaM to the nucleus as shown by Bers and collaborators [70]. It is believed that the presence of CaM binding sites exceeds the amount of available CaM, making diffusion of free CaM difficult and indicating that there must be a selection of targets based on affinities and kinetics [71]. Therefore, subcellular CaM pools could play an important role in target regulation [72]. Another factor regulating the available CaM concentration is the presence of CaM targets that bind in the absence of calcium, e.g. GAP- 43/neuromodulin, which has been proposed to act as CaM buffer [73] to prevent Ca 2+ dependent signaling through CaM. In addition to this CaM storage hypothesis, it has been proposed that CaM could be temporally “masked” and thereby not anymore able to regulate targets by posttranslational modification, e.g. phosphorylation is shown to reduce its activity in regulating targets (reviewed in [13,14]). Moreover, Table 2 The intron/exon boundaries of the CaM genes in human, rat, mouse and chicken. Species Intron/ exon boundary CALM1 CALM2 CALM3 Human 1 ATGgtagg…(þ3 M1) ATGgtgag… (þ3 M1) ATGgtgag… (þ3 M1) 2 …tatagGCTGA … aacagGCTGA …tacagGCTGA 3 TGCTGgtaag… (þ1 E12) TGCAGgtgag… (þ1 E12) TGCAGgtgag… (þ1 E12) 4 …ttcagAATTC …aacagAATTC …cccagAGTTC 5 TGATGgtaag… (þ1 G60) TGATGgtaag… (þ1 G60) AGATGgtgag… (þ1 G60) 6 …tcgagGTAAT …ttcagGTAAT …tccagGGAAC 7 ACAAGgtaat… (þ3 K95) ATAAGgtacg… (þ3 K95) ACAAGgtaag… (þ3 K95) 8 …cacagGATGG …gctagGATGG … cccagGATGG 9 TGAAGgtaaa… (þ1 E141) TGAAGgtaaa… (þ1 E141) TGAAGgtgag… (þ1 E141) 10 …ttcagAATTC …ttcagAGTTT …cacagAGTTT Rat 1 ATGgtagg… (þ3 M1) ATGgtgag… (þ3 M1) ATGgtgag… (þ3 M1) 2 …tgtagGCTGA …tccagGCTGA …tccagGCTGA 3 TGCTGgtaag… (þ1 E12) TGCAGgtaag… (þ1 E12) TGCAGgtgag… (þ1 E12) 4 …ttcagAATTC …ttcagAATTC …cccagAGTTC 5 CGACGgtgag… (þ1 G60) CGACGgtgag… (þ1 G60) TGATGgtgag… (þ1 G60) 6 …taaagGGAAT …taaagGGAAT …gctagGCAAT 7 ACAAGgtaat… (þ3 K95) ACAAGgtaat… (þ3 K95) ACAAGgtaag… (þ3 K95) 8 …cacagGATGG … cacagGATGG … cccagGATGG 9 TGAAGgtaaa… (þ1 E141) TGAAGgtaaa… (þ1 E141) TGAAGgtgtg… (þ1 E141) 10 …ttcagAATTC …ttcagAATTC …ggcagAGTTT Mouse 1 ATGgtagg… (þ3 M1) ATGgtgag… (þ3 M1) ATGgtagg… (þ3 M1) 2 …tgtagGCTGA …tccagGCTGA …tatagGCTGA 3 TGCTGgtaag… (þ1 E12) TGCAGgtgag… (þ1 E12) TGCTGgtaag… (þ1 E12) 4 …ttcagAATTC …cccagAGTTC …ttcagAATTC 5 TGATGgtaag… (þ1 G60) TGATGgtgag… (þ1 G60) TGATGgtaag… (þ1 G60) 6 …taaagGCAAT …gctagGCAAT …tcgagGTAAT 7 ACAAGgtaat… (þ3 K95) ACAAGgtaag… (þ3 K95) ACAAGgtaat… (þ3 K95) 8 …cacagGATGG … cccagGATGG … cacagGATGG 9 TGAAGgtaaa… (þ1 E141) TGAAGgtgag… (þ1 E141) TGAAGgtaaa… (þ1 E141) 10 …ttcagAATTC …ggcagAGTTT …ttcagAATTC Chicken 1 ATGgtacg… (þ3 M1) ATGgtacg… (þ3 M1) – 2 …tgtagGCTGA …tgcagGCTGA – 3 TGCTGgtaag… (þ1 E12) TGCAGgtgag… (þ1 E12) – 4 …tctagAATTC …tacagAATTC – 5 TGATGgtaag… (þ1 G60) TGATGgtaag… (þ1 G60) – 6 …ttaagGTAAT …tgcagGCAAT – 7 ACAAGgtatt… (þ3 K95) ACAAGgtact… (þ3 K95) – 8 …cacagGATGG … cccagGATGG – 9 TGAAGgtaat… (þ1 E141) TGAAGgtaag… (þ1 E141) – 10 …ttcagAATTC …tgcagAGTTT – The transcripts in the databases Ensemble (https://www.ensembl.org/index. html) or NCBI (https://www.ncbi.nlm.nih.gov/) used in the analysis were as follows: From human: CALM1, ENST00000356978.9; CALM2, ENST00000272298.12; and CALM3, ENST00000291295.14; from rat: Calm1, ENSRNOT00000064679.2; Calm2, ENSRNOT00000022603; and Calm3, GenBank X14265.1; from mouse: Calm1, ENSMUST00000110082.11; Calm2, ENSMUST00000040440.7; and Calm3, ENSMUST00000019514.10; and from chicken: Calm1, ENSGALT00000045488.3; Calm2, ENSGALT00000016279.5. Exon sequence segments are highlighted in capital letters. The site of splicing in the codon and the amino acid involved are shown in parenthesis. M. Munk et al. Cell Calcium 107 (2022) 102656 7 as CaM contains two Ca 2+ binding lobes, each with two EF-hands with different Ca 2+ affinities and binding kinetics that could act in a somewhat independent way, this may contribute to its extraordinary flexibility in Ca 2+ /CaM-dependent signaling. The available information on the presence and especially the significance of cis-regulatory elements controlling the differential expression of the three human CaM genes and their alternatively polyadenylated mRNAs is rather scanty. Obviously, a major obstacle is the