Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/53728
Title: Structural basis of interaction between proline-rich sequence of PNRC2 and EVH1 domain of hDcp1a and its implications in mRNA decapping
Authors: Lai, Ethan Tingfeng
Keywords: DRNTU::Science::Biological sciences::Molecular biology
Issue Date: 2012
Source: Lai, E. T. (2012). Structural basis of interaction between proline-rich sequence of PNRC2 and EVH1 domain of hDcp1a and its implications in mRNA decapping. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance system that maintains the integrity of transcripts by removing aberrant mRNAs harbouring premature termination codons (PTCs). In the absence of NMD, truncated proteins with dominant negative or potentially deleterious gain of function activity may be expressed from these erroneous mRNAs. In addition to its function as a quality control for mRNAs, NMD is involved in the regulation of 3 – 10% of the Saccharomyces cerevisiae, Drosophilia melanogaster, and Homo sapiens transcriptomes. PTC recognition in NMD requires the cross talk between terminating ribosome stalled at stop codon and downstream cis-acting signal that is not conserved across species. In S. cerevisiae and D. melanogaster, the faux 3’ untranslated region (UTR) model has been proposed to be the main mechanism in recognising PTC. In mammals, PTC recognition is mediated through the interaction of the SMG1, Upf1, eukaryotic Release factor 1 and 3 (SURF) complex on the terminating ribosome at a PTC and downstream exon junction complex (EJC) during the pioneer round of translation. Upon recognition of PTC, an evolutionarily conserved surveillance complex consisting of the Upf1, Upf2 and Upf3 proteins is assembled. In mammals, the assembly of the surveillance complex also activates a phosphoinositide-3-kinase related protein kinase, SMG1, which will phosphorylate the Upf1 protein. Till date, three mechanisms of how phosphorylated Upf1 protein leads to decay of mRNAs have been proposed. First, the phosphorylated Upf1 protein recruits the SMG7 protein and C terminal of SMG7 protein targets the associated mRNA for decay through an undetermined mechanism. Second, the phosphorylated Upf1 protein recruitshDcp1a or Dcp2 proteins to decap mRNA for its subsequent 5’-3’ decay. Third, the hyperphosphorylated Upf1 protein associates with the nuclear receptor coregulatory protein 2 (PNRC2), which targets the mRNA to the P body through the interaction between the PNRC2 and hDcp1a proteins. Subsequently, decapping and 5’ - 3’ decay of mRNA occurs in the P body. As the three mechanisms are inferred from the results of coimmunoprecipitation or two-hybrid interactions, no direct interaction between the hyperphosphorylated Upf1 protein and decapping enzymes has been demonstrated till date. Here we present the crystal structure of hDcp1a protein in complex with the PNRC2 peptide. The proline-rich region of the PNRC2 peptide is bound to the EVH1 domain of the hDcp1a protein while isothermal titration calorimetry study demonstrates that NR-box of the PNRC2 protein mediates the direct interaction with hyperphosphorylated Upf1 protein. The mode of the PNRC2 protein interaction with the hDcp1a protein is distinct from those observed in other classes of EVH1/proline-rich ligands interactions. Additionally, PNRC2 mutagenesis study performed by our collaborator showed that disruption of the interaction of the PNRC2 protein with the hDcp1a protein abolishes its P-body localization and ability to promote mRNA degradation when tethered to mRNAs. Furthermore, the PNRC2 protein acts in synergy with the hDcp1a protein to stimulate the decapping activity of the Dcp2 protein by bridging the interaction between the hDcp1a and Dcp2 proteins. The formation of a novel PNRC2-hDcp1a-Dcp2 decapping complex suggests that the PNRC2 protein is a decapping coactivator in addition to its adaptor role in NMD.
URI: https://hdl.handle.net/10356/53728
DOI: 10.32657/10356/53728
Fulltext Permission: open
Fulltext Availability: With Fulltext
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