Molecular cloning and characterization of an endogenous antisense transcript of Nphs1

• Published in: Genomics (San Diego, Calif.) Vol. 83; no. 6; pp. 1134 - 1140
• Main Authors: IHALMO, Pekka, RINTA-VALKAMA, Johanna, MAI, Petra, ASTRÖM, Eva, PALMEN, Tuula, THU TRANG PHAM, FLOSS, Thomas, HOLTHÖFER, Harry
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier 01.06.2004
• Subjects: Animals; Base Sequence; Biological and medical sciences; Blotting, Northern; Brain - metabolism; Cloning, Molecular; DNA, Complementary - genetics; Exons - genetics; Expressed Sequence Tags; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes. Genome; Genetics of eukaryotes. Biological and molecular evolution; Genome; Introns - genetics; Kidney - metabolism; Kidney Glomerulus - metabolism; Lymph Nodes - metabolism; Membrane Proteins; Mice; Mice, Mutant Strains; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Pancreas - metabolism; Proteins - genetics; Proteins - metabolism; RNA, Antisense - genetics; RNA, Antisense - metabolism; RNA, Messenger - analysis; RNA, Messenger - genetics; RNA, Messenger - metabolism; Thymus Gland - metabolism; Knockout mice; Genomics; Rodentia; Antisense RNA; Characterization; Vertebrata; Nephrin; Mammalia; Endogenous; Mouse; RT-PCR; Nphs1; Molecular cloning; Reverse transcription polymerase chain reaction
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1016/j.ygeno.2004.01.001

Mutations of NPHS1, the gene encoding the kidney glomerular filtration barrier protein nephrin, cause congenital nephrotic syndrome of the Finnish type. Nephrin is a component of the interpodocyte-spanning slit diaphragm: it mediates outside-in signaling and forms a nexus for homo- and heterotypic molecular interactions. When studying the nephrin-deficient mouse line generated by random insertional mutagenesis we unexpectedly discovered an endogenous antisense transcript originating from the nephrin-encoding locus. Further evidence of the antisense transcript (Nphs1as) was obtained by searching for Nphs1-like expressed sequence tags. Surprisingly, one clone showed exact complementarity in the antisense orientation. Nphs1as is expressed in the brain, thymus, and peripheral lymph nodes as well as in the embryonic stem cells. However, the mesenteric lymph nodes and the main sites of nephrin expression, the kidney and pancreas, were negative. Nphs1as is a continuous, polyadenylated mRNA that spans Nphs1 exons from 7 to 12 in the reverse orientation. The relative amounts of sense and antisense mRNAs as well as nephrin protein were determined by semiquantitative RT-PCR and immunoblotting, respectively, in various mouse tissues. These results suggest that Nphs1as may be important for the regulation of the appropriate tissue- and cell-type-specific expression of nephrin.