fact that it is not possible to discriminate on the level of protein accounted for each gene namely, which CaM gene is responsible for the final protein product as CaM from all three CALM genes are identical. Whether and how these potential regulatory elements are used by the relevant transcription factors and other regulatory proteins as well as RNAs and whether there is cross-talk among these elements to facilitate or repress the relative expression of a particular CaM gene and its mRNA product over the others, depending on different physiological states of the cell, is only very fragmentarily known so far. As work on CaM gene regulation carried out before and around 2000 has been reviewed by the group of Strehler [12], we will only discuss some of the major conclusions and focus on a few more recent examples of cis-regulatory elements and their functions, as well as cases of epigenetic control of the CaM genes in human and other organisms and list and compare these potential regulatory elements in table form. 3.2.1. Promoters, insulators The promoter regions of the three human CaM genes contain a variable number of regulatory elements, such as TATA, CAAT, GC boxes; AGGGA and CRE motifs, transcription factor-binding sites (e.g. for SP1, AP1, AP2, CREB), as shown in Table 1. This may impinge on the relative efficiency of their transcriptional activity [74]. The function of insulators in vertebrates is mediated upon binding to CTCF, an eleven zinc finger DNA-binding domain protein, which could interfere with the communication between promoters and enhancers or by connecting two CTCF binding sites thereby mediating chromatin looping (reviewed in [75]). Of interest, potential CTCF-binding sites have been identified in all three human CaM genes (see Table 1). Possible functional Fig. 3. a.b. Alignment of nucleotide sequences of the three human and other vertebrate CaM genes. Alignment of the three human CALM genes (a), and alignment of each gene compared to the orthologs from four species (b). Nucleotide sequences were retrieved from NCBI database under the following accession numbers: NM_006888.6, NM_001743.6, and NM_005184.4 for human CALM1, CALM2 and CALM3, respectively; NM_031969, NM_017326.3, and BC063187.1 for rat Calm1, Calm2 and Calm3; NM_001242572.1, NM_001242587.1, and NM_001046249.2 for Cow Calm1, Calm2 and Calm3, and NM_001313934.1, NM_007589.5, and NM_007590.3 for mouse Calm1, Calm2 and Calm3. Only coding sequences were aligned with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) using default settings. The start and stop codons are included in the alignments as well as the nucleotide position numbers. Non-identical nucleotides are highlighted in red. The third position in each codon is marked in red=AT3 or green=GC3. M. Munk et al. Cell Calcium 107 (2022) 102656 8 significance of the cAMP response element found in human, rat, and the Xenopus CaM II promotor has been supported by findings showing that cAMP upregulates CaM I and CaM II in rat PC12 cells [76]. It was demonstrated that a 1218 bp segment of the promoter and the 5′-leading sequence of the chicken Calm2 gene containing CpG islands, typical for housekeeping genes and in many cases not containing TATA boxes, was sufficient for the initiation and regulation of transcription [77]. Gene promoters may contain single or multiple dispersed transcriptional start sites (reviewed in [78]), and in mammals CpG island-containing promoters usually are associated with dispersed transcriptional start sites and housekeeping genes (reviewed in [79]). The above mentioned chicken Calm2 promoter was shown to have lower activity in differentiated cells, as compared to proliferating ones, in agreement with the lower endogenously-produced CaM level observed during differentiation [77]. As the chicken Calm2 promoter shares similarities with mammalian CALM2 promoters, it could be predicted that it works in a similar way as the mammalian promoter. Similar studies were conducted using promoter segments of the rat Calm2 and Calm3 genes in subsequent studies. In these cases, transgenic mice expressing constructs of promoter segments with the β-galactosidase gene as a reporter were used to monitor the expression in different regions and cell types of the central nervous system. In most but not all cases, β-galactosidase expression matched the expression of the endogenous CaM genes in adult mice [80,81]. Additional studies were performed in testis, specifically demonstrating Calm2 expression in spermatocytes, and not in other cellular stages during spermatogenesis, when Calm1 and Calm3 are expressed [82]. 