Amino-terminal Isoform Research Articles

The p63 C-terminus is essential for murine oocyte integrity

The transcription factor p63 mediates distinct cellular responses, primarily regulating epithelial and oocyte biology. In addition to the two amino terminal isoforms, TAp63 and ΔNp63, the 3’-end of p63 mRNA undergoes tissue-specific alternative splicing that leads to several isoforms, including p63α, p63β and p63γ. To investigate in vivo how the different isoforms fulfil distinct functions at the cellular and developmental levels, we developed a mouse model replacing the p63α with p63β by deletion of exon 13 in the Trp63 gene. Here, we report that whereas in two organs physiologically expressing p63α, such as thymus and skin, no abnormalities are detected, total infertility is evident in heterozygous female mice. A sharp reduction in the number of primary oocytes during the first week after birth occurs as a consequence of the enhanced expression of the pro-apoptotic transcriptional targets Puma and Noxa by the tetrameric, constitutively active, TAp63β isoform. Hence, these mice show a condition of ovary dysfunction, resembling human primary ovary insufficiency. Our results show that the p63 C-terminus is essential in TAp63α-expressing primary oocytes to control cell death in vivo, expanding the current understanding of human primary ovarian insufficiency.

Expression of DOCK9 and DOCK11 Analyzed with Commercial Antibodies: Focus on Regulation of Mutually Exclusive First Exon Isoforms.

Dedicators of cytokinesis 9 and 11 (DOCK9 and DOCK11) are members of the dedicator of cytokinesis protein family encoding the guanosine nucleotide exchange factors for Rho GTPases. Together with DOCK10, they constitute the DOCK-D or Zizimin subfamily. Two alternative full-length amino terminal isoforms of DOCK9 are known, which we will call DOCK9.1 and DOCK9.2. In order to investigate the relevance of the presence of the alternative first exon isoforms within this family, and to lay the groundwork for future studies that seek to investigate their potential role as biomarkers of disease, the expression levels of DOCK9 and DOCK11 were measured by qRT-PCR in 26 human tissues and 23 human cell lines, and by Western blot analysis, using commercial antibodies in cell lines. DOCK9.1 and DOCK9.2 were widely distributed. High levels of expression of both isoforms were found in the lungs, placenta, uterus, and thyroid gland. However, only DOCK9.1 was significantly expressed in the neural and hematopoietic tissues. The unique first exon form of DOCK11 was highly expressed in hematopoietic tissues, such as the peripheral blood leukocytes, spleen, thymus, or bone marrow, and in others such as the lungs, placenta, uterus, or thyroid gland. In contrast to tissues, the expression of DOCK9.1 and DOCK9.2 differed from one another and also from total DOCK9 in cell lines, suggesting that the amino terminal isoforms of DOCK9 may be differentially regulated. This study demonstrates the usefulness of antibodies in investigating the regulation of the expression of DOCK9.1, total DOCK9, and DOCK11.

Amino-terminal isoforms of the human glycine transporter GlyT1 exhibit similar pharmacology

Alternative promoter usage and mRNA precursor splicing produce three amino-terminal isoforms of the human glycine transporter type 1 (GlyT1). To enable discovery of pharmacological tools that might distinguish them, each of these isoforms was stably expressed in CHO-K1 cells and clonal isolates were generated by limiting dilution. Glycine uptake assays were validated for two lines for each isoform, one low and one high expressor. The data show a modest trend for lower potency against higher expressing lines. IC50 values for reference GlyT1 inhibitors ALX-5407 (Allelix), (S)-13h (Merck), and SSR504734 (Sanofi-Synthelabo) were similar across isoforms. The greatest variation was observed for ALX-5407, and its IC50 values across isoforms were still within one log unit of each other. Antipsychotics previously shown to be weak inhibitors of GlyT1 likewise had similar potency against all three isoforms. The cell lines validated here are tools for discovering inhibitors that might distinguish among GlyT1 isoforms.

Properties of SEPT9 isoforms and the requirement for GTP binding.

