Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
ReviewControl of alternative pre-mRNA splicing by Ca++ signals
Introduction
Ca++ signalling is important for a variety of cellular processes such as muscle contraction, hormone secretion and gene transcription. In neurons, Ca++-regulation of gene expression is known to be critical for the long-lasting changes associated with learning and memory [1]. Accumulating evidence supports that Ca++ signalling also controls the alternative splicing of precursor messenger RNA (pre-mRNA) transcripts (Fig. 1). Examples of this regulation have been identified in myotube cultures, endocrine cells, and mostly, in neuronal cells (Table 1).
The complex structure and function of the brain underlie the requirement for a high level of proteomic complexity. How can the human genome, with only the approximately 25,000 protein-coding genes [2], encode enough diverse proteins to achieve the high level of complexity to ensure, for example, that individual neurons are precisely “wired” with many others and that their functions are finely tuned? It is now clear that protein diversity can be greatly increased through alternative pre-mRNA splicing [3], [4], [5]. This is a particularly common way for the regulation of gene expression among neuronal genes and likely contributes to the fine-tuning of neuronal functions [6], [7], [8].
The pre-mRNA of most eukaryotic genes contains both expressed regions (exons) that will be included in the mature mRNA and regions that will be excluded (called intragenic regions, i.e. introns) [9]. Removal of introns and joining of exons happen in the cell nucleus through pre-mRNA splicing [10], [11], [12]. After introns were discovered, Walter Gilbert predicted in his short essay “Why genes in pieces” in Nature: “…the splicing need not be a hundred percent efficient; changes in sequence can alter the process so that base pairing and splicing occurs only some of the time” [9]. Soon afterward, alternative transcripts from a common precursor RNA were discovered [13]. Since then, more and more eukaryotic genes have been found to undergo alternative splicing [6], [14], [15]. Currently, the estimated percentage of human genes with alternative splicing is more than 50% [16], [17]. In some cases, a single pre-mRNA transcript has the potential to generate a large number of protein isoforms, for example, transcripts of the Dscam, Neurexin and Slo genes [4], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39] (Fig. 2).
The consequences of alternative splicing include altered mRNA stability or subcellular localization and the addition or deletion of specific protein sequences. Functional differences among protein isoforms range from subtle modulations to on/off switches or antagonistic effects [39], [40], [41]. Defects in alternative splicing factors or aberrant inclusion of alternative exons can result in genetic diseases [42], [43], [44], [45], [46], [47]. During the Cambrian “explosion” of metazoans, alternative splicing likely contributed to the expansion of proteomes [48]. Therefore, this is an important control in the transmission of genetic information from DNA to proteins. Currently with the huge amount of genomic information available from various genome sequencing projects, it is becoming imperative to understand how alternative splicing is controlled, particularly by cell signals, and to elucidate the role of this form of regulation in normal cell function and human genetic diseases.