3.2.2. Epigenetic control Epigenetic labeling of DNA and histones plays a central role in the pattern of gene transcription during development, differentiation and the physiological status of the cell [83–86]. Of interest, the cytosine methylation level of human CALM1 in spermatocytes progressively decreases from 25 to 60 years of age, as observed with other genes (e.g. CDH13 and STMN2 encoding T-cadherin and stathmin-2, respectively) [87], suggesting a differential expression level of CaM during aging. Also, a prospective study in a rather small cohort of type 2 diabetes patients demonstrated hypomethylation of CpG islands in the promoter of CALM2 in peripheral leukocytes, contradicting the hypothesis that methylation of this gene contributes to the onset of type 2 diabetes as previously suspected [88]. 3.2.3. Tissue and cell type specific expression of the three mammalian CaM genes CaM is expressed in all cells of eukaryotes in variable amounts. To determine which CaM gene is expressed in each case in a specific cell type and physiological condition may shed light on how the three CaM genes are contributing to the function of the cell or tissue. Expression of the individual CaM genes can only be measured on the transcript level, because the proteins coded by the three genes are identical. Therefore, transcription data cannot be directly linked to the levels of CaM protein. Human CALM1 expression was detected in all organs tested including heart, placenta, lung, and kidney, though it was found to be particularly abundant in brain and skeletal muscles, and very scarce in liver and pancreas [52]. CALM2 also presents very high expression in human brain, followed by similar expression levels in heart, placenta, lung, liver, skeletal muscle and kidney, and in much lesser extent in pancreas [51]. In the heart, the expression of different CaM genes is not homogeneous through its different tissular structures. Thus, in the Purkinje fibers, specialized to conduct the electrical stimuli to the left and right heart ventricles, the expression of CALM1 is higher than in the working atrial and ventricular non-specialized cardiomyocytes, while the expression of CALM3 is higher in both the left and right epicardium than in the endocardium [89]. This suggests that each cardiac structure may require a different quantitative and/or temporal output of CaM for their correct functioning. The specific downregulation of only one of the three CaM genes could give some hints on the functionality exerted on the cell. Several reports have been published in this context [90,91]. However, these studies do not specifically address whether the knocked-down CaM gene was the only one regulating the cell function under study, but rather that the supply of CaM derived from the untouched genes is unable to fully compensate for the observed deficiencies. This could be due to lack of sufficient temporo-spatial supply of CaM to the targeted CaM-binding proteins. 3.2.4. Differential expression of CaM induced by signaling processes, during cell differentiation and development The expression of the CaM genes has been shown to be dependent on the physiological conditions of the cell. For instance, the half-life of CALM1 and CALM3 mRNAs is longer than the one of CALM2 mRNA in mouse myoblasts during both proliferation and differentiation, while the half-life of all three CALM mRNAs increases during differentiation, but the actual level of these transcripts decreases, as well as the CaM level, although with distinct kinetics [92]. CaM expression is also subjected to endocrine control. Thus, it has been shown that resection of the adrenal glands decreased the level of CALM3 mRNA in the rat cerebral cortex and the hippocampus, without affecting CALM1 and CALM2 mRNAs, while corticosterone administration