doi: 10.1161/01.RES.0000263400.64391.37
© 2007 American Heart Association, Inc.
Editorials |
One Gene, Many Proteins
Alternative Splicing of the Ryanodine Receptor Gene Adds Novel Functions to an Already Complex Channel Protein
From the Department of Physiology, University of Wisconsin Medical School.
Correspondence to Héctor H. Valdivia, MD, PhD, Department of Physiology, University of Wisconsin Medical School, 601 Science Dr. Madison, WI 53711. E-mail valdivia{at}physiology.wisc.edu
See related article, pages 874–883
Key Words: ryanodine receptor • alternative splicing • excitation-contraction coupling • apoptosis
When the human genome sequence was first drafted in 2000, researchers were shocked by the low number of genes found. Humans make 
90 000 different types of protein, so estimates were that at least a similar number of genes would be found. The logic was that, given the structural and functional complexity of the human body, we ought to have more genes than the simpler corn (zea mays, 40 000 genes) or the puny worm Caenorhabditis elegans (19 500 genes). So, when the final number of human genes was established to be fewer than 25 000, researchers immediately realized the gene:protein mismatch, reaffirmed the notion that the axiom "one gene, one protein" was inaccurate, and began looking for the evolutionary mechanisms that increase diversity and complexity from a relatively simple genetic makeup. The answer, it seems, is alternative gene splicing.
When a segment of DNA (gene) is transcribed, the resulting RNA (tRNA) contains meaningful (exons) and nonmeaningful (introns) sequences that must be edited to produce a coherent message (mRNA). This cut-and-paste process where exons are retained and introns are discarded is called gene splicing and constitutes the normal processing of genes. However, as it was first discovered some 25 years ago,1 "alternative splicing" occurs, a highly regulated process that confers sophistication to the manufacturing of proteins by frequently "violating the rules" and leaving pieces of introns or excising parts of exons in the final mRNA. The resultant protein, a splice variant, thus contains segment(s) of distinct amino acid sequence that presumably leads to functional diversity. The relevance of alternative splicing cannot be overemphasized. In one extreme example, alternative splicing is largely responsible for determining whether a cell continues to live or whether it programs its death by expressing a promoter of apoptosis (Bcl-xS) instead of a suppressor (the splice variant Bcl-xL). In equally dramatic but fortunately less frequent examples, faulty alternative splicing may lead to several cancers and congenital diseases. In the daily toiling of cells, alternative splicing is ubiquitous and influences central processes such as intracellular signaling and structural phenotype.2
In cardiac cells, Ca2+ release channels/ryanodine receptors (RyRs), play a well-established role in excitation-contraction (e-c) coupling, the series of events that link an electrical event (depolarization) with a mechanical contraction.3,4 Depolarization of the external membrane and its t-tubules opens voltage-sensitive Ca2+ channels/dihydropyridine receptors, allowing a small influx of extracellular Ca2+ (the inward Ca2+ current, ICa). In mature cardiomyocytes, ICa is insufficient to elevate myoplasmic [Ca2+] to fully contracting levels; however, ICa triggers the opening of RyRs, which produce a rapid and massive release of Ca2+ from the sarcoplasmic reticulum. This amplification process, Ca2+-induced Ca2+ release,5 is a signature event of RyRs, and it greatly influences the intensity and duration of ventricular contractions.6
In this issue of Circulation Research, George et al7 provide evidence that the cardiac RyR (RyR2) participates in processes other than those controlling the strength of heart contractility. Nonsurprisingly, alternative splicing of the ryr2 gene may confer this multifaceted functionality. RyR2 proteins, like the skeletal (RyR1) and the widespread (RyR3) RyR isoforms, are homotetramers of more than 2 megadaltons endowed with more than a fair share of structural elements to produce a bona fide ion channel. They contain a high-conductance Ca2+-selective pore, Ca2+ activation and inactivation domains, several phosphorylation sites, and multiple sites for a myriad of endogenous regulators that include ATP, Mg2+, and calmodulin.8 Still, as if this huge structural assembly were not sufficiently complex, RyRs are also capable of protein-protein interactions that allow them to bind, in some cases steadily and in other cases in a time- and Ca2+-dependent manner, to small and independently regulated accessory proteins.9 Therefore, there already seems to be plenty of room to add plasticity to RyR signals without invoking the need for additional protein diversity. Nevertheless, nature does not seem interested in adhering to our rational pattern of conduct and has endowed humans (and several other species) with more than a few RyR splice variants (so far thirteen are known, but this number is sure to increase). As George et al7 demonstrate here for RyR2 splice variants, this pool of "unorthodox" proteins is significant, in one instance (human embryonic hearts) comprising up to 90% of the total RyR2. Furthermore, these tissue-specific and developmentally regulated RyR2 splice variants are found to: 1) promote intracellular segregation of RyR2, 2) suppress Ca2+ release by the normally-spliced RyR2, acting as dominant negative variants; and 3) protect from apoptotic cell death. In previous studies, splice variants of other RyR isoforms were also found to predominantly suppress Ca2+ release or to contribute to distinct Ca2+ releasing patterns.10–12 More speculative but not less likely, splice variants may be responsible for targeting RyRs to mitochondria,13 Golgi apparatus14 or external membranes.15 The emerging notion, therefore, is that splice variants of RyRs add yet another layer of versatility (and intricacy) to modulation and location of Ca2+ release by these already complex channels. Their functional diversity invites us to exert caution when extracting general properties from a segregated pool of RyR channels or from single channel approaches.
