Purified U5 small nuclear ribonucleoprotein can relieve the inhibition of spliceosome assembly and splicing by snRNP-free nuclear proteins.
As demonstrated by RNase T1 protection assays at 0 degrees C without ATP, U1 and U5 snRNPs purified by isopycnic centrifugation in cesium chloride bind to the 5' and 3' splice sites of human beta-globin pre-mRNA, respectively. We also devised a saturation-complementation assay and have found that this purified U5 snRNP, unlike U1, successfully competes with snRNP-free fractions of nuclear proteins which inhibit spliceosome assembly and splicing. Restoration of activity requires intact U5 snRNA and correlates with the presence of the 100 Kd intron binding protein (IBP) which we have previously characterized (Tazi et al., 1986, Cell 47, 755-766). Our results are compatible with a model in which the recognition of the 3' splice site by IBP-U5 snRNP is one of the earliest events of the spliceosome assembly. It could organize the structure of the 3' splice site region of the human beta-globin like pre-mRNAs. However, on the basis of results showing that beta-globin and major late adenovirus seem to have different requirements with respect to IBP-U5 snRNP, it appears that some pre-mRNAs could have a native structure that necessitates less if at all IBP-U5.
Tazi
J
J
UA CNRS 1191, Laboratoire de Biochimie, Centre Val d'Aurelle-Paul Lamarque, Montpellier, France.
Temsamani
J
J
Alibert
C
C
Rhead
W
W
Khellil
S
S
Cathala
G
G
Brunel
C
C
Jeanteur
P
P
D002499:Centrifugation, Density Gradient; D005914:Globins / Q000235:genetics; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009687:Nuclear Proteins / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic; D012335:RNA, Ribosomal / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear; D013698:Templates, Genetic; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D005914:Globins