Exon 1B Research Articles

The exon‐level biomarker SLC39A14 has organ‐confined cancer‐specificity in colorectal cancer

An alternative transcript variant of SLC39A14, caused by pre-mRNA splicing, was recently suggested as a biomarker for colorectal cancer (CRC). In our study, we have validated the cancer-specific splicing pattern of the mutually exclusive exons 4A and 4B in altogether 244 colorectal tissue samples. Exon-specific quantitative RT-PCR analyses across 136 Stage I-IV CRC samples and 44 normal colonic mucosa samples showed complete cancer-specificity, as well as 94% sensitivity of SLC39A14-exon4B relative to SLC39A14-exon4A expression. However, across 20 samples from a range of healthy tissues, 18 expressed the CRC variant. This was true also for ten benign lymph nodes, demonstrating that the cancer-specificity is mainly confined to the colon and rectum. Hence, clinical use of SLC39A14-exon4B as a detection marker for CRC other than in samples taken from the bowel wall is diminished. Prognostic value by detection of metastasis to lymph nodes is also abated, elucidating an important pitfall to biomarker discovery. However, analyses of ten nondysplastic biopsies from patients with active inflammatory bowel disease showed negative results in seven samples and only weakly positive results in three samples, suggesting value of SLC39A14-exon4B as a marker to distinguish CRC from other pathologic conditions of the colon. In conclusion, the SLC39A14-exon4B transcript variant is a CRC biomarker with high sensitivity and organ-confined specificity. Further use of the transcript and its encoded protein isoform should be explored in an organ-confined context.

D469del-COMP Retention in Chondrocytes Stimulates Caspase-Independent Necroptosis

Mutations in the cartilage oligomeric matrix protein gene (COMP) cause pseudoachondroplasia (PSACH). This dysplasia results from the intracellular retention of mutant COMP protein and premature death of growth-plate chondrocytes. Toward better understanding of these underlying mechanisms, we examined D469del-COMP activation of the unfolded protein response and cell death pathways in rat chondrosarcoma cells. Using an inducible expression system, we examined the effects of D469del-COMP retention after 4 days of mRNA expression and then 5 days without inducing agent. Retention of D469del-COMP stimulated Chop (Ddit3) and Gadd34 (Ppp1r15a) and triggered reactivation of protein translation that exacerbated intracellular retention. High levels of Nox4 and endoplasmic reticulum receptor stress-inducible Ero1β generated reactive oxygen species, causing oxidative stress. Increased expression of Gadd genes and presence of γH2AX indicated that DNA damage was occurring. The presence of cleaved apoptosis inducing factor (tAIF) and the absence of activated caspases indicated that retention of D469del-COMP triggers cell death in chondrocytes by necroptosis, a caspase-independent programmed necrosis. Loss of growth-plate chondrocytes by necroptosis was also found in our pseudoachondroplasia mouse model. These results suggest a model in which D469del-COMP expression induces persistent endoplasmic reticulum stress, oxidative stress, and DNA damage, thus priming chondrocytes for necroptosis. We define for the first time the precise mechanisms underlying D469del-COMP pathology in pseudoachondroplasia and suggest that oxidative stress and AIF may be promising therapeutic targets.

Rac1b Increases with Progressive Tau Pathology within Cholinergic Nucleus Basalis Neurons in Alzheimer's Disease

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.

Expression of pro- and anti-angiogenic isoforms of VEGF in the mouse model of oxygen-induced retinopathy

Retinopathy of prematurity (ROP) has become one of the leading causes of blindness and visual loss in children over the last half century. Vascular Endothelial growth Factor (VEGF-A) is the principal stimulator of angiogenesis. Recently, it has been identified that VEGF was differentially spliced from Exons 8 to Exons 8a and 8b to form two families: the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family. This alternate splicing produced VEGFxxxb proteins of the same length as VEGFxxx family, but with different C terminal amino acid sequences. VEGFxxxb appeared to be able to inhibit VEGFxxx-dependent angiogenesis. In our study, we investigated the protein expression course of VEGFxxx and VEGFxxxb by Western-blot in a mouse model of Oxygen-induced Retinopathy (OIR) from postnatal day 1 (P1) to postnatal day 21 (P21). We also analyzed the relative protein expression level of VEGF165b isoform in the OIR mouse model. We found that both VEGFxxx and VEGFxxxb were present in the mouse retina, among which, VEGF164 and VEGF165b appeared to be predominant VEGFxxx and VEGFxxxb isoforms respectively in the mouse retina. We also found that the two family had different expression pattern correlated with neovascularization development and that the relative expression level of VEGF165b isoform switched during the neovascularization development in the OIR mouse model. In OIR group, the protein level of total VEGF isoforms (a mix of VEGF164 and VEGF165b, detected by pan-VEGF antibody) continuously increased and peaked at P17 while VEGF165b continuously decreased from P9 which was well related with the vessel obliteration and neovascularization development in the mouse model of OIR. The neovascularization development correlates with an increase of total VEGF isoforms and the decrease of VEGF165b, indicating that there is a pro-angiogenic VEGF shift. Therefore, anti-angiogenic therapy that could alter the ratio of VEGFxxxb/VEGFxxx may be more effective.