Splicing happens in two transesterification steps within a large spliceosome complex containing more than 100 proteins assembled stepwise onto pre-mRNA introns [12], [49]. During alternative splicing, the spliceosome assembly is altered so that a splice site(s) is optionally used depending on the cell type, developmental stage or sex, resulting in the inclusion or exclusion of alternative exon sequences in the mature mRNA (Fig. 1). Studies with tissue- or sex-specific exons identified both cis-acting pre-mRNA elements and trans-acting factors that control alternative splicing [15]. Depending on their location and effects on splicing, the RNA elements are called intronic or exonic splicing enhancers (ISE or ESE) or silencers (ISS or ESS). Many of these elements bind specific trans-acting factors (mostly proteins, but in some cases RNA) that enhance (enhancer) or inhibit (repressor) the assembly of the constitutive splicing factors. Well characterized examples of enhancer factors include most members of the arginine/serine-rich SR family that bind purine-rich ESE elements [50], and repressors include the heterogeneous ribonucleoprotein particle proteins hnRNP A1 and hnRNP I that bind purine-rich elements containing UAGG motifs and pyrimidine-rich elements containing UUCU, UCUU or CUCU motifs, respectively [51], [52]. There are also elements/factors that function in a location-dependent way as either an enhancer or repressor, for example, the CA-repeat binding protein hnRNP L [53], the UGCAUG-binding proteins FOX-1 and -2 [54], [55], [56], [57], [58], [59], [60], [61], and the YCAY-binding protein Nova-1 [62], [63], [64], [65], [66]. Binding of the trans-acting factors to the elements is thought to control mostly the early stages of spliceosome assembly before the first transesterification step [15]. For example, PTB prevents the binding of U2AF65, a factor of the E (early) complex, through competition or looping out the target exons [67], [68], [69]. In mammalian systems, splicing regulation is generally dependent on the combinatorial effects of multiple pre-mRNA elements and trans-acting factors [70], [71], [72] (Fig. 1). The relative levels of positive and negative regulatory factors determine the generation of variant mRNA isoforms in specific cell types [73], [74], [75]. Brain-enriched splicing factors include Nova-1 and -2 [62], [63], [64], [5], [66], FOX-1 and -2 [54], [55], [56], [57], [58], [59], [60], [61], PTBP2 (nPTB) and neuronal Hu proteins [76], [77], [78], [79], [80], [81], [82], [83].
In addition to cell type, developmental stage and sex-specific regulation, alternative splicing can also be dynamically regulated in response to extracellular stimuli such as cytokines, hormones or neurotransmitters [24], [84], [85], [86], [87], [88], adding a further dimension to the control of the flow of genetic information [86]. Examining the inducible alternative splicing events will help understand their roles in cell functions as well as provide systems where the dynamic changes of spliceosomal components can be tracked down to facilitate the mechanistic studies of splicing. Moreover, understanding the molecular basis of this process will help develop ways to reverse the aberrant splicing events in genetic diseases using extracellular factors. However, it is not clear in most cases how alternative splicing is controlled by external stimuli and intracellular signalling pathways. Ca++-regulation of alternative splicing is among the regulatory events being intensively studied.
The following sections on the evidence for Ca++-regulation of alternative splicing will be focused on the regulated alternative exons, the splicing factors and the pre-mRNA elements. It should be noted that both constitutive and alternative splicing are coupled with transcription and this topic has been covered in recent reviews [89], [90], [91], [92]. Promoter-dependent regulation of alternative splicing by Ca++ stimuli has also been observed [84]. Readers are referred to these reviews and original papers for this aspect [84], [89], [90], [91], [92].
Section snippets
Alternative exons regulated by Ca++ signals
Table 1 lists some of the alternative exons whose inclusion in the mature mRNA can be changed upon stimulation by extracellular factors that activate Ca++ signalling pathways.
These factors include the ionophore ionomycin [93], the neurotransmitter glutamate or its analogue NMDA (N-methyl-d-aspartate) [94], [95], [96], [97], [98], the muscarinic acetylcholine receptor agonist pilocarpine (inducing seizure) [99], the ionotropic glutamate receptor agonist kainate, the inhibitor of the
Splicing factors regulated by Ca++ signals
Of the cis-acting elements in alternative splicing, most that have been studied to date are bound by protein factors, although, as in constitutive splicing, RNA secondary structures and trans-acting small RNAs have also been described [12], [160], [161], [162]. Protein splicing factors reportedly regulated by Ca++ signals at various levels are listed in Table 3.
The protein level of splicing factors can be controlled by Ca++ signals. In some cases, it takes several hours for the splice variant
Pre-mRNA elements responsive to Ca++ signals in the control of alternative splicing
Since most mammalian exons are controlled in a combinatorial way by multiple positive and negative regulatory elements (Fig. 1), the role of a single RNA element in splicing regulation is usually isolated from its endogeneous gene context and tested in a heterologous mini-gene, as has been done in studying the control of gene transcription [194], [195]. This approach has also proved useful in studying the regulation of splicing by the CaM kinase pathway.
