Lipopolysaccharide-induced expression of multiple alternatively spliced MEFV transcripts in human synovial fibroblasts: a prominent splice isoform lacks the C-terminal domain that is highly mutated in familial Mediterranean fever.
OBJECTIVE
To investigate the expression of the familial Mediterranean fever (FMF) gene (MEFV) in human synovial fibroblasts.


METHODS
MEFV messenger RNA in synovial fibroblasts, chondrocytes, and peripheral blood leukocytes (PBLs) was analyzed by semiquantitative and real-time polymerase chain reaction and ribonuclease protection assay. The subcellular localization of pyrin, the MEFV product, was determined in transfected synovial fibroblasts and HeLa cells with plasmids encoding pyrin isoforms. Native pyrin was detected with an antipyrin antibody.


RESULTS
MEFV was expressed in synovial fibroblasts, but not in chondrocytes. Four alternatively spliced transcripts were identified: an extension of exon 8 (exon 8ext) resulting in a frameshift that predicts a truncated protein lacking exons 9 and 10, the addition of an exon (exon 4a) predicting a truncated protein at exon 5, the in-frame substitution of exon 2a for exon 2, and the previously described removal of exon 2 (exon 2Delta). Exon 8ext transcripts represented 27% of the total message population in synovial fibroblasts. All other alternatively spliced transcripts were rare. Consensus and alternatively spliced transcripts were induced by lipopolysaccharide in synovial fibroblasts and PBLs. In transfected cells, the proteins encoded by all highly expressed splice forms were cytoplasmic. In contrast, native pyrin was predominantly nuclear in synovial fibroblasts, neutrophils, and dendritic cells, but was cytoplasmic in monocytes.


CONCLUSIONS
Several MEFV transcripts are expressed and inducible in synovial fibroblasts. A prominent isoform lacks the C-terminal domain that contains the majority of mutations found in patients with FMF. While recombinant forms of all major pyrin isoforms are cytoplasmic, native pyrin is nuclear in several cell types. Thus, mechanisms in addition to splicing patterns must control pyrin's subcellular distribution.
Diaz
Arturo
A
Department of Cell and Developmental Biology, University of Michigan Medical School, 1301 Catherine Street, Ann Arbor, MI 48109, USA.
Hu
Chunbo
C
Kastner
Daniel L
DL
Schaner
Philip
P
Reginato
Anthony M
AM
Richards
Neil
N
Gumucio
Deborah L
DL
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003199:Computer Systems; D003598:Cytoskeletal Proteins; D005091:Exons / Q000235:genetics; D010505:Familial Mediterranean Fever / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D000071198:Pyrin; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D014018:Tissue Distribution
D003598:Cytoskeletal Proteins; D008070:Lipopolysaccharides; C000604192:MEFV protein, human; D020033:Protein Isoforms; D011506:Proteins; D000071198:Pyrin; D012333:RNA, Messenger; D011994:Recombinant Proteins