Splice variants of RyR2 have been recognized for over a decade,16 but their function was undefined. In the elegant approach of George et al.7 two alternatively spliced variants of the human RyR2 corresponding to a 30bp insertion and a 24bp insertion (variants n and d', respectively, in the current nomenclature) were eGFP-tagged and overexpressed (4-fold over native RyR2) in HL-1 cells to follow their topological distribution and determine their influence on cytosolic and nuclear Ca2+ signaling. Reaffirming the uniqueness of each splice variant, the 30bp and the 24bp splice variants produced different effects on the above parameters. Although all RyR2s were expressed in the sarco(endo)plasmic reticulum (and its nuclear extensions) as expected, the 24bp insertion distinctively targeted RyR2 to an intranuclear Golgi apparatus (INGA) that profoundly modified intracellular Ca2+ signaling. Remarkably, the appearance of INGA in HL-1 cells, an implausible spontaneous phenomenon, strictly correlated with the expression of the 24bp RyR2 variant, suggesting that cellular phenotype may be regulated by alternative splicing of the ryr2 gene. Cells expressing the 24bp variant also produced modified responses to acute application of caffeine, decreasing their cytosolic but increasing their intranuclear Ca2+ transients. Experiments in cardiac and skeletal muscle cells have repeatedly shown that RyRs constantly modulate their activity to accommodate cytosolic and luminal feedback mechanisms; hence, the apparently opposite effect on cytosolic and nuclear Ca2+ signals by expression of the 24bp RyR2 variant, though noteworthy, may not be surprising considering the different lipid makeup, accessory proteins, Ca2+ gradients, etc, present in these two compartments. More intriguing, however, is that on chronic exposure to a submaximal dose of caffeine, expression of the 24bp variant attenuated Ca2+ fluxes in both, cytosolic and nuclear compartments. It might be possible that the 24bp insertion regulates the integration of temporal Ca2+ signals by the RyR2, as is the case of the exquisitely different CaMKII splice variants.17 Another important finding from the above experiments is that the steady Ca2+ flux produced by chronic application of caffeine increased apoptotic cell death (in line with the toxic effect of increased Ca2+ stimulation); however, expression of the 24bp RyR2 splice variant prevented apoptosis by virtue of its negative effect on Ca2+ release. Thus, RyR splice variants may produce Ca2+ signals that directly influence apoptotic cell death.
Where do these findings leave us? The results of George et al7 demonstrate that RyR2 splice variants confer novel functions to RyR2 proteins that are likely to be applicable to variants of other RyR isoforms. The insertion of a small number of amino acids (8 or 10, as shown in this study) into a gigantic protein appears sufficient to re-direct its trafficking to specialized compartments. The capacity of distinct RyR2 splice variants to modulate apoptosis suggests new venues for regenerative treatments where rapid turnover of damaged tissue is imperative. However, it is also important to recognize some limitations of the present study. First, experiments were conducted in HL-1 cells, a cell line derived from the AT-1 mouse atrial cardiomyocyte tumor lineage.18 Researchers still yearn for the day in which a cardiac muscle cell line may proliferate in culture while maintaining its differentiated phenotype; HL-1 cells, though the best available, are still far-off from this objective. They retain the round, unstriated morphology of embryonic muscle lines while simultaneously expressing protein markers of adult atrial cells, hence, correlation of expression of RyR2 variants with developmental regulation cannot be safely inferred from HL-1 cells. Importantly, the capacity of the 24bp RyR2 splice variant to attenuate apoptotic cell death, which was largely ascribed to the appearance of INGA in HL-1 cells, may be a unique feature of atrial cells and has yet to be demonstrated in ventricular cells, which apparently lack these intranuclear "tendrils". There are currently no antibodies against specific RyR2 splice variants and thus, it is not possible to track their expression in mature cells to outline a common pattern of distribution, if any. These limitations notwithstanding, the results of George et al7 argue convincingly that RyR2 splice variants are important players in the spatio-temporal encoding of Ca2+ signals in the heart that greatly influences cellular function and phenotype. These variants expand the cellular signaling repertoire, as already observed with splice variants of the voltage-dependent Na+ channel, the inositol 1,4,5-trisphoshate receptors, the sodium-calcium exchanger, SERCA, and other important ion transporters.
 |
Acknowledgments |
|---|
Sources of Funding
H.H.V.’s research is supported by grants HL55438 and HL76826 from the National Institutes of Health. No other financial incentives from public or private companies were received.