Two CaMK IV-responsive RNA elements
The impact of Ca++-regulated alternative splicing on neuronal functions and diseases
As shown in Table 1 and references therein, alternative exons controlled by Ca++ signalling can affect a broad range of neuronal functions. Based on data from these Ca++ signal-regulated splicing events and work from others [7], [8], I will speculate on the effect of Ca++-regulated alternative splicing on neuronal functions and diseases.
Perspectives
Even with the progress described above, much remains to be learned about Ca++ control of alternative splicing and its role in cellular, particularly neuronal, functions and diseases. For example, what is the nature of the molecular link between CaMK IV and the trans-acting splicing factors that function through the CaRRE elements? What is the physiological impact of a specific stimulus through the regulation of a group of alternative exons? How important is this inducible alternative splicing
Acknowledgements
I thank Mary Lynn Duckworth and Jean Paterson for editing the manuscript. Work in my lab is supported by an operating grant (MOP#68919) and a New Investigator Salary Award from the Canadian Institutes of Health Research (CIHR), by a research grant (#016355) from the National Cancer Institute of Canada (NCIC), and by Manitoba Health Research Council and Canada Foundation for Innovation (CFI) funds.
References (225)
- et al.
Alternative splicing: combinatorial output from the genome
Curr. Opin. Chem. Biol.
(2002) Alternative splicing: increasing diversity in the proteomic world
Trends Genet.
(2001)Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology
Cell
(2000)- et al.
Alternative RNA splicing in the nervous system
Prog. Neurobiol.
(2001) - et al.
RNA binding proteins and the regulation of neuronal synaptic plasticity
Curr. Opin. Neurobiol.
(2006) Neuronal proteins custom designed by alternative splicing
Curr. Opin. Neurobiol.
(2005)- et al.
An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA
Cell
(1977) - et al.
Structure of the adenovirus 2 early mRNAs
Cell
(1978) - et al.
Alternative splicing is an efficient mechanism for the generation of protein diversity: contractile protein genes as a model system
Adv. Enzyme Regul.
(1991) - et al.
Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity
Cell
(2000)
Functional differences among alternatively spliced variants of slowpoke, a Drosophila calcium-activated potassium channel
J. Biol. Chem.
Cloning, expression, and distribution of functionally distinct Ca(2+)-activated K+ channel isoforms from human brain
Neuron
Functionally diverse complement of large conductance calcium- and voltage-activated potassium channel (BK) alpha-subunits generated from a single site of splicing
J. Biol. Chem.
A novel MaxiK splice variant exhibits dominant-negative properties for surface expression
J. Biol. Chem.
A cysteine-rich domain defined by a novel exon in a Slo variant in rat adrenal chromaffin cells and PC12 cells
J. Biol. Chem.
Distribution of Ca2+-activated K+ channel isoforms along the tonotopic gradient of the chicken's cochlea
Neuron
Differential distribution of Ca2+-activated K+ channel splice variants among hair cells along the tonotopic axis of the chick cochlea
Neuron
Expression of Ca(2+)-activated K(+) channel subunits and splice variants in the rat cochlea
Hear. Res.
Identification of a novel tetramerization domain in large conductance K(ca) channels
Neuron
Alternative splicing of potassium channels: a dynamic switch of cellular excitability
Trends Cell Biol.
Two splice variants of the Wilms' tumor 1 gene have distinct functions during sex determination and nephron formation
Cell
Splicing regulation in neurologic disease
Neuron
Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16
J. Biol. Chem.
Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system
Neuron
Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability
Neuron
Polypyrimidine tract binding protein blocks the 5′ splice site-dependent assembly of U2AF and the prespliceosomal E complex
Mol. Cell
Alternative pre-mRNA splicing: the logic of combinatorial control
Trends Biochem. Sci.
Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins
Mol. Cell
The microRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing
Mol. Cell
Crossregulation and functional redundancy between the splicing regulator PTB and its paralogs nPTB and ROD1
Mol. Cell
Coordination between transcription and pre-mRNA processing
FEBS Lett.
Coupling transcription, splicing and mRNA export
Curr. Opin. Cell. Biol.
Promoter usage and alternative splicing
Curr. Opin. Cell. Biol.
Induction of CD45 isoform switch in murine B cells by antigen receptor stimulation and by phorbol myristate acetate and ionomycin
Cell. Immunol.
Dopamine and glutamate induce distinct striatal splice forms of Ania-6, an RNA polymerase II-associated cyclin
Neuron
Calcineurin controls the expression of isoform 4CII of the plasma membrane Ca(2+) pump in neurons
J. Biol. Chem.
The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+
J. Biol. Chem.
Regulation of calcium channel alpha(1A) subunit splice variant mRNAs in kainate-induced temporal lobe epilepsy
Neurobiol. Dis.
Calcium regulation of neuronal gene expression
Proc. Natl. Acad. Sci. U. S. A.
Finishing the euchromatic sequence of the human genome
Nature
Why genes in pieces?
Nature
Spliced segments at the 5′ terminus of adenovirus 2 late mRNA
Proc. Natl. Acad. Sci. U. S. A.
Splicing of precursors to mRNAs by the spliceosome
Mechanisms of alternative pre-messenger RNA splicing
Annu. Rev. Biochem.
Genome-wide detection of alternative splicing in expressed sequences of human genes
Nucleic Acids Res.
Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays
Science
Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation
Pfluegers Arch. Eur. J. Physiol.
Cloning of human pancreatic islet large conductance Ca(2+)-activated K+ channel (hSlo) cDNAs: evidence for high levels of expression in pancreatic islets and identification of a flanking genetic marker
Diabetologia
Identification of Ca2+-activated K+ channel splice variants and their distribution in the turtle cochlea
Proc. R. Soc. Lond., B. Biol. Sci.
Control of alternative splicing of potassium channels by stress hormones
Science (Washington D C)
Cited by (38)
Implications of the thyroid hormone on neuronal development with special emphasis on the calmodulin-kinase IV pathway
2017, Biochimica et Biophysica Acta - Molecular Cell ResearchRecent advances in therapeutic strategies that focus on the regulation of ion channel expression
2016, Pharmacology and TherapeuticsFunctional Role of Mitochondrial and Nuclear BK Channels
2016, International Review of NeurobiologyCitation Excerpt :Some exons are constitutively expressed in all cell types, whereas others are expressed in a subset of cell types. Different exon combinations are related to differences in channel structure and function (Fodor & Aldrich, 2009; Glauser, Johnson, Aldrich, & Goodman, 2011; Lee & Cui, 2010; Li, Al-Khalili, Ramosevac, Eaton, & Denson, 2010; Ma et al., 2007; Saito, Nelson, Salkoff, & Lingle, 1997; Shelley, Whitt, Montgomery, & Meredith, 2013; Singh et al., 2013; Tian et al., 2001; Xie, 2008; Yang, Zhang, & Cui, 2015; Zarei et al., 2004). Four auxiliary β subunits (β1–4) and four auxiliary γ subunits (γ1–4) have been identified to date.
Involvement of BK channel in differentiation of vascular smooth muscle cells induced by mechanical stretch
2015, International Journal of Biochemistry and Cell BiologyCitation Excerpt :The roles of BK channel β-subunits in the hypertension need to be further studied in the future. The ER stores intracellular calcium and plays important roles in controlling the alternative splicing of pre-mRNA transcripts (Xie, 2008). It had been reported that ER stress induces intracellular calcium accumulation, which causes vasoconstriction and contributes to vascular remodeling during hypertension (Dromparis et al., 2013).