Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA.

R J Hanson; J Sun; D G Willis; W F Marzluff

doi:10.1021/bi9521856

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Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA.

R J Hanson·J Sun·D G Willis·W F Marzluff

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Abstract

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.

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Animals Base Sequence Blotting, Northern Blotting, Western Mice Micrococcal Nuclease Molecular Sequence Data Nuclear Proteins Nucleic Acid Conformation Polyribosomes RNA, Messenger RNA-Binding Proteins Tumor Cells, Cultured mRNA Cleavage and Polyadenylation Factors Journal Article Research Support, U.S. Gov't, P.H.S.

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R J Hanson, J Sun, D G Willis, W F Marzluff. 1996-02-20. Efficient extraction and partial purification of the polyribosome-associated stem-loop binding protein bound to the 3' end of histone mRNA.. https://doi.org/10.1021/bi9521856

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