Smg1 Knockdown And Ubiquitin-proteasome Inhibitors Caused Accumulation Of Upf1 And Upf2 Proteins In Human Cells

Part I Smg1 knockdown caused accumulation of Upf1 and Upf2 proteins in human cellsGene expression is regulated by various mechanisms in which RNA decay pathway is one of the most important regulators. Nonsense mediated mRNA decay (NMD) is a highly conserved pathway which degrades the nonsense mutation (also called premature termination codon) containing mRNA selectively. NMD suppresses the expression of nonsense mutation derived truncated proteins which would be deleterious by competing with activity of full-length normal proteins and thus plays an important protective role for heterozygous carriers. In addition to nonsense-mutation in genome, transcription error, alternative splicing and RNA editing can also bear targets of NMD. It is thought to function as an important member of quality control in genetic information.NMD activity requires various RNA metabolic mechanisms such as splicing, capping, and translation. As a result of splicing, exon junction complex (EJC) is assembled 20–24 nt upstream of the exon–exon junction on the mRNA, which is essential for NMD. Upf3, a member of NMD factors, is actually a component of EJC; and Upf2, another NMD factor, is recruited onto mRNA molecule by binding to Upf3. When ribosome stops at premature termination codon present at least 50–55 nt upstream of an EJC, a complex named Smg1-Upf1-eRF (SURF) binds to Upf2. Smg1 is a member of PI-3 kinases and is also an essential component for NMD. It phosphorylates Upf1 helicase during NMD reaction. Finally, this interaction causes decapping of mRNA followed by degradation.Upf1 and Smg1 are also known to participate in DNA metabolism. Actually, knockdown of Upf1 in human cells causes arrest in early S phase and DNA-damage response by ATR. Smg1 is also known to work in cell cycle progression and DNA damage checkpoint pathway. These reports showed the multi-function of NMD components which are required for the stability of genomic information. However, their quantitative regulation of each component is largely unknown.Hela cells were transfected with 5032 siRNA against Smg1 mRNA and their lysates were analyzed by western blotting with specific antibodies. Smg1 was down-regulated by siRNA transfection successfully as compared to control cells transfected with anti-Luciferase siRNA. We observed that both Upf1 and Upf2 proteins increased significantly in Smg1 knockdown cells. Next, we tested various Smg1 siRNA molecules with different target sequences. All of them caused similar accumulations of Upf1 and Upf2 proteins. From these results, we concluded Smg1 knockdown could cause the accumulation of Upf1 and Upf2 proteins in human cells. Similar up-regulation of Upf1 and Upf2 proteins in Smg1 knockdown cells was also observed in A549 and skin derived normal fibroblast HE-4 cells. Then we concluded that this up-regulation might be a general phenomenon in culturing human cells.To determine the cellular fraction where Upf1 and Upf2 accumulated, western blotting was performed with fractionated cellular lysates. By Smg1 siRNA transfection, both Upf1 and Upf2 proteins increased in nuclear and cytoplasmic fractions. Then, it seemed that Smg1 could down-regulate the amounts of Upf1 and Upf2 proteins in nuclear and cytoplasmic fraction. However, we could not detect significant augmentation in mRNAs of Upf1 and Upf2 from our real-time PCR results.To get the morphologic proofs of our results, we preformed immunofluorescence staining to test the location of Upf1 and Upf2 proteins. By using specific antibodies, we observed that Upf1 was evenly distributed throughout the cytoplasm, while Upf2 showed strong perinuclear staining. With Smg1 SiRNA knockdown, the fluorescence signals of Smg1 in cells weakened significantly, which suggested the successful knockdown of Smg1. At the same time, we found the fluorescence signals of Upf1 and Upf2 increased respectively, which confirmed our results.Conclusion:Smg1-knockdown caused accumulation of Upf1 and Upf2 proteins in human cells. PartⅡUbiquitin-proteasome inhibitors caused accumulation of Upf1 and Upf2 proteins in human cellsThe generation and degradation of intracellular proteins must keep dynamic balance, which playsa key role to maintain cellular stable and normal functions. The ubiquitin proteasome system(UPS)is an important pathway for the intracellular proteins target degradation.Polymers of ubiquitin can be covalently attached to protein targets by a three-step(ubiquitin-activating enzymes,ubiquitin conjugating enzymes,ubiquitin-protein ligases)conjugated cascade responses . The resulting ubiquitylated proteins are then recognized and degraded by the 26S proteasome.UPS can degrade intracellular proteins with high effeciency and selectivity,especially for cell cycle regulated proteins, oncoproteins,tumor suppressor proteins,and denaturalized proteins.UPS is assential for many cellular processes,including apoptosis,MHC class I antigen presentation,cell cycle and intracellular signal transferring,which has a closed relationship with the cellular physiology and pathology.Ubiquitin-proteasome system is a principal mechanism for protein catabolism in the mammalian cytosol and nucleus. It plays an important role in a wide variety of cellular processes including cell cycle, differentiation, DNA repair, transcriptional regulation, response to stress, and many other basic cellular processes. MG132, a kind of peptide aldehyde, is a more potent and selective proteasome inhibitor. In our present studies, MG132 treatment caused the accumulation of Upf1 and Upf2 proteins, indicating that ubiquitin-proteasome might participate in the down-regulation of the amount of Upf1 and Upf2.These results prompted us to speculate that Upf1 and Upf2 would be target molecules of ubiquitination. To investigate this possibility, we tried detection of higher band shift caused by polyubiquitination of Upf1 proteins. However, we detected no band shift of either unpurified Upf1 or immuno-precipitated Upf1, and no signal of ubiquitin conjugation was detected even in immuno-purified Upf1. At least, the accumulation of Upf1 could not be accounted by the fraction of very little band shift in MG132 treated cells. In conclusion, our experiments provided no clear evidence for polyubiquitination of Upf1 and proposed that the target molecule(s) of ubiquitin-proteasome might down-regulate the amount of Upf1 directly or indirectly.To test whether Smg1 knockdown and ubiquitin-proteasome inhibitors share the same pathway to cause the accumulation of Upf1 and Upf2, we treated Smg1-knockdowned cells with proteasome inhibitors and the lysates for immunoblot were obtained. If they affect on the increments in Upf1 and Upf2 independently, an additional accumulation should be expected. Smg1 knockdown and proteasome inhibitor MG132 caused accumulation of Upf1 and Upf2 respectively. Moreover, in Smg1 knockdowned cells,we detected additive effects of Upf1 and Upf2 accumulation with proteasome inhibitors treatment, which suggested the possibility that SMG1 and ubiquitin-protesome system shared a different, if not all, pathway that regulated the amounts of Upf1 and Upf2.Conclusion:Ubiquitin-proteasome inhibitors caused accumulation of Upf1 and Upf2 proteins in human cells, which shared a different pathway with Smg1 knockdown.