Review
Control of alternative pre-mRNA splicing by Ca++ signals

https://doi.org/10.1016/j.bbagrm.2008.01.003Get rights and content

Abstract

Alternative pre-mRNA splicing is a common way of gene expression regulation in metazoans. The selective use of specific exons can be modulated in response to various manipulations that alter Ca++ signals, particularly in neurons. A number of splicing factors have also been found to be controlled by Ca++ signals. Moreover, pre-mRNA elements have been identified that are essential and sufficient to mediate Ca++-regulated splicing, providing model systems for dissecting the involved molecular components. In neurons, this regulation likely contributes to the fine-tuning of neuronal properties.

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.

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