CpG methylation at the USF-binding site mediates cell-specific transcription of human ascorbate transporter SVCT2 exon 1a

SVCT2 (sodium-vitamin C co-transporter 2) is the major transporter mediating vitamin C uptake in most organs. Its expression is driven by two promoters (CpG-poor exon 1a promoter and CpG-rich exon 1b promoter). In the present study, we mapped discrete elements within the proximal CpG-poor promoter responsible for exon 1a transcription. We identified two E boxes for USF (upstream stimulating factor) binding and one Y box for NF-Y (nuclear factor Y) binding. We show further that NF-Y and USF bind to the exon 1a promoter in a co-operative manner, amplifying the binding of each to the promoter, and is absolutely required for the full activity of the exon 1a promoter. The analysis of the CpG site located at the upstream USF-binding site in the promoter showed a strong correlation between expression and demethylation. It was also shown that exon 1a transcription was induced in cell culture treated with the demethylating agent decitabine. The specific methylation of this CpG site impaired both the binding of USF and the formation of the functional NF-Y-USF complex as well as promoter activity, suggesting its importance for cell-specific transcription. Thus CpG methylation at the upstream USF-binding site functions in establishing and maintaining cell-specific transcription from the CpG-poor SVCT2 exon 1a promoter.

Adhesion Protein VSIG1 Is Required for the Proper Differentiation of Glandular Gastric Epithelia

VSIG1, a cell adhesion protein of the immunoglobulin superfamily, is preferentially expressed in stomach, testis, and certain gastric, esophageal and ovarian cancers. Here, we describe the expression patterns of three alternatively spliced isoforms of mouse Vsig1 during pre- and postnatal development of stomach and potential function of Vsig1 in differentiation of gastric epithelia. We show that isoforms Vsig1A and Vsig1B, which differ in the 3′untranslated region, are expressed in the early stages of stomach development. Immunohistochemical analysis revealed that VSIG1 is restricted to the adherens junction of the glandular epithelium. The shorter transcript Vsig1C is restricted to the testis, encodes an N-terminal truncated protein and is presumably regulated by an internal promoter, which is located upstream of exon 1b. To determine whether the 5′ flanking region of exon 1a specifically targets the expression of Vsig1 to stomach epithelia, we generated and analyzed transgenic mice. The 4.8-kb fragment located upstream of exon 1a was sufficient to direct the expression of the reporter gene to the glandular epithelia of transgenic stomach. To determine the role of VSIG1 during the development of stomach epithelia, an X-linked Vsig1 was inactivated in embryonic stem cells (ESCs). Although Vsig1−/Y ESCs were only able to generate low coat color chimeric mice, no male chimeras transmitted the targeted allele to their progeny suggesting that the high contribution of Vsig1−/Y cells leads to the lethality of chimeric embryos. Analysis of chimeric stomachs revealed the differentiation of VSIG1-null cells into squamous epithelia inside the glandular region. These results suggest that VSIG1 is required for the establishment of glandular versus squamous epithelia in the stomach.

Age-related changes in expression of CTL2/SLC44A2 and its isoforms in the mouse inner ear

The membrane glycoprotein CTL2/SLC44A2 is expressed by supporting cells in the inner ear and has been identified as a target of antibodies that may induce auto-immune hearing loss. To determine if CTL2/SLC44A2 also has roles in inner ear development and to distinguish between isoform-specific roles, we assessed age-related changes in expression of CTL2/SLC44A2 isoforms and protein in the developing murine inner ear. We determined that both isoform p1 and isoform p2 (named for the upstream p1 and proximal p2 promoters that control alternate exons 1a and 1b) were robustly expressed as early as E14 and persisted during embryonic development, but after birth the p1 isoform fell to barely detectable levels while isoform p2 levels were maintained. This trend continued and became even more apparent later in post-natal development and remained in mature ears until at least 6 weeks of age. In aged (18mo old) mice, the level of isoform p1 transcripts rose again to levels similar to the p2 isoform like that seen early in development. At the earliest stage examined, CTL2/SLC44A2 protein was expressed in both immature supporting cells and immature sensory cells, but after birth expression in the sensory cells declined in both the utricle and cochlea and by day P1 expression of CTL2/SLC44A2 was restricted to supporting cells. The changes we observed in isoform distribution are indicative of differential developmental roles and age related changes between the two isoforms of CTL2/SLC44A2 in the inner ear.

