A molecular approach to mRNA translation regulation and protein quality control
- 주제(키워드) mRNA translation , protein quality control
- 발행기관 고려대학교 대학원
- 지도교수 김윤기
- 발행년도 2021
- 학위수여년월 2021. 8
- 학위구분 박사
- 학과 대학원 생명과학과
- 원문페이지 116 p
- UCI I804:11009-000000251967
- DOI 10.23186/korea.000000251967.11009.0001253
- 본문언어 영어
초록/요약
When the pre-mature messenger RNAs (mRNAs) are synthesized in the nucleus, they are processed with multiple steps; 5’ capping, 3’ polyadenylation, and splicing. While processing, the nuclear cap-binding protein 80/20 complex (CBC) binds to 5’ cap of mRNA and exon junction complex (EJC) binds to 20 ~ 24 nucleotides upstream of exon-exon junction (1-4). Mature mRNAs are exported to the cytoplasm for translation and mRNA translation can be divided into two steps depend on its 5’ cap-binding protein; CBC-dependent translation (CT) and eukaryotic translation initiation factor 4E (eIF4E)-dependent translation (ET) (1). In CT, CBC-bound translation initiation factor (CTIF) acts as a scaffold protein between CBC and eukaryotic translation initiation factor 3 (eIF3) that recruits ribosome to mRNA to elicit CT (5). CT has been suggested as a key step for mRNA surveillance since it is the pioneer (first) round of translation that occurs right after mRNA export from the nucleus to the cytoplasm. ET is a step for massive protein production and, unlike CT, eukaryotic translation initiation factor 4G (eIF4G) binds to eIF3 to elicit ET (6). It is known that CBC to eIF4E replacement is independent to translation (7). When the premature termination codon (PTC) is addressed to mRNA for various reasons, such as alternative splicing, point mutation, or ribosomal frameshift, PTC-containing mRNAs are degraded because faulty transcripts can arise accumulation of defective polypeptides. When a ribosome encounters PTC, the recruited eukaryotic peptides chain release factor 1/3 (eRF1/3) interacts with UPF1 and SMG1 forming a SURF complex (SMG1-UPF1-eRF1/3) (8). The interaction of the SURF complex and downstream EJC leads to the nonsense-mediated mRNA decay (NMD) for degradation of PTC-containing mRNA. NMD is a post-transcriptional and translation-dependent mRNA surveillance pathway that is well-conserved in many species. Accumulation of misfolded polypeptides can be deleterious to cell since it can cause proteotoxicity that leads to apoptosis (9). The ubiquitin-proteasome system (UPS) is one of the ways to eliminate misfolded polypeptides (10,11). However, when the UPS is overloaded or malfunctions, the aggresome can be used as an alternative pathway (12). Aggresome is a non-membranous organelle that locates in the cytoplasm close to the nucleus. When the UPS is blocked, the misfolded polypeptides are transported to aggresome in retrograde along the microtubule (13,14). Though aggresome is known to be degraded ultimately by autophagy, the detailed mechanism of aggresome biogenesis is still vague. For easier inspection of aggresome, proteasome inhibitors, such as MG132 or Bortezomib, are used for blocking the UPS and further stimulation of aggresome formation (15). In Chapter 1, there is an auto-commentary about “Misfolded polypeptides are selectively recognized and transported toward aggresomes by a CED complex” in Nature Communications, 2017, 8:15730. This chapter was published as “Crosstalk between translation and the aggresome–autophagy pathway” in Autophagy, 2018, 14(6):1079-1081. According to “Misfolded polypeptides are selectively recognized and transported toward aggresomes by a CED complex” that is published in 2017, Nature Communications, 8:15730, CTIF localizes at the perinuclear region and is concentrated in one or two puncta that are colocalized with γ-tubulin. Considering that (i) γ-tubulin is a component of microtubule organizing center (MTOC) where the aggresome forms, and (ii) the treatment of nocodazole, microtubule disruption chemical, hindered CTIF bodies’ integration, CTIF-concentrated bodies are aggresome. Clarifying the role of CTIF in aggresome, LC-MS/MS was proceeded for finding the novel interactor of CTIF. DCTN1 and eukaryotic translation elongation