Functional implications of novel human acid sphingomyelinase splice variants

Background

Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity.

Methodology/Principal Findings

We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models.

Conclusions/Significance

These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity.     

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.     

Identification and characterization of two splice variants of human diacylglycerol kinase eta

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGK eta (eta 1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGK eta isoform (eta 2, 135 kDa) that shared the same sequence with DGK eta 1 except for a sterile alpha motif (SAM) domain added at the carboxyl terminus. The DGK eta 1 mRNA was ubiquitously distributed in various tissues, whereas the DGK eta 2 mRNA was detected only in testis, kidney, and colon. The expression of DGK eta 2 was suppressed by glucocorticoid in contrast to the marked induction of DGK eta 1. DGK eta 2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (delta 1 and delta 2). Interestingly, DGK eta 1 and DGK eta 2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGK eta 1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGK eta 2 exhibited sustained endosomal association. The experiments using DGK eta mutants suggested that the oligomerization of DGK eta 2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGK eta 2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGK eta gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.     

Linear motifs confer functional diversity onto splice variants

The pre-translational modification of messenger ribonucleic acids (mRNAs) by alternative promoter usage and alternative splicing is an important source of pleiotropy. Despite intensive efforts, our understanding of the functional implications of this dynamically created diversity is still incomplete. Using the available knowledge of interaction modules, particularly within intrinsically disordered regions (IDRs), we analysed the occurrences of protein modules within alternative exons. We find that regions removed or included by pre-translational variation are enriched in linear motifs suggesting that the removal or inclusion of exons containing these interaction modules is an important regulatory mechanism. In particular, we observe that PDZ-, PTB-, SH2- and WW-domain binding motifs are more likely to occur within alternative exons. We also determine that regions removed or included by alternative promoter usage are enriched in IDRs suggesting that protein isoform diversity is tightly coupled to the modulation of IDRs. This study, therefore, demonstrates that short linear motifs are key components for establishing protein diversity between splice variants.     

Functional differences between TRPC4 splice variants.

Functional characterizations of heterologously expressed TRPC4 have revealed diverse regulatory mechanisms and permeation properties. We aimed to clarify whether these differences result from different species and splice variants used for heterologous expression. Like the murine beta splice variant, rat and human TRPC4beta both formed receptor-regulated cation channels when expressed in HEK293 cells. In contrast, human TRPC4alpha was poorly activated by stimulation of an H(1) histamine receptor. This was not due to reduced expression or plasma membrane targeting, because fluorescent TRPC4alpha fusion proteins were correctly inserted in the plasma membrane. Furthermore, currents through both human TRPC4alpha and TRPC4beta had similar current-voltage relationships and single channel conductances. To analyze the assembly of transient receptor potential channel subunits in functional pore complexes in living cells, a fluorescence resonance energy transfer (FRET) approach was used. TRPC4alpha and TRPC4beta homomultimers exhibited robust FRET signals. Furthermore, coexpressed TRPC4alpha and TRPC4beta subunits formed heteromultimers exhibiting comparable FRET signals. To promote variable heteromultimer assemblies, TRPC4alpha/TRPC4beta were coexpressed at different molar ratios. TRPC4beta was inhibited in the presence of TRPC4alpha with a cooperativity higher than 2, indicating a dominant negative effect of TRPC4alpha subunits in heteromultimeric TRPC4 channel complexes. Finally, C-terminal truncation of human TRPC4alpha fully restored the channel activity. Thus, TRPC4beta subunits form a receptor-dependently regulated homomultimeric channel across various species, whereas TRPC4alpha contains a C-terminal autoinhibitory domain that may require additional regulatory mechanisms.     

Alternative splice variants encoding unstable protein domains exist in the human brain

Alternative splicing has been recognized as a major mechanism by which protein diversity is increased without significantly increasing genome size in animals and has crucial medical implications, as many alternative splice variants are known to cause diseases. Despite the importance of knowing what structural changes alternative splicing introduces to the encoded proteins for the consideration of its significance, the problem has not been adequately explored. Therefore, we systematically examined the structures of the proteins encoded by the alternative splice variants in the HUGE protein database derived from long (>4 kb) human brain cDNAs. Limiting our analyses to reliable alternative splice junctions, we found alternative splice junctions to have a slight tendency to avoid the interior of SCOP domains and a strong statistically significant tendency to coincide with SCOP domain boundaries. These findings reflect the occurrence of some alternative splicing events that utilize protein structural units as a cassette. However, 50 cases were identified in which SCOP domains are disrupted in the middle by alternative splicing. In six of the cases, insertions are introduced at the molecular surface, presumably affecting protein functions, while in 11 of the cases alternatively spliced variants were found to encode pairs of stable and unstable proteins. The mRNAs encoding such unstable proteins are much less abundant than those encoding stable proteins and tend not to have corresponding mRNAs in non-primate species. We propose that most unstable proteins encoded by alternative splice variants lack normal functions and are an evolutionary dead-end.     