A wide methodological approach to identify a large duplication in CFTR gene in a CF patient uncharacterised by sequencing analysis

Background PCR-based diagnostic procedures are not able to characterise 6% of CF alleles. Recently, the application of array-CGH and of CFTR mRNA analysis has allowed the identification of new copy number mutations and splicing defects, that account for 2% and 13% of CF alleles, respectively, in the Italian population. Methods Here, we report the characterisation of a large duplication in CFTR gene through different methods: MLPA assay, RT-PCR and high-resolution array-CGH. Results We identified a large duplication, involving exons 6b–16, in a patient heterozygous for F508del mutation. This duplication produces an abnormal transcript with an out of frame addition of 2244 nucleotides and leads to the insertion of 8 amino-acid residues in the protein, followed by a stop codon. Conclusions We propose a wide methodological approach based on MLPA assay, RT-PCR and high-resolution array-CGH to routinely analyse CF patients uncharacterised for one or both CFTR alleles.

Increased complexity of Tmem16a/Anoctamin 1 transcript alternative splicing

BackgroundTMEM16A (Anoctamin 1; ANO1) is an eight transmembrane protein that functions as a calcium-activated chloride channel. TMEM16A in human exhibits alternatively spliced exons (6b, 13 and 15), which confer important roles in the regulation of channel function. Mouse Tmem16a is reported to consist of 25 exons that code for a 956 amino acid protein. In this study our aim was to provide details of mouse Tmem16a genomic structure and to investigate if Tmem16a transcript undergoes alternative splicing to generate channel diversity.ResultsWe identified Tmem16a transcript variants consisting of alternative exons 6b, 10, 13, 14, 15 and 18. Our findings indicate that many of these exons are expressed in various combinations and that these splicing events are mostly conserved between mouse and human. In addition, we confirmed the expression of these exon variants in other mouse tissues. Additional splicing events were identified including a novel conserved exon 13b, tandem splice sites of exon 1 and 21 and two intron retention events.ConclusionOur results suggest that Tmem16a gene is significantly more complex than previously described. The complexity is especially evident in the region spanning exons 6 through 16 where a number of the alternative splicing events are thought to affect calcium sensitivity, voltage dependence and the kinetics of activation and deactivation of this calcium-activated chloride channel. The identification of multiple Tmem16a splice variants suggests that alternative splicing is an exquisite mechanism that operates to diversify TMEM16A channel function in both physiological and pathophysiological conditions.

Regulatory Roles of Heterogeneous Nuclear Ribonucleoprotein M and Nova-1 Protein in Alternative Splicing of Dopamine D2 Receptor Pre-mRNA

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.

Identification of CD3ɛ, CD4, CD8β splice variants of Atlantic salmon

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ɛ, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ɛ is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.

Differentially expressed three non-coding alternate exons at 5′ UTR of regulatory type I beta subunit gene of mouse

Prkar1b gene encodes regulatory type I, beta subunit (RIβ)ofcAMP dependent protein kinase A in mouse. Among the various isoforms ofregulatory and catalytic subunits that comprise mammalian PKA, RIβ subunit is considered to be one of the important subunits for neuronal functions. This is involved in multiple forms of synaptic plasticity, and influences memory and learning by maintaining hippocampal long-term potentiation (LTP). Deficient expression of this gene has been implicated in autoimmune disease systemic lupus erythematosus (SLE). We have identified two novel non-coding exons of the Prkar1b gene (designated as exon 1A and exon 1B), which are spliced to the canonical exon 2 and constitute the 5' untranslated region giving rise to three alternative transcript isoforms. We have also confirmed the expression of the previously known first exon (designated as exon 1C) with known transcript published earlier. The transcripts containing exons 1A, 1B and 1C are differentially regulated during the development and tissue types. In silico study of more than 20kb nucleotide sequence upstream of known translational initiation codon revealed three distinct promoter regions named as PA, PB, and PC upstream of the exon 1A, exon 1B and exon 1C respectively. PB is non-CpG related promoter but PA and PC are CpG related promoters, however all three promoters are TATA less. Further analysis showed that these promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Prkar1b gene.

