Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly.
The in vitro interaction of recombinant hnRNP A1 with purified snRNPs and with pre-mRNAs was investigated. We show that protein A1 can stably bind U2 and U4 snRNP but not U1. Oligo-RNAse H cleavage of U2 nucleotides involved in base pairing with the branch site, totally eliminates the A1-U2 interaction. RNase T1 protection and immunoprecipitation experiments demonstrate that recombinant protein A1 specifically binds the 3'-end regions of both beta-globin and Ad-2 introns. However, while on the beta-globin intron only binding to the polypyrimidine tract was observed, on the Ad-2 intron a 32 nt fragment encompassing the branch point and the AG splice-site dinucleotide was bound and protected. Such protection was drastically reduced in the presence of U2 snRNP. Altogether these results indicate that protein A1 can establish a different pattern of association with different pre-mRNAs and support the hypothesis that this protein could play a role in the annealing of U2 to the branch site and hence in the early events of pre-splicing complex assembly.
Buvoli
M
M
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Cobianchi
F
F
Riva
S
S
D000260:Adenoviruses, Human / Q000235:genetics*; D001483:Base Sequence; D005914:Globins / Q000235:genetics*; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D005914:Globins; D016914:Ribonuclease H; D006163:Ribonuclease T1