engleski

Optimal conditions for in vitro reconstitution of small nuclear ribonucleoprotein particles

Aim. Optimization of conditions required for the in vitro reconstitution of small nuclear ribonucleoprotein particles (snRNPs) of the U family using different parameters. Method. Individual snRNPs were reconstituted by adding each small nuclear RNA of the "U" family to the snRNP protein pool obtained from S 100 HeLa cytoplasmic extract. The mixture was then immunoprecipitated with anti-trimethylguanosine (aTMG) monoclonal antibody, followed by polyacrylamide gel electrophoresis and autoradiography. Results. U1 required 0.5 nuclear extract equivalents of U1 snRNA, 6 mM MgCl2 and incubation at 30oC for 30 minutes ; U2 required 0.5 nuclear extract equivalents of U2 snRNA, the same conditions as above but without Mg++ ions. U4 was reconstituted by incubating it for 20 minutes at 30oC and for another 10 at 37oC without Mg++ ions. U5 required 2 nuclear extract equivalents of U5 snRNA, 6 mM MgCl2 and incubation for 30 minutes at 30oC. U4/U6 needed 0.5 nuclear extract equivalents of each snRNA, and the same time and temperature as U5. U4/U5/U6 was reconstituted employing 0.5 nuclear extract equivalents of each snRNA and incubation at 30oC for 30 minutes with 6 mM MgCl2. None of the snRNPs required ATP for the reconstitution. Conclusion. The optimal conditions reported in this paper should prove useful in the elucidation of the function of snRNP specific polypeptides and eventual reconstitution of snRNPs active in splicing.

ribonucleoproteins; small nuclear; RNA splicing; RNA; small nuclear

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