Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons.     

Alternative splicing of the FMR1 gene in human fetal brain neurons

The alternative splicing expression of the FMR1 gene was reported in several human and mouse tissues. Five regions of FMR1 gene can be alternatively spliced, but the combination of them has not been investigated fully. We reported here the analysis of alternative splicing pattern of the FMR1 gene in cultured fetal human neurons, using a RT-PCR and cloning strategy. Eleven splicing types were cloned and different isoforms were not equally represented. The dominant isoform represents nearly 40%, and the other isoforms were relatively rare. One isoform has a different carboxyl-terminus. Most of the alternative spliced regions appear hydrophilic; thus, they may locate on the surface of the FMR1 protein. © 1996 Wiley-Liss, Inc.     

Alternative RNA splicing in the nervous system

Tissue-specific alternative splicing profoundly effects animal physiology, development and disease, and this is nowhere more evident than in the nervous system. Alternative splicing is a versatile form of genetic control whereby a common pre-mRNA is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. In the nervous system, thousands of alternatively spliced mRNAs are translated into their protein counterparts where specific isoforms play roles in learning and memory, neuronal cell recognition, neurotransmission, ion channel function, and receptor specificity. The essential nature of this process is underscored by the finding that its misregulation is a common characteristic of human disease. This review highlights the current views of the biological phenomenon of alternative splicing, and describes evidence for its intricate underlying biochemical mechanisms. The roles of RNA binding proteins and their tissue-specific properties are discussed. Why does alternative splicing occur in cosmic proportions in the nervous system? How does it affect integrated cellular functions? How are region-specific, cell-specific and developmental differences in splicing directed? How are the control mechanisms that operate in the nervous system distinct from those of other tissues? Although there are many unanswered questions, substantial progress has been made in showing that alternative splicing is of major importance in generating proteomic diversity, and in modulating protein activities in a temporal and spatial manner. The relevance of alternative splicing to diseases of the nervous system is also discussed.     

Distribution of fibronectin isoforms in human renal disease

Fibronectin (FN) is an extracellular matrix component which appears in different isoforms, due to alternative mRNA splicing of the ED-A, ED-B, and IIICS regions, and subsequent post-translational modifications. The FN isoforms, some of which occur specifically during fetal development and in fibrogenic diseases, have been reported to play a role in various biological functions, such as regulation of the matrix assembly, adhesion, and proliferation. The contribution of these FN isoforms to the pathogenesis of chronic renal diseases, which are also fibrogenic disorders, is not well known. This study therefore examined the distribution of FN isoforms in renal diseases by immunohistochemistry, with a panel of isoform-specific monoclonal antibodies (MAbs), applied to 63 abnormal renal biopsies and ten normal controls. Normal kidneys contained total FN (MAb IST4) both in the mesangial and in the interstitial extracellular matrix (ECM), but only traces of ED-A-positive FN (MAb IST9), and no ED-B-positive FN (MAb BC1) or oncofetal FN (MAb FDC6) was found in normal renal tissue. All patients with renal disease demonstrated increased total FN staining of the interstitium and the mesangium. Periglomerular fibrotic lesions and fibrous crescents showed massive accumulation of total FN, whereas the amount of total FN in the ECM of obsolescent glomeruli was decreased, compared with that in normal mesangial ECM. Oncofetal (FDC6), EDB-negative (MAb IST6), ED-A-positive, and ED-B-positive FN isoforms were found in glomerular ECM accumulations and in fibrous crescents. Tubulointerstitial fibrotic lesions predominantly contained the ED-A-positive FN isoform, whereas in globally sclerotic glomeruli, predominantly ED-B-positive FN was observed. The expression of FN isoforms was similar in all renal diseases studied. These results show that in various renal diseases, oncofetal (FDC6) FN and ED-A- and ED-B-positive isoforms of FN accumulate at locations of chronic lesions, independently of the aetiology of the disease. The deposition of these isoforms in human renal tissue may play a role in the modulation of the immune response by attracting monocytes and lymphocytes to the injured kidney. Furthermore, because the ED-B-positive FN isoform is highly susceptible to proteolytic degradation, its accumulation may play a role in scar formation and tissue repair. ED-B-positive FN forms a temporary scaffold supporting the cells, which can easily be cleared by proteolytic degradation once new tissue has been produced at the site of injury.     

Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation

The formation of base-pairing between the branch-site (BS) sequence and the U2 snRNP is an important step in mRNA splicing. We developed a new algorithm to identify both the BS sequence and the polypyrimidine tract (PPT) and validated its predictions experimentally. To assess BS conservation between human and mouse, we assembled and analyzed 46 812 and 242 constitutively and alternatively spliced orthologs of human-mouse intron pairs, respectively. Combinations of BSs and PPTs can be found in most of the constitutive and alternative introns. The average distance between the BS and the 3' splice site (3'ss) is 33-34 nt. Acceptor-like AG dinucleotides that resided between the predicted BS and the 3'ss were found to appear mostly within 5 nt, but not more than 19 nt, downstream of the BS. However, although 32% of homologous alternatively spliced BS sequences were fully conserved between human and mouse, only a small fraction (3%) of homologous constitutive counterparts was fully conserved. This indicates that the full sequence of the BS is under weak purifying selection in constitutively spliced introns and further strengthens the view that the BS sequence is just one of several factors determining the ability of the splicing machinery to identify the BS location. Mutations in the putative BS revealed a shift from constitutive to alternative splicing, and it also controls the inclusion/skipping ratio in alternative splicing. This suggests a role for BS sequences in regulated splicing.     