Disclosures
None.
|
Footnotes |
|---|
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
|
References |
|---|
 Top References |
|---|
1. Rogers J, Wall R. A mechanism for RNA splicing. Proc Natl Acad Sci U S A. 1980; 77 (4): 1877–9.
2. Ast G. The alternative genome. Scientific Am. 2005; 292 (4): 58–65.
3. Fill M, Copello JA. Ryanodine receptor calcium release channels. Physiol Rev. 2002; 82 (4): 893–922.
4. Meissner G. Molecular regulation of cardiac ryanodine receptor ion channel. Cell Calcium. 2004; 35: 621–628.[CrossRef][Medline] [Order article via Infotrieve]
5. Fabiato A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac Purkinje cell. J Gen Physiol. 1985; 85: 247–290.
6. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415: 198–205.[CrossRef][Medline] [Order article via Infotrieve]
7. George CH, Rogers SA, Bertrand BMA, Tunwell REA, Thomas NL, Steele DS, Cox EV, Hazeel CJ, Claycomb WC, Pepper C, Lai FA. Alternative splicing of ryanodine receptors (RyR2) modulates cardiomyocyte Ca2+ signaling and susceptibility to apoptosis. Circ Res. 2007; 100: 874–883.
8. Franzini-Armstrong C, Protasi F. Ryanodine receptors of striated muscle: a complex channel capable of multiple interactions. Physiol Reviews. 1997; 77: 699–729.
9. Bers DM. Macromolecular complexes regulating cardiac ryanodine receptor function. J Mol Cell Cardiol. 2004; 37 (2): 417–29.[CrossRef][Medline] [Order article via Infotrieve]
10. Kimura T, Nakamori M, Lueck JD, Pouliquin P, Aoike F, Fujimura H, Dirksen RT, Takahashi MP, Dulhunty AF, Sakoda S. Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1. Hum Mol Genet. 2005; 14: 2189–2200.
11. Jiang D, Xiao B, Li X, Chen SRW. Smooth muscle tissues express a major dominant negative splice variant of the type 3 Ca2+ release channel (ryanodine receptor). J Biol Chem. 2003; 278: 4763–4769.
12. Dabertrand F, Morel JL, Sorrentino V, Mironneau J, Mironneau C, Macrez N. Modulation of calcium signaling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells. Cell Calcium. 2006; 40: 11–21.[CrossRef][Medline] [Order article via Infotrieve]
13. Beutner G, Sharma VK, Giovannucci DR, Yule DI, Sheu SS. Identification of ryanodine receptor in rat heart mitochondria. J Biol Chem. 2001; 276: 21482–21488.
14. Cifuentes F, Gonzalez CE, Fiordelisio T, Guerrero G, Lai FA, Hernandez-Cruz A. A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca2+ signaling. Cell Signal. 2001; 13: 353–362.[CrossRef][Medline] [Order article via Infotrieve]
15. Zaidi M, Shankar VS, Tunwell R, Adebanjo OA, Mackrill J, Pazianas M, O’Connell D, Simon BJ, Rifkin BR, Venkitaraman AR, Huang CL-H, Lai FA. A ryanodine receptorlike molecule expressed in the osteoclast plasma membrane functions in extracellular Ca2+ sensing J Clin Invest. 1995; 96: 1582–1590.[Medline] [Order article via Infotrieve]
16. Tunwell REA, Wickenden C, Bertrand BMA, Shevchenko VI, Walsh MB, Allen PD, Lai FA. The human cardiac muscle ryanodine receptor-calcium release channel: identification, primary structure and topological analysis. Biochem J. 1996; 318: 477–487.[Medline] [Order article via Infotrieve]
17. Xu X, Yang D, Ding JH, Wang W, Chu PH, Dalton ND, Wang HY, Bermingham JR Jr, Ye Z, Liu F, Rosenfeld MG, Manley JL, Ross J Jr, Chen J, Xiao RP, Cheng H, Fu XD. ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle. Cell. 2005; 120 (1): 59–72.[CrossRef][Medline] [Order article via Infotrieve]
18. Claycomb WC, Lanson NA, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci U S A. 1998; 95: 2979–2984.
Related Article:
Circ. Res. 2007 100: 874-883.
This article has been cited by other articles:
 |
|
 |
|
  V. Srivastava, M. K. Srivastava, K. Chibani, R. Nilsson, N. Rouhier, M. Melzer, and G. Wingsle Alternative Splicing Studies of the Reactive Oxygen Species Gene Network in Populus Reveal Two Isoforms of High-Isoelectric-Point Superoxide Dismutase Plant Physiology, April 1, 2009; 149(4): 1848 - 1859. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Eisenreich, V. Y. Bogdanov, A. Zakrzewicz, A. Pries, S. Antoniak, W. Poller, H.-P. Schultheiss, and U. Rauch Cdc2-Like Kinases and DNA Topoisomerase I Regulate Alternative Splicing of Tissue Factor in Human Endothelial Cells Circ. Res., March 13, 2009; 104(5): 589 - 599. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||