Abstract:
Quality control mechanisms have evolved to guard the cell against defects in gene expression. The nonsense mediated mRNA decay (NMD) pathway is one of the most well studied surveillance mechanisms. It detects and triggers degradation of aberrant mRNAs that contain pre-mature termination codons (PTCs), preventing the accumulation of truncated polypeptides that are potentially deleterious to the cell. PTC recognition in eukaryotes results in the assembly of a surveillance complex on the mRNA, which triggers the degradation of the PTC-containing mRNA. The surveillance complex consists of the Up-frameshift (UPF) proteins (1 to 3) and in metazoa, the Suppressor with morphological effects on genitalia (SMG) proteins (1, 5 to 9). UPF1 undergoes a phosphorylation/dephosphorylation cycle that is a key event driving NMD. Upon PTC recognition, UPF1 is phosphorylated by the SMG1 kinase, initiating mRNA decay through the recruitment of the 14-3-3 domain-containing proteins SMG5, SMG6 and SMG7. SMG6 was shown to possess endonuclease activity that cleaves the target mRNA in the vicinity of the PTC. On the other hand, SMG5 and SMG7 form a heterodimer and recruit general cellular decay enzymes. SMG5 was shown to recruit the decapping enzyme DCP2 and its co-factors through PNRC2. SMG7 was shown to decay mRNA efficiently through its Proline-rich C-terminus (PC) region, which is necessary and sufficient for this activity. However, which decay factors are recruited to the target mRNA has remained unknown.
The major part of my doctoral work focused on elucidating the significance of the SMG5:SMG7 heterodimer formation in NMD and understanding how SMG7 elicits decay of a NMD-targeted mRNA. Here, I could show that SMG5:SMG7 heterodimer formation is necessary for functional NMD and that SMG5 and SMG7 use distinct mechanisms to degrade NMD-targeted mRNA. SMG5 promotes decapping independently of deadenylation while SMG7 promotes deadenylation-dependent decapping through a direct interaction with POP2, a catalytic subunit of the CCR4-NOT deadenylase complex. This interaction is specific, as SMG7 did not bind to CAF1, a paralog of POP2. I could further demonstrate that POP2 contributes to NMD target degradation in human cells and that the SMG7-POP2 interaction was critical for NMD in cells depleted of SMG6. This indicated that SMG6 and SMG7 act redundantly to degrade NMD targets. Taken together, my work demonstrated how NMD employs diverse and partially redundant decay mechanisms to ensure that aberrant mRNAs are efficiently degraded.
The CCR4-NOT deadenylase complex is the major component involved in the first step of cellular mRNA degradation. This complex catalyzes the removal of the poly(A) tail from the 3’-end of the mRNA, hence causing translational repression and committing the mRNA to degradation. The core components of the CCR4-NOT complex consist minimally of two modules, i.e. the NOT module that includes NOT1, NOT2 and NOT3, and the catalytic module that involves two deadenlyases, CCR4 and POP2/CAF1 bound to NOT1. In human cells, additional components have been identified, namely, CAF40/CNOT9, CNOT10 and CNOT11. These components are conserved in Drosophila cells, however the role of these proteins and how they are integrated into the complex remained unknown. Thus, the next part of my studies addressed the molecular characterization of the CCR4-NOT complex in Drosophila cells. In this part of my work, I could show that NOT10 binds directly to NOT11 and forms a novel module of the CCR4-NOT complex. This module docks directly on the N-terminus of NOT1 that was hence named the NOT10/11 Binding Domain (NOT10/11 BD). This direct interaction to NOT1 was mediated by NOT11 and is conserved in human cells.
Decay
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