snRNPs from Mouse Teratocarcinoma Cells Reacting with Polyclonal anti-Sm and anti-m^{2, 2, 7}₃G Antibodies and Biochemical Characterization of the snRNPs

Abstract

Highly purified Sm small nuclear ribonucleoproteins (snRNPs) in the nuclear extract of embryoid bodies and F9 cell extract that contained small nuclear RNAs (snRNAs) U1a, U1b, U2, U4, U5, U6 and X (about 90 nucleotides) were recovered from anti-Sm antibody column. Sm snRNPs were also tried to isolate immunochemically from chromatin/nucleolar digest and the post-mitochondrial cytoplasmic fraction. It was demonstrated that selective leakages of any major U-snRNPs to those fractions during aqueous cell fractionation seem unlikely. The high proportion of U1a-snRNP to U1b-snRNP in embryoid bodies and F9 cells was reproduced, using anti-Sm column. X snRNA could not be detected in the Sm snRNP fraction from human KB cell and 129 male mouse liver nuclei. Anti-2, 2, 7-trimethylguanosine (m^{2, 2, 7}₃G) antibody column was also demonstrated to be useful for the fractionation of U-snRNPs from embryoid bodies. Only U1a, U1b and U2-snRNP were retained by and eluted from the column. However, when the deproteinized RNA isolated from the nuclear extract of embryoid bodies was loaded onto the column, other U snRNAs (U4 and U5 snRNA) as well as the three U snRNAs were also retained by and eluted from the column. The partially purified U1-snRNP(s) from embryoid bodies was eluted at 0.18 M NH₄Cl and U2+U4+U5 as well as U1-snRNP(s) eluted at approximately 0.3 M NH₄Cl from a DEAE column. The U6 and U7/10-snRNPs were eluted at approximately 0.35 M NH₄Cl from the column.