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Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization.

ABSTRACT: Mutations in RPGRIP1 cause Leber congenital amaurosis. The human and bovine RPGRIP1 undergo alternative splicing. A single murine rpgrip1 transcript has been reported, but distinct expression profiles of RPGRIP1 isoforms exist between species in the retina. To elucidate the heterogeneity of RPGRIP1 isoforms and the degree of functional redundancy among these, we extended the analysis of RPGRIP1 to the region between exons 12 and 14, which undergoes significant alternative splicing.Identification of alternative splice transcripts of murine and human RPGRIP1 was performed by reverse transcription-polymerase chain reaction (RT-PCR). The murine rpgrip1 isoforms were analyzed by immunoblot and immunocytochemistry analysis of murine retinas and transient transfected cultured cells.A novel murine-specific transcript, rpgrip1b was identified. It arises from the extension of exon 13, leading to the premature truncation of rpgrip1 and deletion of its C2 and RID domains. It is predominantly expressed in the retina, where it is more abundant than the transcript(s) encompassing the constitutive exons 12 to 14. Conversely, the human retina lacks rpgrip1b, and the constitutive transcript is the major isoform. The subcellular distribution of rpgrip1b is distinct from its larger isoform, rpgrip1. In the photoreceptor inner segments and cells expressing enhanced green fluorescent protein (EGFP)-rpgrip1b, rpgrip1b is dispersed as punctate foci throughout the perikarya, where it colocalizes with a subpopulation of lysosomes.These data support the RPGR-independent function of the isotype- and species-specific rpgrip1b in lysosome-related processes. The results further strengthen the model of the selective participation of distinct RPGRIP1 isoforms in different subcellular processes and molecular pathogenesis of RPGRIP1-allied diseases.

SUBMITTER: Lu X

PROVIDER: S-EPMC1769349 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization.

Lu Xinrong X Ferreira Paulo A PA

Investigative ophthalmology & visual science 20050601 6

<h4>Purpose</h4>Mutations in RPGRIP1 cause Leber congenital amaurosis. The human and bovine RPGRIP1 undergo alternative splicing. A single murine rpgrip1 transcript has been reported, but distinct expression profiles of RPGRIP1 isoforms exist between species in the retina. To elucidate the heterogeneity of RPGRIP1 isoforms and the degree of functional redundancy among these, we extended the analysis of RPGRIP1 to the region between exons 12 and 14, which undergoes significant alternative splicin ...[more]

PMID: 15914599

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Project description:Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, Itg?V and Pdgfr?, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and cellular distribution in transfected COS7 fibroblasts. We conclude that mesothelin is a marker of activated murine liver myofibroblasts. Mesothelin gene expression and regulation may be critical in liver myofibroblasts functions and fibrosis progression.

Dataset Information

Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization.

Publications

Identification of novel murine- and human-specific RPGRIP1 splice variants with distinct expression profiles and subcellular localization.

Similar Datasets

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