Members of the evolutionarily conserved septin family of genes are emerging as key components of several cellular processes including membrane trafficking, cytokinesis, and cell-cycle control events. SEPT9 has been shown to have a complex genomic architecture, such that up to 15 different isoforms are possible by the shuffling of five alternate amino termini and three alternate carboxy termini. Genomic and transcriptional alterations of SEPT9 have been associated with neoplasia. The present study has used a Sept9-specific antibody to determine the pattern of isoform expression in a range of tumour cell lines. Western blot analysis indicated considerable variation in the relative amounts and isoform content of Sept9. Immunofluorescence studies showed a range of patterns of cytoplasmic localization ranging from mainly particulate to mainly filamentous. Expression constructs were also generated for each amino terminal isoform to investigate the patterns of localization of individual isoforms and the effects on cells of ectopic expression. The present study shows that the epsilon isoform appears filamentous in this overexpression system while the remaining isoforms are particulate and cytoplasmic. Transient transfection of individual constructs into tumour cell lines results in cell-cycle perturbation with a G2/M arrest and dramatic growth suppression, which was greatest in cell lines with the lowest amounts of endogenous Sept9. Similar phenotypic observations were made with GTP-binding mutants of all five N-terminal variants of Sept9. However, dramatic differences were observed in the kinetics of accumulation of wild-type versus mutant septin protein in transfected cells. In conclusion, the present study shows that the expression patterns of Sept9 protein are very varied in a panel of tumour cell lines and the functional studies are consistent with a model of septin function as a component of a molecular scaffold that contributes to diverse cellular functions. Alterations in the levels of Sept9 protein by overexpression of individual isoforms can clearly perturb cellular behaviour and may thus provide a mechanistic explanation for observations of deranged septin expression in neoplasia.

Characterization of crab EcR and RXR homologs and expression during limb regeneration and oocyte maturation

We report here complete coding sequences for the Uca pugilator homologs of the ecdysteroid (UpEcR) and retinoid-X receptors (UpRXR). Library screenings recovered cDNA clones containing a unique amino terminal open-reading frame (A/B domain) for each gene, most similar to insect B1 EcR and USP1/RXR isoforms. Splicing variants in the UpRXR ligand-binding domain were also identified, in a region critical for folding of Drosophila and lepidopteran USP. UpEcR and UpRXR proteins were able to associate, and both are required for binding to an ecdysteroid HRE; these interactions were not hormone-dependent. Ribonuclease protection assays (RPA) were conducted using A/B domain and ‘common’ (C or E) domain probes on RNA isolated from various stages of regenerating limb buds and ovaries. For several of the limb bud and ovarian stages examined, the relative level of A/B domain sequence protected was significantly less than common domain suggesting alternative amino terminal isoforms other than those isolated through cloning. This is the first report of UpEcR and UpRXR transcription during ovarian maturation, implicating the ovary as a potential target for hormonal control in Crustacea.

Evidence for the presence of two amino-terminal isoforms of neurofibromin, a gene product responsible for neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder, primarily affecting cells of neural crest origin, and is characterized by café-au-lait skin spots, multiple neurofibromas, and higher incidence of malignancy. A gene linked to NF1 encodes neurofibromin, an established function of which is to stimulate intrinsic GTPase activity of ras protein. The neurofibromin gene gives rise to multiple transcripts generated by alternative splicing, that encode various neurofibromin isoforms. In this study, we have cloned a cDNA encoding a newly identified species of a putative amino-terminal isoform which lacks a large portion of neurofibromin, including the domain related to GTPase-activating protein. This clone carries the insert of 2.7 kb, coding for a protein of 593 amino acid residues, tentatively termed N-isoform 11, whose amino-terminal 574 residues are identical to those of authentic neurofibromin encoded by the eleven exons located at the 5' portion of the gene. Previously, we cloned a cDNA coding for a similar isoform of 551 amino acid residues, termed N-isoform 10, whose amino-terminal portion is encoded by the first ten exons. The molecular weights of these two deduced N-isoforms are consistent with the values determined by in vitro translation of the mRNA transcribed from each N-isoform cDNA. The presence of the amino-terminal isoform(s) suggests the physiological significance of the amino-terminal portion of neurofibromin.