Molecular characterization of 9p21 deletions shows a minimal common deleted region removing CDKN2A exon 1 and CDKN2B exon 2 in diffuse large b‐cell lymphomas

Diffuse large B cell lymphoma represent the most frequent subtype of non Hodgkin's lymphoma, accounting for 30-40% of cases. Several studies have shown that CDKN2A and CDKN2B deletions are frequent in these lymphomas. These genes encode the P14ARF, CDKN2A, and CDKN2B proteins which play a key role in the control of the G1/S transition of the cell cycle. Using array CGH and a quantitative multiplex PCR method, we have previously identified such deletions in 36% of cases at diagnosis. Using a walking strategy to approach the breakpoints of these deletions we could identify the breakpoints junction of thirteen deletions in 11 patients and of two unbalanced translocation leading to a loss of these genes. A minimal common deleted region of 22.4 kb containing exon 1β of CDKN2A encoding P14ARF and exon 2 of CDKN2B encoding CDKN2B was present in all cases but one. Analysis by quantitative RT PCR showed that the expression level of these genes was decreased in these patients as compared with non deleted cases and that this level was correlated with the deletion status of P14ARF, CDKN2A, and CDKN2B. Analysis of the breakpoint sequences showed that some of them were clustered within a few hundred base-pairs suggesting, even if we failed to identify any clear recombination prone sequences, that in these deletions the rearrangement results from non-random mechanisms.

Melanoma candidate genes CDKN2A/p16/INK4A, p14ARF, and CDK4 sequencing in patients with uveal melanoma with relative high-risk for hereditary cancer predisposition

The reported frequencies of germline mutations in the melanoma candidate genes are low in patients with uveal melanoma (UM). However, the number of families studied is limited and the majority of the published reports used low-sensitivity techniques for mutational screening. Identifying the frequency of alterations in any of the melanoma genes in patients with UM with increased hereditary cancer risk is important for proper counseling of these patients. We studied a total of 47 patients with UM including three with a family history of UM, 18 with a family and/or personal history of cutaneous melanoma (CM), three with early age at diagnosis (<30), 11 with increased risk for a known familial cancer syndrome, and 12 with a second primary tumor. Germline screening for mutations in CDKN2A, p14ARF, and exon 2 of CDK4 was carried out by direct sequencing. We identified a variant (IVS1-69 C>T) of uncertain significance in exon 1b of p14ARF in one of the patients with UM and his mother who also had UM. The variant was neither detected in any of the other patients with UM nor in 146 controls. We did not identify pathogenic mutations in CDKN2A nor exon 2 of CDK4 gene. Our study supports the low frequency of germline mutation of the CM candidate genes in patients with UM with family histories suggestive of a high risk for hereditary cancer. Germline testing for CDKN2A might be reserved for patients with UM with a family history of two or more CM.

Structure and polymorphisms of the 5′ regulatory region of porcine adenylate kinase 3-like 1 gene and effect on trait of meat quality

The porcine adenylate kinase 3-like 1 (AK3L1) gene is considered as a potential candidate gene among the encoded isoforms of adenylate kinase for meat quality based on the functional role of the AK3L1 gene in energy metabolism via high-energy phosphoryl transfer reaction. The genomic position of the AK3L1 gene is located in the QTL region, which codes for traits of meat quality. We characterized the genomic structure of the porcine AK3L1 gene and found polymorphisms in the 5′ regulatory region. Typical transcription factor binding sites, such as the TATA box and initiator sequence, were not found. However, the AK3L1 gene had two different transcription start points with different transcripts from Exon 1a and 1b isoforms within the high G+C content region without changing the translation initiation site. The SNPs around two alternative transcription start points in the porcine AK3L1 gene were significantly associated with the following traits of meat quality: back fat thickness (SNP1, P < 0.01), IMF (SNP5, P < 0.06), muscle pH (SNP4, P < 0.05), meat color (SNP1, P < 0.03), and drip loss (SNP1, 2, 4, and 5, P < 0.04). We suggest that SNPs of the AK3L1 gene can induce variation in meat formation and may be potential markers for the quality of pig meat.

