Alternative splicing generates a smaller assortment of CaV2.1 transcripts in cerebellar Purkinje cells than in the cerebellum

2006 ◽  
Vol 24 (2) ◽  
pp. 86-96 ◽  
Author(s):  
Srinivasan Kanumilli ◽  
Elizabeth W. Tringham ◽  
C. Elizabeth Payne ◽  
Jonathan R. B. Dupere ◽  
Kanamarlapudi Venkateswarlu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Calcium Channels ◽  
Purkinje Cells ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Brain Regions ◽  
Rt Pcr ◽  
Brain Neurons ◽  
Cerebellar Purkinje Cells ◽  
P Type

P/Q-type calcium channels control many calcium-driven functions in the brain. The CACNA1A gene encoding the pore-forming CaV2.1 (α1A) subunit of P/Q-type channels undergoes alternative splicing at multiple loci. This results in channel variants with different phenotypes. However, the combinatorial patterns of alternative splice events at two or more loci, and hence the diversity of CaV2.1 transcripts, are incompletely defined for specific brain regions and types of brain neurons. Using RT-PCR and splice variant-specific primers, we have identified multiple CaV2.1 transcript variants defined by different pairs of splice events in the cerebellum of adult rat. We have uncovered new splice variations between exons 28 and 34 (some of which predict a premature stop codon) and a new variation in exon 47 (which predicts a novel extended COOH-terminus). Single cell RT-PCR reveals that each individual cerebellar Purkinje neuron also expresses multiple alternative CaV2.1 transcripts, but the assortment is smaller than in the cerebellum. Two of these variants encode different extended COOH-termini which are not the same as those previously reported in Purkinje cells of the mouse. Our patch-clamp recordings show that calcium channel currents in the soma and dendrites of Purkinje cells are largely inhibited by a concentration of ω-agatoxin IVA selective for P-type over Q-type channels, suggesting that the different transcripts may form phenotypic variants of P-type calcium channels in Purkinje cells. These results expand the known diversity of CaV2.1 transcripts in cerebellar Purkinje cells, and propose the selective expression of distinct assortments of CaV2.1 transcripts in different brain neurons and species.

Identification of Pore-forming Subunit of P-type Calcium Channels: an Antisense Study on Rat Cerebellar Purkinje Cells in Culture

1997 ◽  
Vol 36 (3) ◽  
pp. 405-409 ◽  
Author(s):  
S.E GILLARD ◽  
S.G VOLSEN ◽  
W SMITH ◽  
R.E BEATTIE ◽  
D BLEAKMAN ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Purkinje Cells ◽  
Cells In Culture ◽  
Cerebellar Purkinje Cells ◽  
P Type

P-type calcium channels in the somata and dendrites of adult cerebellar purkinje cells

Neuron ◽  
1992 ◽  
Vol 9 (6) ◽  
pp. 1185-1199 ◽  
Author(s):  
Maria M. Usowicz ◽  
Mutsuyuki Sugimori ◽  
Bruce Cherksey ◽  
Rodolfo Llinás
Keyword(s):  
Calcium Channels ◽  
Purkinje Cells ◽  
Cerebellar Purkinje Cells ◽  
P Type

scholarly journals Courtship and Visual Defects of cacophony Mutants Reveal Functional Complexity of a Calcium-Channel α1 Subunit in Drosophila

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1407-1426 ◽  
Author(s):  
Lee A Smith ◽  
Alexandre A Peixoto ◽  
Elena M Kramer ◽  
Adriana Villella ◽  
Jeffrey C Hall
Keyword(s):  
Alternative Splicing ◽  
Calcium Channels ◽  
Calcium Channel ◽  
Stop Codon ◽  
Biological Functions ◽  
Visual Defects ◽  
Transcript Diversity ◽  
Functional Complexity ◽  
Α1 Subunit ◽  
Subtle Abnormality

Abstract We show by molecular analysis of behavioral and physiological mutants that the Drosophila Dmca1A calcium-channel α1 subunit is encoded by the cacophony (cac) gene and that nightblind-A and lethal(1)L13 mutations are allelic to cac with respect to an expanded array of behavioral and physiological phenotypes associated with this gene. The cacS mutant, which exhibits defects in the patterning of courtship lovesong and a newly revealed but subtle abnormality in visual physiology, is mutated such that a highly conserved phenylalanine (in one of the quasi-homologous intrapolypeptide regions called IIIS6) is replaced by isoleucine. The cacH18 mutant exhibits defects in visual physiology (including complete unresponsiveness to light in certain genetic combinations) and visually mediated behaviors; this mutant (originally nbAH18) has a stop codon in an alternative exon (within the cac ORF), which is differentially expressed in the eye. Analysis ofthe various courtship and visual phenotypes associated with this array ofcac mutants demonstrates that Dmca1A calcium channels mediate multiple, separable biological functions; these correlate in part with transcript diversity generated via alternative splicing.

Abstract 293: Tcf7l2 Expression And Isoform Switching In Mouse Hearts

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
LU XIAO ◽  
Haiqing Bai ◽  
James Boyer ◽  
Bo Ye ◽  
Ning Hou ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Wnt Signaling ◽  
Stop Codon ◽  
Medical Center ◽  
Premature Stop Codon ◽  
Beta Catenin ◽  
Canonical Wnt Signaling ◽  
Cardiovascular Research ◽  
Reading Frame ◽  
Canonical Wnt

Lu Xiao, Haiqing Bai, James Boyer, Bo Ye, Ning Hou, Haodong Xu, and Faqian Li Department of Pathology and Laboratory Medicine and Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA Backgrounds: Canonical Wnt signaling appears to have multiphasic and often antagonistic roles in cardiac development. The molecular mechanism for these opposing actions is not clear. We hypothesized that alternative splicing of TCF7L2, a nuclear interaction partner of beta-catenin is involved in the specificity of canonical Wnt signaling. Methods: RT-PCR were performed on embryonic (E16.5) and neonatal (day 8) hearts with primers spanning the end of first exon and the beginning of last exon and the products were cloned and sequenced. Result: There are totally 18 exons identified so far in TCF7L2. We sequenced 56 clones and 53 clones (29 from day 8) and (24 from E16.5) contained TCF7L2 sequences. No exon 6 or exon 17 was found in TCF7L2 transcripts of mouse hearts. Most clones (more than 80%) from E16.5 and day 8 hearts excluded exon 4. Both E16.5 and day 8 hearts had one clone with exon 9 deletion which does not change reading frame and another with alterations in exon 3 that lead to reading frame shift and premature stop codon. As reported in other organs, there were extensive alternative splicing in the C-terminal exons 14, 15 and 16. The inclusion of exon 14 was more frequently in day 8 (18 of 29, 62%) than in E16.5 (8 of 24, 33%) hearts. The peptide encoded by exon 14 has conserved functional motif. Additionally, this alternative exon usage can change the C-terminus of TCF7L2 to include or exclude the so-called E tail with two binding motifs for C-terminal binding protein. Conclusion: The isoform switch of TCF7L2 occurs in neonatal mouse hearts and may have a role in the terminal differentiation of cardiac myocytes during this period.

scholarly journals Activation of MAP3K DLK and LZK in Purkinje Cells Causes Rapid and Slow Degeneration Depending on Signaling Strength

2020 ◽  
Author(s):  
Yunbo Li ◽  
Erin M Ritchie ◽  
Christopher L. Steinke ◽  
Cai Qi ◽  
Lizhen Chen ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Purkinje Cell ◽  
Purkinje Cells ◽  
Leucine Zipper ◽  
Activity Levels ◽  
Cell Type ◽  
Cell Degeneration ◽  
Mechanistic Basis ◽  
Cerebellar Purkinje Cells ◽  
Jnk Activation

SummaryThe conserved MAP3K Dual leucine zipper kinases can activate JNK via MKK4 or MKK7. Vertebrate DLK and LZK share similar biochemical activities and undergo auto-activation upon increased expression. Depending on cell-type and nature of insults DLK and LZK can induce pro-regenerative, pro-apoptotic or pro-degenerative responses, although the mechanistic basis of their action is not well understood. Here, we investigated these two MAP3Ks in cerebellar Purkinje cells using loss- and gain-of function mouse models. While loss of each or both kinases does not cause discernible defects in Purkinje cells, activating DLK causes rapid death and activating LZK leads to slow degeneration. Each kinase induces JNK activation and caspase-mediated apoptosis independent of each other. Significantly, deleting CELF2, which regulates alternative splicing of Mkk7, strongly attenuates Purkinje cell degeneration induced by activation of LZK, but not DLK. Thus, controlling the activity levels of DLK and LZK is critical for neuronal survival and health.