prevented CALM3 downregulation as expected [93]. Evidence that differential transcription of the CALM genes in rats provide a means to control the spatial and temporal availability of CaM pools was reviewed in [12]. The CALM mRNAs exhibit cell type and cell state-specific mRNA abundance. In rats CALM1 and CALM2 appear in neurites and dendrites, while CALM3 predominates in the cell body. Humans express a long CALM1 transcript (4.1 kb) as well as a shorter 1.8 kb version. The longer CALM1 (4.1 kb) human mRNA has been demonstrated to have increased stability. It was suggested that this protects the transcripts from degradation during trafficking to distal locations (reviewed in [12]). We searched for evidence of differential expression of the two CALM1 RNA isoforms, in the RNA sequence database GTExPortal (gtexportal.org). It appears that the 4.1 kb version is more specific to the brain (Fig. 4). The shorter version appears to be the most abundant in all tissues sequenced, except in brain, where the long form dominates. This may reflect the need for Fig. 4. RNA isoforms from alternative polyadenylation in CALM1. RNA- sequencing reads from representative tissues. The figure shows RNA sequencing results of CALM1. The 3 ′UTR starts at position 0.6 Kb of the transcript. Evidence is seen of reverse transcriptase activity starting from d-TTT primers at position 1.8 Kb (red arrow a) and at position 4.1 Kb (red arrow b). https://www.gtexportal.org/). M. Munk et al. Cell Calcium 107 (2022) 102656 9 stable CALM mRNA when trafficking to distal dendrites in brain. 3.2.5. 5′-UTR and 3′-UTR The 5′-UTR of mRNAs may be involved in transcriptional or translational regulation whereas the 3′-UTR of mRNAs may act, among other functions, in transport and location of the mRNA to specific intracellular locations where translation is required, as well as controlling mRNA stability by promoting or inhibiting its degradation. In mammals, both UTRs are unique in the three CaM mRNAs, presenting highly conserved sequence homology (76–95%) among human, rat and mouse, and 85–96% between human and several cetaceans. The importance of the 5′-UTRs was demonstrated in the three human CaM genes in experiments testing transcriptional activity with the use of the luciferase reporter gene in teratoma cells, as its absence strongly decreased transcription [51] and discussed in Toutenhoofd and Strehler [12]. The least conserved 3′-UTR is found in the CALM3 transcript (79.7% and 78.2% comparing human with rat and mouse, respectively), while the most conserved corresponded to the CALM2 transcript (90.2% and 92.7% comparing human with rat and mouse, respectively), closely followed by the CALM1 transcript (89.3% and 86.2% comparing human with rat and mouse, respectively) [94,95]. This is close to the somewhat higher conservation (91.1–92.7%) of the CDSs of the three CaM genes comparing human with rat and mouse transcripts indicating an important function of the 3´non-coding region [94]. There are putative CPEB binding sites in the 3′-UTR of human CALM1 and CALM2 mRNAs (see Table 1). The CPEB protein, first identified in Xenopus oocyte, binds to the 3′-UTR at specific sites and regulates mRNA polyadenylation and translation (reviewed in [96]). CPEB activity is regulated by phosphorylation with diverse protein kinases, including CaMK-II [97,98]. Nevertheless, CPEB phosphorylation does not affect RNA binding, a process requiring Zn 2+ , and which is mediated by two RNA recognition motifs and the Cys/His-rich C-ter- minus of CPEB needed for interaction with the consensus sequence 5′-UUUUUAU-3′[98] (see Table 1). The 3′-UTR of the human CALM3 transcript contains three polyadenylation sites, accounting for the identified 0.9, 1.9 and 2.3 kb transcripts [94]. The close homology of these UTRs denotes close phylogenic relationship as well. The 3′-UTR in the long mRNA of mouse Calm3, an isoform of the canonical Calm3 mRNA, contains an intron forming a hairpin duplex where the double-stranded RNA-binding protein, denoted Staufen2, binds. This binding promotes the location of the long mRNA molecule in dendritic terminals, only in activated synapses in mouse hippocampal neurons without affecting its stability [99]. Among the transcripts of Calm1 in mouse, isoforms containing short and long 3′-UTRs were found. Knocking down the mRNA isoform with the long 3′-UTR, but not the one with the short 3′-UTR, blocks the development of axon of dorsal root ganglion neurons, restricts their rostral migration to the hindbrain in embryos, and impairs activation of hippocampal neurons in the adult [100]. The presence of 3 CaM genes in mammals each with several variants due to differential polyadenylation with different regulatory properties may allow for a high level of orchestration and adaptation to different physiological conditions, pinpointing any specific function or targeted CaM-binding protein(s) involved. 3.2.6. Regulation by miRNAs MicroRNAs usually downregulate the expression of proteins by inducing the degradation of the mRNAs via the dicer/RNA-induced silencing complex. In transgenic mice, it was shown that overexpression of the miRNA miR-1 downregulates CaM after binding to the 3′-UTR of CALM1 and CALM2 [101,102]. Subsequent downregulation of CaM translation leads to the negative control of transcription factors involved in cardiac hypertrophy. This anti-hypertrophic effect is produced by the inhibition of the CaM-dependent calcineurin/NFAT and CaMK/Mef2a pathways [101]. Studies performed in a mouse model have shown that miR-1 is downregulated during heart failure [101]. On the other hand, in conjunction with MLCK repression, low CaM levels contribute to sarcomere damage and impaired cardiac contraction [102]. The miRNA Let7a has also been shown to hybridize with the 3′-UTR of human CALM1 at the 5′-UACCUC-3′sequence, a segment that is conserved in rat and mouse. Thus, this interaction also produces the downregulation of CaM translation leading to a cardiac anti-hypertrophic effect [103]. Another miRNA downregulating CaM expression was shown to be miR-335, although the authors of this work did not specify on which CALM gene it was acting [104]. The microRNA hsa-miR-4709-3p was identified to act on CALM3 in kidney, and this microRNA strongly downregulated CaM mRNA expression in patients suffering chronic kidney disease [105]. Curiously, the decreased plasma levels of miR-335 in patients suffering an acute ischemic stroke inversely correlated with the plasma levels of CaM [104], indicating the importance of other factors regulating the level of plasma CaM levels. Moreover, the miRNA miR-4661-3p potentially interacts with the 3′-UTR of CaM mRNA, although this miRNA stabilizes the CaM mRNA, instead of inducing its degradation. This was due to increasing the stability of the AU-rich elements, where miR-4661-3p is expected to bind. Suppression of miR-4661-3p by peptide nucleic acid inhibitors lowered CaM levels and suppressed neuroinflammation mediated by microglial cells [106]. 3.2.7. Differential subcellular localization of CaM mRNAs Establishment of local mRNA pools, which could be far away from the cell body in neurons, is believed to contribute to the posttranscriptional regulation leading to temporal and spacial fine-tuning of CaM gene expression. The initial discovery of specific subcellular localization for several CaM RNAs in differentiated PC12 cells in 1993 by Zhang et al. [107] showed that CALM1 and CALM2 mRNAs mostly localized to the neurites and that CALM3 mRNA was positioned in the cell body or close to it. The group of Gulya investigated the differential subcellular localization of the different mRNAs of the 3 CaM genes in neuronal rat tissue, which contains the highest CaM concentration in the mammalian body (reviewed in [108]). The presence of different patterns of mRNAs derived from the same CaM gene, detected on Northern blots from different tissues or cell populations as well as degradation sequence elements in certain transcripts of the same CaM gene but not in others, pointed to posttranscriptional regulation on the level of mRNA stability due to differential polyadenylation (see also chapter 3.2.8 on codon usage). Targeting specific mRNA to dendritic sublocalization, where CaM mRNA is colocalized together with other transcripts, including alpha CaMKII, and assumed to be translated there, has been shown in a number of articles indicating an