Dynamic exchanges of RNA interactions leading to catalytic core formation in the U12-dependent spliceosome.
Important general insights into the mechanism of pre-mRNA splicing have emerged from studies of the U12-dependent spliceosome. Here, photochemical cross-linking analyses during U12-dependent spliceosome assembly have surprisingly revealed that an upstream 5' exon region is required for establishing two essential catalytic core interactions, U12/U6atac helix Ib and U6atac/5' splice site contacts, but not for U5/5' exon interactions or partial unwinding of U4atac/U6atac. A novel intermediate, representing an alternative pathway for catalytic core formation, is a ternary snRNA complex containing U4atac/U6atac stem II and U12/U6atac helix Ia that forms even without U6atac replacing U11 at the 5' splice site. A powerful oligonucleotide displacement method suggests that the blocked complexes analyzed to deduce the interdependence of these multiple RNA exchanges are authentic intermediates in U12-dependent spliceosome assembly.
Frilander
M J
MJ
Department of Molecular Biophysics, and Biochemistry, Yale University School of Medicine, Howard Hughes Medical Institute, New Haven, CT 06536-0812, USA.
Steitz
J A
JA
D020134:Catalytic Domain / Q000235:genetics*; D005091:Exons / Q000235:genetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics* / Q000378:metabolism*; D017381:Spliceosomes / Q000235:genetics*
D009841:Oligonucleotides; D012333:RNA, Messenger; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C552126:U11-U12 small nuclear ribonucleoprotein