Identification Of The Subcellular Localization Motifs In Gdown1 And Dissection Of Its Transcriptional Regulatory Effects

Transcription is the first step of the central dogma and plays a key role in regulating gene expression in eukaryotes.RNA polymerase Ⅱ(Pol Ⅱ)catalyzes the transcription of most protein-coding genes and numerous non-coding genes,so that studying the regulatory mechanisms of Pol Ⅱ is one of the most important topics in the field of molecular and cellular biology.Gdown1 was discovered in 2006 as an interacting protein of Pol Ⅱ.Biochemical experiments have proved that Gdown1 inhibits the interaction between a series of transcriptional regulatory proteins(including TFⅡF,PAF1,and TTF2)and Pol Ⅱ,and leads to a significant interference on transcription initiation and elongation.However,it is not clear whether and how GDOWN1 participates in transcription in live cells.On the other hand,it has been reported that the subcellular localization of GDOWN1 changed from the nucleus to the cytoplasm during the embryonic development in Drosophila,but the subcellular localization of GDOWN1 and its functions in mammals have not been reported.Based on the above unknown scientific questions,this thesis investigated the subcellular localization of human GDOWN1 and its functions during Pol Ⅱ transcription.The results showed that GDOWN1 was strictly localized in the cytoplasm in human somatic cells.Two classical NESes(Nuclear Export Signals)and a novel subcellular localization signal with a strong cytoplasmic retention activity,namely CAS(Cytoplasmic Anchoring Signal),were identified in human GDOWN1.The cytoplasmic retention activity of CAS did not depend on CRM1,but it anchored GDOWN1 at the cytoplasmic side of the NPC(Nuclear Pore Complex)and inhibited the cytoplasmicnuclear shuttling of GDOWN1,instead.Further studies showed that GDOWN1 interacted to many components of the stress granules and had a positive effect on the cellular responses to stresses.Additionally,knockout of GDOWN1 resulted in the cytoplasmic accumulation of the largest subunit of Pol Ⅱ,RPB1,suggesting that GDOWN1 may regulate the assembly or nuclear export of Pol Ⅱ.We mutated the subcellular localization motifs and generated a GDOWN1 mutant that was able to localize in the nucleus,without affecting its known interactions.The results showed that the long-lasting accumulation of GDOWN1 in the nucleus led to a loss the cell hemostasis,resulting in a significant decrease of RPB1 and further inhibiting global transcription.In summary,this thesis identified a novel type of the subcellular localization motif in GDOWN1 and uncovered its novel functions in cellular responses to the oxidative stress.We also found that the continuous accumulation of GDOWN1 in the nucleus led to global transcription repression by decreasing the level of RPB1.These results provide reliable evidence for further understanding of the mechanisms of GDOWN1 in transcriptional regulation,physiological function,and shed lights on its potential application.