increased need of CaM signaling for postsynaptic activities (reviewed in [108]) and [109]. Differential transport of mRNAs from the 3 CaM genes to functional different compartments in neurons and glia cells of the rat hippocampus [110] as well as in the midbrain-brain stem region has been shown by the Guyla group [111]. The intracellular distribution of the mRNAs derived from the 3 CaM genes also varies in isolated mouse ventricular cardiomyocytes. It was shown that the mRNA from the CALM2 gene is more concentrated in the central part of the cell, close to the nucleus, and the CALM3 mRNA is concentrated in the cell periphery, while the Calm1 mRNA is mostly located between the other two [112]. It is possible that the differential intracellular distribution of the three mRNAs is correlated with their translation in specific cellular regions where CaM is transiently required. Moreover, treatment of animals with isoproterenol, an agonist of adrenergic-β receptors, enhanced the level of Calm3 mRNA, but not Calm1 and Calm2 mRNAs, coinciding with enhanced overlap of this mRNA with Ryr2 mRNA at the intercalated discs [112]. This is in agreement with the expected increase of Ca 2+ mobilization required for heart contraction during sympathetic stimulation, and therefore suggestive of a specialized role of a given CaM gene for particular cell functions. M. Munk et al. Cell Calcium 107 (2022) 102656 16 perhaps because a disease variant with higher expression is more likely to lead to severe symptoms. However, due to the small number of patients with CaM mutations this remains speculative. Even if a clearer picture of mRNA levels will arrive, it is still an open question whether this would relate to the level of proteins, as mutant CaM may differ from the wild-type one in stability, subcellular location, target specificity, and might be post-translationally modified (e.g. phosphorylation, methylation, acetylation, oxidation) in different ways. Even though a potential connection between the type of CALM mutations and the caused pathology has emerged, as outlined above, a clear correlation between phenotype and genetic alteration or properties of the resulting proteins has not been established yet. As CaM has many important targets in cardiomyocytes, it is likely that the pathophysiological effect of mutant CaM is the result of improper regulation of several critical players. 4.2. CaM mutations potentially leading to neural diseases Ion channels are essential for neural function, and CaM has an important role in the regulation of many of these ion channels, including Ca 2+ -dependent L-type Ca 2+ channels [148] (reviewed in [134,149]), voltage-gated Na + channels [150] (reviewed in [134,151]), voltage-gated K + channels (reviewed in [134]), Ca 2+ -activated K + channels (reviewed in [152]), among others (reviewed in [35,134,149, 152]) Additionally, other CaM-dependent systems in neurons and/or glial cells, including the important signaling molecules CaMK-II and calcineurin, could also be affected likewise when CaM mutations occur and contribute to neural pathogenesis. Patients with CALM1 and CALM2 (CALM3 have 29% average relative expression in brain tissue, but only 8% across 11 glial and neuronal single cell RNA measurements) mutations affecting residues at EF-hands III and IV, showing decreased Ca 2+ affinity, exhibited in addition to cardiac arrhythmias as described above, neurodevelopmental delay, epilepsy, cognitive, and motor deficiencies. This was first ascribed to be secondary to brain injury after transient anoxic episodes, resulting from arrhythmias leading to syncope and cardiac arrest during early life, that was successfully resuscitated [123]. However, the authors of this report could not exclude primary neuronal affectation associated to circulatory insufficiency. The same group hypothesized thereafter that perhaps only in cases where multiple cardiac arrests occurred, post-anoxic neural sequelae remain [153]. Nevertheless, this hypothesis was based considering only few cases with divergent neural clinical features, ranging from mental retardation, autism, developmental delay and seizures, and without history of cardiac arrest (as recorded in the International Calmodulinopathy Registry) [132,135,154]. This suggests, not unexpectedly, that CaM mutations may directly affect brain functions. 