Initial recognition of U12-dependent introns requires both U11/5' splice-site and U12/branchpoint interactions.
We have investigated the formation of prespliceosomal complex A in HeLa nuclear extracts on a splicing substrate containing an AT-AC (U12-type) intron from the P120 gene. Using an RNase H protection assay and specific blocking oligonucleotides, we find that recognition of the 5' splice-site (5'ss) and branchpoint sequence (BPS) elements by U11 and U12 snRNPs, respectively, displays strong cooperativity, requiring both sites in the pre-mRNA substrate for efficient complex formation. Deletion analysis indicates that beside the 5'ss and BPS, no additional elements in the pre-mRNA are necessary for A-complex formation, although 5' exon sequences provide stimulation. Cross-linking studies with pre-mRNAs containing the 5'ss or BPS alone indicate that recognition of the BPS by the U12 snRNP is stimulated at least 20- to 30-fold by the binding of the U11 snRNP to the 5'ss in the same pre-mRNA molecule, whereas recognition of the 5'ss by U11 is stimulated approximately fivefold by the U12/BPS interaction. These results argue that intron recognition in the U12-dependent splicing pathway is carried out by a single U11/U12 di-snRNP complex, suggesting greater rigidity in the intron recognition process than in the major spliceosome.
Frilander
M J
MJ
Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, Howard Hughes Medical Institute, New Haven, Connecticut 06536-0812 USA.
Steitz
J A
JA
D000256:Adenoviridae / Q000235:genetics; D015152:Blotting, Northern; D004305:Dose-Response Relationship, Drug; D019143:Evolution, Molecular; D005363:Ficusin / Q000378:metabolism; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D008957:Models, Genetic; D009841:Oligonucleotides / Q000378:metabolism; D012326:RNA Splicing / Q000502:physiology*; D016914:Ribonuclease H / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D013997:Time Factors
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009841:Oligonucleotides; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C098762:U11 small nuclear ribonucleoprotein, human; C552126:U11-U12 small nuclear ribonucleoprotein; D016914:Ribonuclease H; D005363:Ficusin