factor 1A1 (eEF1A1) were distinctive factors related to aggresome. EEF1A1 was known as a translation elongation factor but recent study suggested that eEF1A1 functions in aggresome by selectively recognize the misfolded polypeptides and generates aggresome formation signal. DCTN1 is a subunit of Dynein complex which is a motor protein complex for transport cargoes in the cell. The interaction of CTIF-eEF1A1-DCTN1 (CED complex) was RNA-independent and MG132-independent. For demonstration of the role of CED complex in aggresome formation, the percentile of aggresome formation was calculated under downregulation of each CED complex components. Downregulation of CED complex disrupted aggresome indicating that CED complex has active role in aggresome formation. The role of CED complex in aggresome formation can be critical for cell survival since the accumulation of the misfolded polypeptides can increase proteotoxicity which leads to cell apoptosis. Analysis of apoptotic level induced by the accumulation of misfolded polypeptides showed that downregulation of CED complex increased cell apoptosis Considering the role of CTIF in translation, the relation between aggresome formation and translation was needed to be clarified. The polysomal distribution of full length or N-terminal deleted CTIF 54-598 that lost aggresomal function was inspected with or without treatment of MG132. While treatment of MG132 inhibited participation of full length CTIF in translation, N-terminal deleted CTIF 54-598 did not show distribution change, suggesting sequestration of CTIF in aggresome inhibits participation of CTIF in translation. In Parkinson’s diseases, the Lewy bodies are hallmark of pathogenesis which has similar physical features with aggresome. Interestingly, CTIF co-localized with α-synuclein in the brain section of Parkinson’s disease patients. Considering that α-synuclein is a marker of Lewy body, CTIF possibly take an action in Lewy body formation in neurons. In Chapter 2, there is a study about the relationship between NMD and aggresome formation. Considering that the NMD majorly occurs during CT and the aggresome formation is related to CT, there can be connection between NMD and aggresome formation. While there are numerous studies about the mechanism of NMD, it was still vague how the truncated polypeptides are degraded that is generated from PTC-introduced NMD target mRNA (NMD-polypeptides). This study showed that the NMD-polypeptides go to aggresome when the UPS malfunctions. Downregulation of CED complex hinders the aggresome formation of NMD-polypeptides. Since the NMD is an mRNA quality control mechanism and aggresome is protein surveillance pathway, the hypothesis was the NMD factor might functions in aggresome formation as well. While the downregulation of various NMD factors (PNRC2, SMG5, SMG6, and SMG7) did not affect aggresome formation, only the downregulation of UPF1 inhibited aggresome formation. NMD is well-conserved mRNA surveillance pathway across the species and UPF1 is a common key NMD factor. Since the phosphorylation of UPF1 is a trigger for NMD, the localization of UPF1 was inspected according to its phosphorylation level. Interestingly, the hyperphosphorylated UPF1 (UPF1-HP) specifically enriched in aggresome under MG132 treatment while less phosphorylated mutant UPF1-HP-12A was overlapped with aggregation but not enriched in aggresome. This data suggest that phosphorylation of UPF1 is important to aggresome formation as well. Of note, the E3 ligase activity disrupted form of UPF1 (UPF1-E3 mut) was not localized in aggresome regardless of MG132 treatment indicating that E3 ligase activity of UPF1 is irrelevant with aggresome formation. Indeed, while the downregulation of UPF1 dispersed aggresome, the introduction of UPF1-HP restored aggresome formation and the introduction of UPF1-HP-12A could not, suggesting that hyperphosphorylated UPF1 specifically functions in aggresome formation. Supporting to this, the UPF1-HP preferentially binds to CED complex while UPF1-HP-12A showed reduced interaction. Downregulation of UPF1 showed lower interaction between