Mechanistic regulation of selenoprotein mRNAs during selenium deficiency

Abstract

The essential trace element selenium is present in selenoproteins via the unique amino acid selenocysteine (Sec). Most often, selenoproteins have a single Sec residue that utilizes the electrochemical properties of selenium to catalyze crucial biochemical reactions. Characterized selenoproteins possess antioxidant functions that play integral roles in various aspects of human health. The molecular biology of Sec incorporation is also unique because UGA serves as its triplet. In order to recode this UGA codon, all selenoprotein mRNAs have a specialized secondary structure in their 3'un-translated region (UTR) which facilitates the placement of Sec into the ribosome. In addition, Sec is synthesized directly on its tRNA and selenoprotein synthesis is thus sensitive to selenium availability. Since UGA is a common stop codon, selenoprotein mRNAs are potential targets of the nonsense-mediated decay pathway (NMD). This pathway targets aberrant transcripts with premature termination codons (PTCs) for degradation in order to prevent the production of potentially toxic truncated proteins. Several groups have observed a hierarchy of selenoprotein mRNA abundance during selenium deficiency whereby the levels of certain transcripts decline while others do not. Since the cellular machinery cannot distinguish Sec codons from UGA stop codons, it is generally postulated that NMD is involved in this response to selenium deficiency. While this assumption is logical, there has been little evidence to support it. This primary aim of this dissertation is to evaluate the role of the NMD pathway in the regulation of selenoprotein mRNAs during selenium deficiency. The overarching hypothesis of this thesis is that selenoprotein mRNA that are predicted sensitive to NMD will decrease in abundance during selenium deficiency. The established rules that govern the mammalian model for NMD were utilized to assess the susceptibility of the human selenoprotein transcriptome to the NMD pathway. About half the mRNAs were predicted sensitive to NMD while the other half were predicted resistant. Those that were predicted sensitive decreased significantly in abundance during selenium deficiency while those that were predicted resistant did not. In addition, the mRNAs that were sensitive to NMD and likewise responded to selenium status, were also more abundantly bound to central NMD regulator UPF1 during selenium deficiency. Furthermore, siRNA depletion of SMG1, the kinase responsible for UPF1 phophorylation and NMD activation, abrogated the selenium response of the NMD-sensitive transcripts. These results strongly suggest that NMD is involved in the decrease of selenoprotein transcript abundance observed during selenium deficiency. The stability of GPx4 mRNA presents an exception to the expected responses to selenium status and NMD predictions. GPx4 mRNA is predicted to be sensitive to NMD but does not respond to selenium deficiency and likewise remains stable with knockdown of SMG1. A consensus sequence in the 5'UTR of GPx4 was previously shown to facilitate the translation of its mRNA. The 5'UTR of GPx4 was thus analyzed in order to investigate its potential role in GPx4s enigmatic response to cellular selenium status. Our analysis of this consensus sequence suggests that it is not involved in the observed stability of GPx4 mRNA during selenium deficiency although it may be increasing the translatability of the transcript.