Chapter 25 Proteins of Nuclear Ribonucleoprotein Subcomplexes

Abstract

Publisher Summary This chapter describes the isolation of proteins of nuclear ribonucleoprotein (RNP) subcomplexes. Heterogeneous nuclear RNA (hnRNA) and some messenger RNA (mRNA) molecules contain internal base methylation at the N 6 -position of adenosine. The substrate for the processing of hnRNA by cleavage, polyadenylation, capping, methylation, and selection for transport to the cytoplasm is not a naked RNA molecule, but the RNP fibril. The nascent hnRNA in chromatin spreads appears in shortened RNP fibrils still attached to the chromatin template. Ribonucleoprotein complexes containing rapidly labeled RNA have also been isolated from purified nuclei and characterized biochemically. Proteins characteristic of RNP comprise a significant fraction of the total chromatin nonhistone proteins. A number of procedures release RNP from purified nuclei. Nuclear rupture by sonication or lysis with detergents can be used; however, contamination by preribosomal RNP and chromatin fragments may obscure subsequent analysis. High salt lysis combined with DNase treatment eliminate the entrapment of RNP in the chromatin gel giving larger yields than by sonication; however, released DNA-binding proteins that are maintained in solution by high salt, may interact with RNP. Gentle extraction procedures with buffered salt solutions, leaving nuclei intact and avoiding exposure to high salt, should minimize rearrangements of nuclear proteins; however they suffer the disadvantage of being lengthy, resulting in considerable cleavage of hnRNA.