Identification of the Protein That Interacts with the 3′ End of Histone mRNA

Publisher Summary This chapter discusses the methods used to isolate and characterize the factor(s),that is, the stem-loop binding protein (SLBP), that interact with the 3 ′ end of histone messenger ribonucleic acid (mRNA). The replication-dependent histone mRNAs are unique among metazoan mRNAs in that they are the only mRNAs that do not have poly(A) tails. The replication-dependent histone genes for all five histone proteins are tightly linked and in mammals are present in two distinct clusters. There are a total of 50–60 genes for the replication-dependent histone mRNAs. The chapter discusses in detail about the detecting specific protein–RNA interactions. The chapter also explains the concepts related to in vitro processing of histone pre-mRNA, and isolation of polyribosomes. The chapter presents an overview of the isolation and characterization of RNA-binding proteins using the yeast three-hybrid system. β-galactosidase filter assay and elimination of the false positives are elaborated in depth. The chapter also discusses about in vitro translation and mobility shift assays using the reticulocyte lysate.