Four nucleotides are the minimal requirement for RNA recognition by rotavirus non-structural protein NSP3.
The interaction of the group A rotavirus non-structural protein NSP3 (NSP3A) with RNA has been studied in vitro. Using semi-purified NSP3A protein expressed by a recombinant baculovirus and in vitro synthesized RNA, we determined by UV cross-linking and gel retardation assays that NSP3A binds, in a sequence-specific manner, the consensus sequence (AUGUGACC) present on the 3' ends of all group A rotavirus mRNAs. Using short oligoribonucleotides, we established that the minimal RNA sequence required for binding of NSP3A is GACC. Modifications of the UGACC oligonucleotide sequence impaired binding of the protein to the RNA. Furthermore, the recombinant NSP3 protein from rotavirus group C showed specificity for the 3' end consensus sequence (AUGUGGCU) of only group C mRNAs. Sequence analysis of the NSP3 proteins did not reveal significant homologies with other RNA binding proteins, thus the NSP3 proteins of rotaviruses are the prototypes of a new kind of sequence-specific RNA binding protein.
Poncet
D
D
Laboratoire INRA de Virologie et d'Immunologie Moléculaires, Jouy-en-Josas, France.
Laurent
S
S
Cohen
J
J
D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D016384:Consensus Sequence; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; C082740:NSP3 protein, Rotavirus; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins