A Potential New Mechanism of Arsenic Carcinogenesis: Depletion of Stem-Loop Binding Protein and Increase in Polyadenylated Canonical Histone H3.1 mRNA.

ABSTRACT

Canonical histones are synthesized with a peak in S-phase, whereas histone variants are formed throughout the cell cycle. Unlike messenger RNA (mRNA) for all other genes with a poly(A) tail, canonical histone mRNAs contain a stem-loop structure at their 3'-ends. This stem-loop structure is the binding site for the stem-loop binding protein (SLBP), a protein involved in canonical histone mRNA processing. Recently, we found that arsenic depletes SLBP by enhancing its proteasomal degradation and epigenetically silencing the promoter of the SLBP gene. The loss of SLBP disrupts histone mRNA processing and induces aberrant polyadenylation of canonical histone H3.1 mRNA. Here, we present new data supporting the idea that the lack of SLBP allows the H3.1 mRNA to be polyadenylated using the downstream poly(A) signal. SLBP was also depleted in arsenic-transformed bronchial epithelial cells (BEAS-2B), which led us to hypothesize the involvement of SLBP and polyadenylated H3.1 mRNA in carcinogenesis. Here, for the first time, we report that overexpression of H3.1 polyadenylated mRNA, and knockdown of SLBP enhances anchorage-independent cell growth. A pcDNA-H3.1 vector with a poly(A) signal sequence was stably transfected into BEAS-2B cells. Polyadenylated H3.1 mRNA and exogenous H3.1 protein levels were significantly increased in cells containing the pcDNA-H3.1 vector. A soft agar assay revealed that cells containing the vector formed significantly higher numbers of colonies compared to wild-type cells. Moreover, small hairpin RNA for SLBP (shSLBP) was used to knockdown the expression of SLBP. Cells stably transfected with the shSLBP vector grew significantly more colonies in soft agar than cells transfected with a control vector. These data suggest that upregulation of polyadenylated H3.1 mRNA holds potential as a mechanism to facilitate carcinogenesis by toxicants such as arsenic that depletes SLBP.