Intronic sequences flanking alternatively spliced exons are conserved between human and mouse

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.     

atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes

SR proteins are nuclear phosphoproteins with a characteristic Ser/Arg-rich domain and one or two RNA recognition motifs. They are highly conserved in animals and plants and play important roles in spliceosome assembly and alternative splicing regulation. We have now isolated and partially sequenced a plant protein, which crossreacts with antibodies to human SR proteins. The sequence of the corresponding cDNA and genomic clones from Arabidopsis revealed a protein, atSRp30, with strong similarity to the human SR protein SF2/ASF and to atSRp34/SR1, a previously identified SR protein, indicating that plants possess two SF2/ASF-like proteins. atSRp30 expresses alternatively spliced mRNA isoforms that are expressed differentially in various organs and during development. Overexpression of atSRp30 via a strong constitutive promoter resulted in changes in alternative splicing of several endogenous plant genes, including atSRp30 itself. Interestingly, atSRp30 overexpression resulted in a pronounced down-regulation of endogenous mRNA encoding full-length atSRp34/SR1 protein. Transgenic plants overexpressing atSRp30 showed morphological and developmental changes affecting mostly developmental phase transitions. atSRp30- and atSRp34/SR1-promoter–GUS constructs exhibited complementary expression patterns during early seedling development and root formation, with overlapping expression in floral tissues. The results of the structural and expression analyses of both genes suggest that atSRp34/SR1 acts as a general splicing factor, whereas atSRp30 functions as a specific splicing modulator.     

Urinary oncofetal ED-A fibronectin correlates with poor prognosis in patients with bladder cancer

The extracellular matrix protein fibronectin (FN) contributes to the structural integrity of tissues as well as the adhesive and migratory functions of cells. While FN is abundantly expressed in adult tissues, the expression of several alternatively spliced FN isoforms is restricted to embryonic development, tissue remodeling and cancer. These FN isoforms, designated ED-A and ED-B, are frequently expressed by cancer cells, tumor-associated fibroblasts and newly forming blood vessels. Using a highly sensitive collagen-based indirect ELISA, we evaluated the correlation of urinary ED-A and ED-B at time of cystectomy with overall survival in patients with high-grade bladder cancer (BCa). Detectable levels of total FN as well as ED-A and ED-B were found in urine from 85, 73 and 51 % of BCa patients, respectively. The presence of urinary ED-A was a significant independent predictor of 2-year overall survival (OS) after adjusting for age, tumor stage, lymph node stage, and urinary creatinine by multivariable Logistic Regression (p = 0.029, OR = 4.26, 95 % CI 1.16–15.71) and improved accuracy by 3.6 %. Furthermore, detection of ED-A in the urine was a significant discriminator of survival specifically in BCa patients with negative lymph node status (Log-Rank, p = 0.006; HR = 5.78, 95 % CI 1.39–24.13). Lastly, multivariable Cox proportional hazards analysis revealed that urinary ED-A was an independent prognostic indicator of 5-year OS rate for patients with BCa (p = 0.04, HR = 2.20, 95 % CI 1.04–4.69). Together, these data suggest that cancer-derived, alternatively spliced FN isoforms can act as prognostic indicators and that additional studies are warranted to assess the clinical utility of ED-A in BCa.     

Changes in the pattern of fibronectin mRNA alternative splicing in acute experimental mesangioproliferative nephritis

Fibronectins (FN) regulate cell migration, proliferation, and matrix formation during tissue injury. In humans, up to 20 different FN isoforms are generated by alternative splicing in three regions called EIIIA, EIIIB, and V, which have been implicated in the process undergoing wound healing and embryonic development. Specifically, EIIIA- and EIIIB-containing isoforms have been implicated in the regulation of cell proliferation and migration, whereas FN isoforms containing the full-length V region (named V120) are ligands to the VLA-4 integrin. To study the changes in the expression of FN isoforms in the anti-Thy-1 nephritis, an acute and self-resolutive model of mesangioproliferative nephritis, we analyzed the FN splicing patterns by means of ribonuclease protection assays. At Day 7 after anti-Thy-1 monoclonal injection, time of the maximal matrix expansion and glomerular hypercellularity, EIIIA+, EIIIB-, and V120 FN mRNA isoforms were increased. In accordance with the mRNA studies, FN proteins, including the EIIIA and V120 regions, increased in the mesangium of nephritic rats, as assayed by immunohistochemistry. Coinciding with the EIIIA and V120 isoforms up-regulation, there was an increase in mesangial cell proliferation and in the number of VLA-4+ infiltrating cells. At Day 14, in parallel with a remission of the above-mentioned changes, there was a decline in the mRNA and protein FN isoforms increased in the previous phase. The marked and reversible changes in the pattern of FN isoforms and their temporal association with other indicators of glomerular injury suggest that certain FN isotypes are important and coordinated components of the mechanisms attempting to reverse glomerular damage.