An Autosomal-Recessive GFI1B Mutation Defines the Splice Isoform p37 As Essential for Biogenesis of Functional Human Platelets, but Dispensable for Erythropoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2644-2644
Author(s):  
Harald Schulze ◽  
Axel Schlagenhauf ◽  
Georgi Manukjan ◽  
Christine Beham-Schmid ◽  
Oliver Andres ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Growth Factor ◽  
Autosomal Recessive ◽  
Stop Codon ◽  
Zinc Fingers ◽  
Premature Stop Codon ◽  
Index Patient ◽  
Homozygous Mutation ◽  
Recessive Trait ◽  
Erythroid Progenitor

Abstract Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner. We report on a Chechen family from eastern Georgia whose affected family members all present with severe, life-threatening bleeding diathesis. The female index patient had recurrent hematomata and multiple petechiae since childhood. Both of her children (age 9 and 7) present with very low platelet counts (below 45/nL) and a similar cutaneous bleeding pattern like her mother. The brother also had thrombocytopenia and died at age 33 in reponse to a spontaneous cerebral hemorrhage. In contrast, the index patient's husband, her parents and the children of the deceased brother were clinically unaffected. Blood smears of affected patients showed macrothrombocytopenia with reduced May-Grünwald-Giema staining and decreased staining for alpha-granule markers von Willebrand factor (vWF) and P-selectin (CD62P). Platelet function testing revealed reduced responses to ADP, collagen, TRAP-6 and arachidonic acid. White and red blood cell parameters were overall normal in the index patient and the two affected children. We analyzed DNA from the index patient by targeted next generation sequencing for 59 genes relevant for platelet formation or function. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), which was confirmed by Sanger sequencing and is expected to cause a premature stop-codon. The homozygous mutation co-segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, suggesting a local founder variant and an unexpected autosomal-recessive trait. Bone marrow analysis showed unaffected myeloid and erythroid cells, but dysplastic micromegakaryocytes with increased CD34 staining. Peripheral blood platelets were also positive for CD34. We performed quantitative real-time PCR of platelet RNA and found residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of the p32 variant. The p37 transcript was markedly reduced in context with an increased p32/p37 ratio compared to controls. Our findings indicate that the mutated transcript was not completely degraded by nonsense-mediated decay, but mostly subjected to alternative splicing skipping the mutated exon 9. Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and the production of functional platelets. While previous mutations affect both isoforms, the insertion variant presented here, results in a premature stop-codon and affects only the p37 isoform due to alternative splicing. This splice variant defines an important node at the megakaryocytic-erythroid progenitor stage and we conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B. Disclosures No relevant conflicts of interest to declare.

scholarly journals The neuronal splicing factor Nova controls alternative splicing in N-type and P-type CaV2 calcium channels

Channels ◽  
2010 ◽  
Vol 4 (6) ◽  
pp. 483-489 ◽  
Author(s):  
Summer E. Allen ◽  
Robert B. Darnell ◽  
Diane Lipscombe
Keyword(s):  
Alternative Splicing ◽  
Calcium Channels ◽  
Splicing Factor ◽  
P Type

scholarly journals Alternative REST Splicing Underappreciated

2017 ◽  
Author(s):  
Guo-Lin Chen ◽  
Gregory M. Miller
Keyword(s):  
Alternative Splicing ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Protein Isoforms ◽  
Multiple Protein ◽  
Mrna Variants ◽  
Cellular Context ◽  
Active Transcription ◽  
Context Dependent ◽  
Ageing Brain

As a major orchestrator of the cellular epigenome, the repressor element-1 silencing transcription factor (REST) can either repress or activate thousands of genes depending on cellular context, suggesting a highly context-dependent REST function tuned by environmental cues. While REST shows cell-type non-selective active transcription1, an N-terminal REST4 isoform caused by alternative splicing – inclusion of an extra exon (N3c) which introduces a premature stop codon – has been implicated in neurogenesis and tumorigenesis2-5. Recently, in line with established epigenetic regulation of pre-mRNA splicing6,7, we demonstrated that REST undergoes extensive, context-dependent alternative splicing which results in the formation of a large number of mRNA variants predictive of multiple protein isoforms8. Supported by that immunoblotting/-staining with different anti-REST antibodies yield inconsistent results, alternative splicing allows production of various structurally and functionally different REST protein isoforms in response to shifting physiological requirements, providing a reasonable explanation for the diverse, highly context-dependent REST function. However, REST isoforms might be differentially assayed or manipulated, leading to data misinterpretation and controversial findings. For example, in contrast to the proposed neurotoxicity of elevated nuclear REST in ischemia9 and Huntington’s disease10,11, Lu et al. recently reported decreased nuclear REST in Alzheimer’s disease and neuroprotection of REST in ageing brain12. Unfortunately, alternative REST splicing was largely neglected by Lu et al., making it necessary for a reevaluation of their findings.

scholarly journals SO094HUMAN URINE-DERIVED RENAL EPITHELIAL CELLS PROVIDE INSIGHTS INTO THE PATHOGENICITY OF PKHD1 VARIANTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Elisa Molinari ◽  
Shalabh Srivastava ◽  
Becky Dewhurst ◽  
John Sayer
Keyword(s):  
Epithelial Cells ◽  
Stop Codon ◽  
Rna Extraction ◽  
Premature Stop Codon ◽  
Missense Variant ◽  
Rt Pcr ◽  
Mrna Isoforms ◽  
Renal Epithelial Cells ◽  
Frame Shift ◽  
Synonymous Variant

Abstract Background and Aims A 26 year old woman was referred for the investigation of polycystic kidney disease. There was no family history of renal disease and no obvious extra-renal manifestations. A GEMINI ciliopathy gene panel was performed which identified two heterozygous sequence changes (segregating from each parent) in PKHD1: a missense variant c.7964A>C, p.(His2655Pro), listed as a pathogenic variant in association with ARPKD on the Human Gene Mutation Database and a synonymous variant c.6900C>T, p.(Asn2300Asn). The pathogenicity of the synonymous PKHD1 variant is not clear. It is reported as a variant of unknown significance on ClinVar and has a low population frequency in the gnomAD cohort. The altered nucleotide is weakly conserved, but the same heterozygous change has been previously reported in an individual prenatally presenting with multicystic kidneys together with a missense variant on the other allele. We aim to confirm the pathogenicity of this variant in our patient, analysing its effects on the splicing of PKHD1 mRNA. Method PKHD1 is expressed at low levels in the leukocytes, therefore it can be difficult to analyse the splicing of PKHD1, using routine methods that involve RNA extraction from the blood. Urine-derived renal epithelial cells were isolated and cultured from two wild type individuals and from the patient and total RNA was extracted from these cells. RT-PCR and RT-qPCR were carried out to analyse the effects of the synonymous variant on the splicing of the PKHD1 gene in renal epithelial cells. Results RT-PCR revealed that PKHD1 is alternatively spliced both in the controls and in the patient and Sanger sequencing following T-cloning of PCR products revealed that both controls and the patient express an in-frame transcript and a shorter transcript with a 47 nucleotide loss in the exon 43 of PKHD1, that leads to a frame-shift and to the formation of a premature stop codon. We hypothesized that, although both mRNA isoforms are expressed in controls as well as in the patient URECs, the variant p.(Asn2300Asn) may shift the expression ratio between the two transcript isoforms in favour of the shorter, out-of-frame transcript. We designed and tested two sets of transcript-specific primers and performed a SYBR-green based RT-qPCR on controls and patient URECs cDNA. RT-qPCR, revealed that PKHD1 is expressed at lower levels in patient URECs, compared to controls. Specifically, the in-frame PKHD1 isoform is expressed at lower levels in patient URECs, compared to controls, whereas the levels of the shorter transcript leading to a frame-shift are higher in patient cells. Conclusion Using urine-derived renal epithelial cells as a source of kidney-specific RNA, we confirmed the pathogenicity of the PKHD1 synonymous variant p.(Asn2300Asn), which leads to an increased expression of an out-of-frame PKHD1 transcript, predicted to result in a truncated protein and expressed at lower levels also in control cells. The significance of this isoform in control renal epithelial cells is unclear.

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