Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor of the serine/arginine protein family.
By adopting a monoclonal antibody approach, we have identified a novel splicing factor of 35 kDa which we have termed 9G8. The isolation and characterization of cDNA clones indicate that 9G8 is a novel member of the serine/arginine (SR) splicing factor family because it includes an N-terminal RNA binding domain (RBD) and a C-terminal SR domain. The RNA binding domain of 9G8 is highly homologous to those of the SRp20 and RBP1 factors (79-71% identity), but the homology is less pronounced in the cases of SF2/ASF and SC35/PR264 (45-37% identity). Compared with the other SR splicing factors, 9G8 presents some specific sequence features because it contains an RRSRSXSX consensus sequence repeated six times in the SR domain, and a CCHC motif in its median region, similar to the zinc knuckle found in the SLU7 splicing factor in yeast. Complete immunodepletion of 9G8 from a nuclear extract, which is accompanied by a substantial depletion of other SR factors, results in a loss of splicing activity. We show that a recombinant 9G8 protein, expressed using a baculovirus vector and excluding other SR factors, rescues the splicing activity of a 9G8-depleted nuclear extract and an S100 cytoplasmic fraction. This indicates that 9G8 plays a crucial role in splicing, similar to that of the other SR splicing factors. This similarity was confirmed by the fact that purified human SC35 also rescues the 9G8-depleted extract. The identification of the 9G8 factor enlarges the essential family of SR splicing factors, whose members have also been proposed to play key roles in alternative splicing.
Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
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Y
Cavaloc
M
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Popielarz
J P
JP
Fuchs
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R
Gattoni
J
J
Stévenin
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001120:Arginine; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D014176:Protein Biosynthesis; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D016415:Sequence Alignment; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012694:Serine; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D001120:Arginine