Bilateral (opercular and paracentral lobular) polymicrogyria and neurofibromatosis type 1

Anecdotal cases of polymicrogyria (PMG; a malformation of cortical development consisting of an excessive number of small gyri with abnormal lamination) in patients with neurofibromatosis type 1 (NF1) have been described; however, the cases were unilateral and had negative NF1 genetic testing. We describe an 11-year-old girl with NF1 manifesting as a complex epileptic syndrome, including partial seizures secondarily generalized and status epilepticus, who had in association, bilateral, asymmetrical (opercular and paracentral lobular) PMG. She had a 1-bp deletion (c.1862delC) in exon 12b of the NF1 gene. It is notable that, given the key role played by the NF1 gene product, neurofibromin, in normal brain development, and the relatively high frequency of other brain findings in NF1, there are not more NF1 cases with brain malformations manifesting as PMG.

Regulation of the human ascorbate transporter SVCT2 exon 1b gene by zinc-finger transcription factors

The sodium-dependent vitamin C transporter (SVCT) 2 is crucial for ascorbate uptake in metabolically active and specialized tissues. This study focused on the gene regulation of SVCT2 exon 1b, which is ubiquitously expressed in human and mouse tissues. Although the human SVCT2 exon 1b promoter does not contain a classical TATA box, we found that it does contain a functional initiator that binds Yin Yang-1 (YY1) and interacts with upstream Sp1/Sp3 elements in the proximal promoter region. These elements in turn play a critical role in regulating YY1-mediated transcription of exon 1b. Formation of YY1/Sp complexes on the promoter is required for its optional function. YY1 with Sp1 or Sp3 synergistically enhanced exon 1b promoter activity as well as the endogenous SVCT2 protein expression. Further, in addition to Sp1/Sp3, both EGR-1 and EGR-2 were detected in the protein complexes that bound the three GC boxes bearing overlapping binding sites for EGR/WT1 and Sp1/3. The EGR family factors WT1 and MAZ were found to differentially regulate exon 1b promoter activity. These results show that differential occupancy of transcription factors on the GC-rich consensus sequences in the SVCT2 exon 1b promoter contributes to the regulation of cell and tissue expression of SVCT2.

Species-specific 5′-genomic structure and multiple transcription start sites in the chicken Pax5 gene

Master differentiation transcription factors (MDFs) play decisive roles in cell lineage commitment. Paired box 5 (Pax5) is one of MDFs essential for differentiation of pre-B cells into mature B cells. Here, we analyzed the 5′-genomic structure and transcription of the chicken Pax5 (c Pax5) gene in the chicken mature B cell line, DT40. We showed that the c Pax5 gene has two first exons: exon 1A contains long AG repeats, while exon 1B has high GC contents. The exons 1A and 1B had one and three major transcription start sites, respectively. Semi-quantitative RT-PCR revealed that comparable amounts of mRNA are transcribed from the exons 1A and 1B. Interestingly, the transcription start site of the c Pax5 exon 1A was chicken-specific. In addition, the c Pax5 promoter upstream of the exon 1A had no homology with the human and mouse Pax5 promoters. Thus, the mechanisms regulating transcription of the Pax5 exon 1A might not be conserved among species. Furthermore, we determined the physical structure of the exons 1A, 1B, and 2 in the genome of DT40 cells. Our results will be useful for elucidating mechanisms that control c Pax5 transcription and B cell lineage commitment, which is conserved or not conserved among different species.

Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway

Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10(-10)) and cancers (p = 9.4 × 10(-11)), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear β-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of β-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10(-5)) and cancer samples (p = 5 × 10(-4)). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.

CD5 Expression in B Cells from Patients with Systemic Lupus Erythematosus

The recently recognized importance of B cells in systemic lupus erythematosus (SLE) raises the question as to whether those expressing CD5 predominate over the remaining B lymphocytes in the pathophysiology of this disease. Owing to their B B-cell receptor (BCR) polyspecificity, autoantibody production has been originally ascribed to CD5-positive B1 lymphocytes. Instead, it has since been established that high-affinity autoantibodies derive from CD5-negative B2 cells. Even worse, in the light of current findings, CD5-positive B cells have been considered to play a paradoxical role in preventing, rather than inducing, autoimmunity. In this context, there is evidence that the membrane expression of CD5 is regulated, and, to this end, a genetic mechanism has been described, based on the selection between exon 1A (E1A) and exon 1B (E1B). The full-length protein variant, encoded by E1A-cd5, translocates the phosphatase SHP-1 to the vicinity of the BCR, raises its threshold, and thereby limits the response of autoreactive B cells. In contrast, the truncated variant, encoded by E1B-cd5, remains in the cytoplasm, along with SHP1. Normally, EIB E1B is silenced by methylation and its product degraded in the proteosomes. Hence, a defect in the DNA methyl transfer favors the development of SLE, by preventing the effects of SHP-1.