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.
Cavaloc
Y
Y
Unité 184 de Biologie Moléculaire et de Génie Génétique de l'INSERM, Faculté de Médecine, Strasbourg, France.
Popielarz
M
M
Fuchs
J P
JP
Gattoni
R
R
Stévenin
J
J
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