Animal replication-dependent histone mRNAs are the only eukaryotic mRNAs that lack a polyA tail ending instead in a conserved stemloop. In contrast mRNAs for histone variants, e.g. H3.3 and H2A.Z, are encoded by polyadenylated mRNAs. The genes for all five histone proteins are clustered in metazoan genomes, and factors required for histone gene expression are concentrated near the histone genes. A nuclear body, the histone locus body (HLB) forms at the histone genes and contains factors essential for histone mRNA biosynthesis. Our goal is to understand the detailed mechanisms unique to histone mRNA metabolism and regulation, which occurs primarily at the posttranscriptional level, both regulating histone pre-mRNA processing and histone mRNA degradation. The stemloop is the major cis element responsible for both these regulatory steps in the cell cycle regulation of histone mRNAs. The three aims of this proposal are: 1. Using an in vitro processing system composed primarily of recombinant proteins and synthetic RNAs to understand how the histone pre-mRNA is cleaved by a set of factors also used in cleavage and polyadenylation. These factors specifically assemble on the U7 snRNP. In particular we will determine the specific interactions between the U7 snRNP, the substrate and SLBP which activate the endonuclease CPSF73 for cleavage. 2. Understand the biochemical details, including the mechanism of regulation, of the novel pathway of histone mRNA degradation. Using CRISPR-Cas9 to edit the factors required for degradation, we will determine the mechanism by which the uridyl transferase TUT7 is recruited to modify histone mRNA, and the role of Upf1 in this process. 3. Understand how histone mRNA expression is regulated by factors present in the HLB, focusing on the changes that occur as cells progress from G1 to S-phase. NPAT, the major scaffolding factor, and FLASH, the factor required for assembly of the active U7 snRNP, are large, largely unstructured proteins, that contain small domains that interact with specific factors. We are characterizing the factors that interact with these proteins to determine factors involved in coordinate regulation of histone gene expression.
Histone proteins are critical components of the chromosome which must be synthesized in appropriate amounts whenever the cell replicates its DNA and divides. We are determining the mechanism which regulate histone genes and mRNAs to ensure proper cell growth. Since histone mRNAs differ from all other mRNAs in the cell, we have determined several novel regulatory strategies unique to histone genes.
Showing the most recent 10 out of 111 publications