4.3. CaM mutations potentially leading to endocrine-associated illness A female patient affected with sinus bradycardia presenting heterozygous mutation of CALM3 (E141K) suffered as well a serious hypoglycemic condition, that was the ultimate cause of her death, and this was attributed to hyperinsulinemia [138]. The authors of this study suggested that this CaM mutation could be responsible for the dysregulated insulin secretion by affecting L-type Ca 2+ channels in pancreatic β-cells [138]. This report opens the possibility that CaM mutations could cause or contribute to some endocrine disorders. 4.4. CaM polymorphisms potentially associated with osteoarthropathies Some single nucleotide polymorphisms (SNP) in CALM1 have been associated with osteopathies. Mototani et al. [155] reported that a SNP in intron 3 (IVS3 -293C>T), as well as another SNP in the core promoter region of CALM1 (rs12885713 -16C>T) strongly correlated with hip osteoarthritis. Particularly, these authors claimed that the latter SNP decreased the rate of transcription of this gene, which lowers the CaM level and reduces the expression of other genes essential for cartilage matrix formation. These include COL2A1, encoding the pro-a1 chain of type II collagen [156], and AGC1, encoding chondroitin sulfate proteoglycan 1, also denoted aggrecan [157]. The study of Mototani et al. [155] was performed with Japanese patients. However, analogue studies with British and Greek Caucasian patients did not find a correlation between the occurrence of polymorphisms in the core promoter region of CALM1 and susceptibility to hip or knee osteoarthritis [158, 159]. Hoaglund [160], commented that this apparent inconsistency could be explained by the different underlying pathology in the two patient groups, being mostly primary or idiopathic osteoarthritis in the Caucasian group, while being congenital hip dislocations or developmental acetabular dysplasia in the Japanese group, therefore giving validity to both studies. Nevertheless, two SNPs (i-CALM2-5 rs10153674 A>G; and i-CALM2-6 novel G>A) both located in intron 1 of CALM2 were significantly associated with hip osteoarthritis without acetabular dysplasia in the Japanese patients [161]. The authors of this study pointed out that this was observed only after stratification of the patients according to their acetabular dysplasia status, otherwise no significant correlation was found. In addition, SNP rs12885713 -16C>T occurrence in the core promoter region of CALM1, and SNP rs5871 located in the 3′-UTR, were associated with idiopathic scoliosis in adolescents Chinese patients [162]. Nevertheless, the genetic basis of these pathological conditions are multifactorial and many SNPs associated with idiopathic scoliosis have been found in other genes (reviewed in [163]). Furthermore, the association of SNP rs12885713 (−16C>T) in the core promoter region of CALM1 with osteoarthritis predisposition has been further questioned in a study carried out in Han Chinese patients and in a meta-analysis pooling Asian and Caucasian patients [164,165]. These findings suggest that ethnicity may play a role in the pathogenesis of these osteoarthropathies, although further studies are required to clarify this issue. 5. Concluding remarks Based on a multiplicity of discoveries described earlier in [108], and extended in this article, it can be concluded that the three CaM genes in mammals cannot solely function as redundant copies replacing each other in case of need. The following issues would argue against this possibility: (1) Non-coding regions including promoter/enhancers 5´- and 3´- noncoding mRNA sequences, may lead to different stabilities of the RNA, e.g., due to target sequences of proteins and miRNA. The importance of these sequences is highlighted by the fact that they are almost as highly conserved among the corresponding genes in different vertebrates as the coding sequences. (2) Every individual CaM gene has highly conserved non-coding sequences, when different vertebrate species are compared. The conservation of these