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Nucleic Acids Research, 1989, Vol. 17, No. 13 5223-5244
© 1989

MOLECULAR BIOLOGY

Purified U5 small nuclear ribonucleoprotein can relieve the inhibition of spliceosome assemblyand spiking by snRNP-free nuclear proteins

Jamal Tazi+, Jamal Temsamani§, Christine Aliber, William Rhead{pi}, Samy Khellil, Guy Cathala, Claude Brunei and Philippe Jeanteur*

UA CNRS 1191, Laboratoire de Biochimie, Centre Val d'Aurelle-Paul Lamarque, 34094 Montpellier Cedex 2 and Laboratoire de Biologie Moleculaire, University des Sciences et Techniques du Languedoc 34060 Montpellier, France

*To whom correspondance to be addressed

Received April 14, 1989. Revised May 31, 1989. Accepted May 31, 1989.

As demonstrated by RNase T1 protection assays at 0°C without ATP, U1 and U5 snRNPs purified by isopycnic centrifugation in cesium chloridebind to the 5' and 3' splice sites of human ß-globin pre-mRNA,respectively. We also devised a saturation-complementation assay and havefound that this purified US snRNP, unlike U1, successfully competes withsnRNP-free fractions of nuclear proteins which inhibit spliceosome assemblyand splicing. Restoration of activity requires intact U5 snRNA andcorrelates with the presence of the 100 Kd intron binding protein (IBP)which we have previously characterized (Tazi et al., 1986, Cell 17,755-766). Our results are compatible with a model in which the recognitionof the 31 splice site by IBP-U5 snRNP is one of the earliest events of thespliceosome assembly. It could organize the structure of the 31 splice siteregion of the human B-globin like pre-mRNAs. However, on the basis ofresults showing that B-globin and major late adenovirus seem to havedifferent requirements with respect to IBP-U5 snRNP, it appears that somepre-mRNAs could have a native structure that necessitates less if at allIBP-U5.

+Present addresses: Institute of Molecular Pathology, 7 Dr Bohrgrasse, A 1030, Vienna, Austria

§Worcester Foundation for Experimental Biology,Shrewsbury, MA 01545

{pi}Pediatrics, University of Lowa Hospital and Clinics Iowa City, IA 52242


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