Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA.
Replication-dependent histone mRNAs end in a highly conserved stem-loop sequence rather than a polyA sequence. A 45-kDa stem-loop binding protein (SLBP), which specifically binds the stem-loop of histone mRNA, is present in both polyribosomes and nuclei. An identical 45-kDa protein, as determined by partial protease digestion, is cross-linked to a 30 nt RNA containing the 3' stem-loop from both nuclei and polyribosomes. The SLBP can also be detected by a Northwestern blot procedure using the 30 nt RNA as a probe. As judged from the Northwestern assay, more than 90% of the SLBP in the cell is found in the polyribosomes with the remaining SLBP localized to the nucleus. Only 5-10% of the SLBP could be extracted from the polyribosomes with salt. Treatment of the polyribosomes with micrococcal nuclease prior to salt extraction solubilized 5-10 times more SLBP as an RNA-protein complex. The SLBP could be subsequently partially purified from this complex.
Hanson
R J
RJ
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599, USA.
Sun
J
J
Willis
D G
DG
Marzluff
W F
WF
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D051379:Mice; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011132:Polyribosomes / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D039221:mRNA Cleavage and Polyadenylation Factors
D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C490494:Slbp protein, mouse; D039221:mRNA Cleavage and Polyadenylation Factors; D008836:Micrococcal Nuclease