Nucleic Acids Research, 1989, Vol. 17, No. 12 4817-4828
© 1989
CHEMISTRY |
U2 small nuclear RNP assembly in vitro
Cell Biology Group, Worcester Foundation for Experimental Biology Shrewsbury, MA 01545, USA
*To whom correspondence should be addressed
Received January 19, 1989. Revised May 19, 1989. Accepted May 19, 1989.
Incubation of a SP6-transcribed human U2 RNA precursor molecule in a HeLa cell SlOO fraction resulted in the formation of ribonucleoprotein complexes. In the presence of ATP, the particles that assembled had several properties of native U2 snRNP, including resistance to dissociation in Cs2SO4 gradients, their buoyant density, and pattern of digestion by micrococcal nuclease. These particles also reacted with Sm monoclonal antibody and a human autoantibody with specificity for the U2 snRNP-specific proteins A' and B", but not with antibodies for U1 snRNP-specific proteins. In contrast, the particles that formed in the absence of ATP did not have these properties. ATP analogs with non–hydrolyzable ß-
bonds did not substitute for ATP in U2 snRNP assembly. Additional experiments with a mutant U2 RNA confirmed that nucleotides 154–167 of U2 RNA are required for binding of the U2 snRNP-specific proteins but not of the "Sm" core proteins. Pseudouridine formation, a major post-transcriptional modification of U2 RNA, was enhanced under assembly permissive conditions.
+Present address: Department of Pathology, University of South Carolina Medical School, Columbia, SC 29208, USA
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