Efficient Extraction and Partial Purification of the Polyribosome-Associated Stem−Loop Binding Protein Bound to the 3‘ End of Histone mRNA

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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.