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Nucleic Acids Research, 1981, Vol. 9, No. 4 815-830
© 1981

MOLECULAR BIOLOGY

Purification and characterization of a simple ribonucleoprotem particle containing small nucleoplasmic RNAs (snRNP) as a subset of RNP containing heterogenous nuclear RNA (hnRNP) from HeLa cells

Claude Brunel, Joannes Sri Widada, Marie-Noelle Lelay, Philippe Jeanteur and Jean-Pierre Liautard

Laboratoire de Biochimie, Centre Paul Lamarque B.P.No 5054, Montpellier Cedex, and Laboratoire de Biologie Moléculaire, Université des Sciences et Techniques du Languedoc Place Eugéne Bataillon, 34060 Montpellier Cedex, France

Correspondence should be addressed to : Laboratoire de Biochimie, Centre Paul Lamarque, B.P. N° 5054, 34033 MONTPELLIER CEDEX - FRANCE -

Received December 15, 1980. A ribonucleoprotein complex whose RNA complement consists exclusively of small nuclear RNA species (snRNA) has been purified from particles containing heterogenous nuclear RNA (hnRNP) from HeLa cells. This was accomplished by taking advantage of their ability to band at a density of about 1.43 g/cm3 in plain cesium chloride as well as in cesium chloride gradients containing 0.5% sarkosyl without prior aldehyde fixation. After these two steps of equilibrium density centrifugation, these snRNPs were still largely contaminated by free proteins (and especially phosphoproteins). A final step of purification by velocity sedimentation in a sucrose gradient containing 0.5 M cesium chloride and 0.5% sarkosyl was efficient in completely eliminating all free proteins. U1, U2, U4, U5 and U6 species according to the nomenclature of Lerner et al. (Nature, (1980) 283, 220–224) were found in these purified snRNPs, while a significant part U6 and a small amount of U2 were found in the bottom fraction. 5S species behaved entirely as free RNA and is presumably a contaminant of cyteplasmic origin. Electrophoresis of proteins from snRNP labeled in vivo with (35S) methionine, revealed four bands with migrations corresponding to molecular weights ranging between 10,000 and 14,000 daltons.


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