Functional diversity of microRNA variants in plants

MicroRNAs (miRNAs) are small noncoding RNAs that play a crucial role in plant growth, development, and stress responses by regulating target gene expression. With the development of high-throughput sequencing technology, it has been facilitated to identify new miRNAs, as well as diversity and variability of miRNA variants. miRNA variants share the sequences with other closely related miRNAs and contain length and/or sequence variations at the 5’-, 3’-ends, as well as internal positions. They originate from the same miRNA precursor or from the diversity of members in the same miRNA family. It has been reported that tissue- or condition-specific variation in the relative abundance of different miRNA variants could contribute to differential functions of those in development or stress responses. In addition, the diversity of miRNA variants affects stability, loading efficiency onto AGO, and target selection. In this review, the diversification of miRNA sequences and the evidences of the distinct functional role of miRNA variants will be discussed.     

The N and C termini of the splice variants of the human mitogen-activated protein kinase-interacting kinase Mnk2 determine activity and localization

The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.     

Functional properties of four splice variants of a human pancreatic tandem-pore K+ channel,TALK-1

TALK-1a, originally isolated from human pancreas, is a member of the tandem-pore K⁺ channel family. We identified and characterized three novel splice variants of TALK-1 from human pancreas. The cDNAs of TALK-1b, TALK-1c, and TALK-1d encode putative proteins of 294, 322, and 262 amino acids, respectively. TALK-1a and TALK-1b possessed all four transmembrane segments, whereas TALK-1c and TALK-1d lacked the fourth transmembrane domain because of deletion of exon 5. Northern blot analysis showed that among the 15 tissues examined, TALK-1 was expressed mainly in the pancreas. TALK-1a and TALK-1b, but not TALK-1c and TALK-1d, could be functionally expressed in COS-7 cells. Like TALK-1a, TALK-1b was a K⁺-selective channel that was active at rest. Single-channel openings of TALK-1a and TALK-1b were extremely brief such that the mean open time was <0.2 ms. In symmetrical 150 mM KCl, the apparent single-channel conductances of TALK-1a and TALK-1b were 23 ± 3 and 21 ± 2 pS at –60 mV and 11 ± 2 and 10 ± 2 pS at +60 mV, respectively. TALK-1b whole cell current was inhibited 31% by 1 mM Ba²⁺ and 71% by 1 mM quinidine but was not affected by 1 mM tetraethylammonium, 1 mM Cs⁺, and 100 µM 4-aminopyridine. Similar to TALK-1a, TALK-1b was sensitive to changes in external pH. Acid conditions inhibited and alkaline conditions activated TALK-1a and TALK-1b, with a K_1/2 at pH 7.16 and 7.21, respectively. These results indicate that at least two functional TALK-1 variants are present and may serve as background K⁺ currents in certain cells of the human pancreas. tandem-pore potassium channel; background potassium channel; pancreas; pH     

Cloning and characterization of diacylglycerol kinase iota splice variants in rat brain

Diacylglycerol kinase (DGK) catalyzes phosphorylation of a second messenger diacylglycerol (DG) to phosphatidic acid in cellular signal transduction. Previous studies have revealed that DGK consists of a family of isozymes including our rat clones. In this study we isolated from rat brain cDNA library the cDNA clones for a rat homologue of DGKiota (rDGKiota-1) that contains two zinc finger-like sequences, the highly conserved DGK catalytic domain, a bipartite nuclear localization signal, and four ankyrin repeats at the carboxyl terminus. In addition, we found novel splice variants, which contain either insertion 1 (71 bp) or insertion 2 (19 bp) or both in the carboxyl-terminal portion. Each of the insertions causes a frameshift, and the resultant premature stop codons produce two truncated forms (termed rDGKiota-2 and -iota-3), the former lacking the ankyrin repeats at the carboxyl terminus and the latter lacking a part of the catalytic domain and the ankyrin repeats. Truncation of the carboxyl-terminal portion clearly exerts effects on the detergent solubility and enzymatic activity of the splice variants, although all three variants showed similar cytoplasmic localization in cDNA-transfected cultured neurons despite the continued presence of the nuclear localization signal sequence. Immunoblot analysis using anti-rDGKiota antibody raised against the common amino-terminal portion clearly shows that these rDGKiota variants are indeed expressed in the brain. These results suggest that the carboxyl-terminal truncated forms of rDGKiota-2 and -iota-3 that exhibit reduced enzymatic activities might show a dominant negative effect against the intact rDGKiota-1, and that the modulation of signal transduction by the splice variants may play some roles in the physiologic and/or pathologic conditions of neurons.     

Interaction of human GTP cyclohydrolase I with its splice variants

Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.