sequences among genes in the same species is lower, allowing the synthesis of mRNAs in different cell types with different stabilities and subcellular localizations. This can be responsible, at least in part, of multitude modes of fine-tuning regulation. (3) Another factor not previously focused on, but highlighted in this article, is the potential significance of differential codon usage by the 3 CaM genes, which may lead to different stabilities and translational efficiency. It is captivating to observe how the three CALMs have differentiated in their GC3 usage, considering that they originated from one single CALM gene by duplication. The pattern of two low GC3 CALM1,2 genes, and one high GC3 CALM3 gene in humans is also found in seven other mammals investigated. Especially, CALM2 appears to be highly selected for instability throughout its coding region. CaM is a very important protein, as highlighted by its many interacting partners, and uniquely 100% sequence conservation in all investigated M. Munk et al. Cell Calcium 107 (2022) 102656 17 vertebrates. Higher organisms may have evolved to take advantage of three CALM genes with different mRNA stabilities. A low stability mRNA can be in some cell types where fast regulation of the available mRNA pool is especially advantageous, but can be a trade-off in other cell types. With three CALM genes evolution may have more freely tailored the three mRNAs. Following this hypothesis, it is interesting to observe how the high stability CALM3 is the main mRNA in late spermatids, where transcription is very low, and high stability is therefore needed. The organism ability to use an available stable CALM3 mRNA in spermatids may then have allowed the CALM1,2 mRNAs to evolve in a less stable direction, without evolutionary pressure selecting in opposite direction. (4) Every individual CaM gene seems to be irreplaceable, as deduced from pathological conditions in human. First, mutations in a single CaM gene can lead to illness, despite co-expression of nonmutated paralogs. Second, the specific expression patterns of CaM genes change in cancer cells. It would be interesting to know whether different subcellular pools of CaM created by the presence of specific mRNA pools would be prone to different post-translational modifications, such as phosphorylation, methylation, carboxymethylation or acetylation [13,166–169]. It can be imagined that the cellular location where the mRNA is translated, and in addition the speed of the translation machinery could result in different modifications of CALM1,2,3 products. Codon usage predict faster translation of the CALM3 compared to the other CaM genes. This could be relevant to determine the contribution of a specific CaM gene mutation or an isoform for the development of a pathology. The advanced gene editing methods, using CRISPR technology, would allow to slightly modify CaM genes, for example adding different tags. This can be the basis for gene discrimination in mammals in order to study the differential expression of CaM from the 3 genes. However, this may of course be difficult, as the modification of CaM, even if minor, could lead to changes in function. Data Availability Data will be made available on request. Acknowledgments This work was funded by grants from the Danish Research Council (DFF-4004-00560) the Wedell Wedellsborg Foundation and the Candys Foundation, Denmark, to MWB; grants from the program Consolidaci´ on de Grupos de la Junta de Andalucía (research group BIO-210) and from the Plan Estatal 2017–2020 (PID2019-103850GB-I00), Spain, to EV; and grant from the Consejería de Educaci´ on, Juventud y Deportes – Comunidad de Madrid (B2017/ BMD-36), Spain, involving contributions from the European Funds for Regional Development and the Social European Fund, to AV. References [1] E. Carafoli, J. Krebs, Why calcium? How calcium became the best communicator, J. Biol. Chem. 291 (2016) 20849–20857. [2] R.H. Kretsinger, E. Carafoli, F. Clementi, W. Drabikowski, A. Margreth, Hypothesis: calcium modulated proteins contain EF hands. Calcium Transport, Contraction, Secretion, Elsevier, Amsterdam, 1975. [3] D. Chin, A.R. 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