Different complexes are formed on the 3′ end of histone mRNA with Nuclear and polyribosomal proteins

Abstract

Specific protein-RNA complexes are formed by incubating a synthetic histone mRNA 3' end (a 30 nucleotide stem-loop structure) RNA with extracts of either nuclei or polyribosomes. The complex formed between the stem-loop and nuclear proteins has a lower electrophoretic mobility than the complex formed between the stem-loop and polyribosomal proteins. Binding of the synthetic 3' end by both polyribosomal and nuclear proteins is abolished when two of the conserved uridine residues in the loop are replaced with adenosines. UV crosslinking of the protein complexes to the synthetic RNA resulted in transferring radiolabel to similar sized proteins, 50 kD, in both the nuclear and polyribosomal extracts.

The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing.

“…We have shown here specifically that the SLBP we cloned is the major protein that binds to the 3' end of histone mRNA as detected by the RNA-binding assay. Using antibodies specific for the carboxyl terminus of the SLBP, we have demonstrated that the SLBP is the major binding activity present in both the nucleus and the polyribosomes, in agreement with our previous conclusion that the same polypeptide functions in both compartments (Pandey et al 1991;Dominski et al 1995;Hanson et al 1996). There are two forms of SLBP detected by Western blotting (Figs.…”

“…A similar size was obtained when the human SLBP was synthesized in frog oocytes after injection of the mRNA or in a wheat germ translation system (not shown). This mobility is consistent with the cloned cDNA representing the previously characterized SLBP (Pandey et al 1991;Hanson et al 1996), and suggests that the discrepancy in molecular mass is attributable to an anomalous mobility of the SLBP on SDS--polyacrylamide gels (Fig. 4B, lane 1).…”

“…The complete h u m a n SLBP contains 269 amino acids (Fig. 3A) with a predicted molecular mass of 31 kD, significantly smaller than the 45 kD estimated for the SLBP by SDS-gel electrophoresis (Pandey et al 1991;Hanson et al 1996). The reason for this discrepancy is discussed below.…”

“…We have detected a specific protein, termed SLBP, that interacts with the 3' end of histone mRNA. The SLBP is present in both nuclei and polyribosomes (Pandey et al 1991;Williams and Marzluff 1995;Hanson et al 1996), with most of the SLBP present in polyribosomes, presumably as part of the histone mRNP {Hanson et al 1996). The SLBP binds the stem-loop as a monomer (Hanson et al 1996), requiring the 26 nucleotides at the 3' end of histone mRNA for efficient binding {Williams .…”

“…Mobility-shift assays were performed using a radiolabeled 30-nucleotide stem-loop RNA prepared by transcription of oligonucleotide templates as described previously (Pandey et al 1991). Twenty-five microliters of reticulocyte lysate programmed with the appropriate RNAs were used for the binding reactions.…”