Detection and analysis of an alternatively spliced isoform of interleukin-6 mRNA in peripheral blood mononuclear cells.
Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transciptase-polymerase chain reaction (RT-PCR) studies to examine lL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PCR product, a second 0.45-kb product was observed. Cloning and dideoxy sequence analysis of this product revealed evidence for an alternatively spliced lL-6 transcript lacking exon II. Further RT-PCR analysis using forward primers ending at or one base before the exon I donor splice site again yielded both products. Additional primers were designed and successfully used to selectively distinguish the two forms of IL-6 transcript. Both transcripts were prominent in peripheral blood monocytes and lymphocytes, whereas only the 0.64-kb, full-length transcript was prominent in the lL-6-producing 5637 (human bladder carcinoma) cell line. Northern analysis revealed, in addition to the predominant 1.3-kb transcript, several minor transcripts at 1.9 to 4.8 kb that hybridized with the alternatively spliced cDNA probe but not with an exon II probe. Western analysis revealed lL-6 polypeptides of predicted size (26 to 29 kD) in culture medium from PBMC, while showing an immunoreactive band at 17 kD in cell lysates. These findings suggest the existence of an alternatively spliced form of lL-6 mRNA, which would encode for a polypeptide missing the gp130 interactive (signal-transducing) domain contained in exon II while retaining the lL-6 receptor (p80) domain. Such a molecule could in theory function as a natural antagonist of lL-6, as it would be expected to bind to the IL-6 receptor but not lead to signal transduction.
Department of Medicine, University of Tennessee Medical Center at Knoxville 37920, USA.
D P
DP
Kestler
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Agarwal
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J
Cobb
K M
KM
Goldstein
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Hall
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002277:Carcinoma / Q000235:genetics / Q000473:pathology; D017931:DNA Primers; D005091:Exons / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D018123:Receptors, Interleukin / Q000037:antagonists & inhibitors / Q000378:metabolism; D019947:Receptors, Interleukin-6; D016415:Sequence Alignment; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology
D015703:Antigens, CD; D017931:DNA Primers; D015850:Interleukin-6; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6