Full Project B: The Role of Phosphrylation in the NMD RNA Surveillance Mechanism

Co-Investigators

Miles Wilkinson, Ph.D., Professor, Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas

Carlos Gonzalez, Ph.D., Associate Professor, Department of Biology, University of Puerto Rico - Rio Piedras, Rio Piedras, Puerto Rico

One third of inherited human genetic diseases are caused by genes harboring nonsense or frameshift mutations. These aberrant genes transcribe mRNAs containing premature termination codons (PTCs) and hence are targeted for decay by the nonsense-mediated decay (NMD) RNA surveillance pathway. By rapidly degrading PTC-bearing mRNAs, NMD reduces the synthesis of truncated proteins, some of which possess deleterious gain-of-function or dominant-negative effects.

PTCs are common features of mRNAs. First, they arise as a result of nonsense and frameshift mutations acquired at increased frequency in some types of tumor cells. Second, PTCs arise very frequently (two-thirds of the time) during the normal programmed rearrangement events that occur in the immunoglobulin (Ig) and T-cell receptor (TCR) gene loci. Lastly, PTCs are often acquired as a result of biosynthetic errors, including aberrant RNA splicing. In addition to degrading aberrant mRNAs, NMD regulates the level of a subset of normal wild-type mRNAs. While the physiological consequences of this regulation of normal mRNAs is not yet clear, it suggests that NMD also serves as a regulator of specific cell functions. In agreement with this possibility, NMD factors have recently been shown to be necessary for oxidative-stress responses and provide protection against genotoxic stress. Both of these findings suggest that NMD has a role in tumor progression.

While increasing evidence supports the notion that NMD is a physiological relevant pathway, the underlying mechanism of NMD remains poorly understood. This is an important issue to address, as solving this problem may drive the generation of novel strategies to ameliorate human diseases caused by nonsense and frameshift mutations. For example, this information may lead to approaches to upregulate NMD and thereby decrease the levels of toxic truncated proteins expressed from PTC-bearing mRNAs.

Many of the genes essential for NMD were first identified in S. cerevisiae. In addition, the functions of the encoded proteins have been studied in great detail in S. cerevisiae. One of them, Upf1p, possesses RNA binding and RNA-dependent ATPase/helicase activities. It interacts with another NMD factor, Upf2p, a central adaptor protein that interacts with other NMD factors. It is not known when these two proteins interact during NMD, nor how they trigger rapid mRNA decay. We hypothesize that a key step important for their function in NMD and other PTC-induced events is their phosphorylation. In agreement with this hypothesis, many studies have provided evidence that both the phosphorylation and dephosphorylation of the higher eukaryote orthologue of Upf1p (UPF1) has a role in NMD in many organisms. Indeed, four NMD factors (SMG1, SMG5, SMG6 and SMG7) are devoted to controlling the phosphorylation status of UPF1 in higher eukaryotes.

Despite the likely importance of UPF1 phosphorylation, the sites of UPF1 that are phosphorylated and their functional role in NMD have only begun to be studied. Using yeast and mammalian cell lines, the goal of this proposal is to understand the biochemistry of UPF1 and UPF2 phosphorylation and what role it has in NMD. Towards this goal, our preliminary mass spectrometry analysis has identified 11 phosphorylated residues in Upf1p. An amino-acid substitution of at least one of these residues impairs the function of Upf1p in NMD. Another issue that we intend to address is the functional role of Upf2p phosphorylation. We recently published that some of the residues in the Upf2p N-terminal domain that are likely to be phosphorylated are required for an interaction between Upf2p and an RNA-binding protein involved in NMD: Hrp1p. Mutations that affect the phosphorylation of these residues reduce Upf2p/Hrp1p interaction and cause an impairment of NMD. This evidence suggests that Upf2p phosphorylation has a role in NMD, a hypothesis we will pursue in more depth in this proposal. By combining both of our laboratories’ expertise in studies of NMD in both yeast and mammals, we anticipate we will make significant progress in understanding the role of phosphorylation in NMD in both single-celled and multicellular eukaryotes. 

Specific Aims

• To identify the phosphorylation sites in yeast Upf2p
• To define the functional role(s) of phosphorylated sites in yeast Upf1p and Upf2p
• To identify and determine the functional role(s) of conserved phosphorylated residues in mammalian UPF1 and UPF2