CTIF and eEF1A1 or DCTN1, respectively, suggesting that UPF1 is important to CED complex integrity and to its selective recognition of the misfolded polypeptides. To test the active role of UPF1 in aggresome, this study observed the movement of CTIF aggregates and also check the apoptotic effect by proteotoxicity under downregulation of UPF1. Live cell imaging in single particular level showed the active movement of CTIF aggregates toward aggresome along the microtubule. Interestingly, this movement was disorientated and its frequency toward aggresome was reduced under UPF1 depletion implying the role of UPF1 in aggresome formation. Since the aggresome formation is closely related to cell apoptosis because the accumulation of the misfolded polypeptides are toxic to cell, the apoptotic level were measured under depletion of UPF1. Downregulation of UPF1 increased the cell apoptosis while the introduction of UPF1-HP restored the phenomenon. This data shows that UPF1 helps the cells resistant to cell apoptosis induced by proteotoxicity. Overall, this study showed that the role of UPF1 in aggresome formation. This chapter was published as “Nonsense-mediated mRNA decay factor UPF1 promotes aggresome formation” in Nature Communications, 2020, 11(1):3106. Chapter 3 is about the mechanism that how CT is regulated. Despite its importance as a stage where mRNA surveillance occurs, how CT is regulated is still vague. Because CTIF is a translation initiation factor in CT, the yeast two-hybrid system was applied for searching the novel interactor of CTIF. The dead box helicase 19B (DDX19B) was selected as a candidate for CT regulator. DDX19B is the RNA-helicase that localizes around the nucleus and attached to NUP214 which is a component of the nuclear pore complex (NPC). The interaction of CTIF-DDX19B was independent to mRNA and DDX19B did not interact with other translational factors, such as CBP80 or eIF4E. For demonstration of the role of DDX19B in CT, the expression of DDX19B was inhibited and it showed enhanced interaction between CTIF and CBP80. When the expression of CBP80 was downregulated, the interaction between CTIF and DDX19B was increased, implying the competition of DDX19B and CBP80 toward CTIF. The mutation of DDX19B (DDX19B-W6A/V10A) showed decreased interaction to CTIF compare to its wild type and the mutation of CTIF (CTIF-F460A) showed decreased interaction to DDX19B. Interestingly, CTIF-F460A showed increased interaction to translational factors, such as CBP80 and eIF3b, indicating that CTIF-DDX19B interaction inhibits the participation of CTIF in CT. Previous studies showed that CTIF localizes at the perinuclear region and the aggresome. However, the introduction of mutation on CTIF (CTIF-F460A) changed its localization into the cytoplasm and the downregulation of DDX19B affected the localization of endogenous CTIF from perinuclear region to cytoplasm indicating that the localization of CTIF was mainly restricted to the perinuclear region because of its interaction with DDX19B. Of note, the localization of endogenous CBP80 also altered when CTIF-F460A was overexpressed or the expression of DDX19B was downregulated. As a translational factor, polysome fractionation showed that most of the CTIF resides in the monosomal fraction compared to polysome. However, the introduction of F460A mutation changed its distribution from monosome to polysome showing increased participation in translation. Consistently, overexpression of CTIF-F460A distinctively changed the distribution of CBP80 in translation from monosome to polysome while eIF4E distribution did not change indicating that translation regulation by DDX19B-CTIF interaction is restricted to CT. For clarifying what is the key for restricted participation of CTIF in CT either the CTIF-DDX19B interaction itself or restricted localization of CTIF to perinuclear region by DDX19B, the Klarsicht, ANC-1, and syne homology1 (KASH1) domain of nesprin-1 was conjugated to CTIF and CTIF-F460A. As expected, the conjugation of KASH1 to CTIF-F460A alters its localization from the cytoplasm to perinuclear region since KASH1 domain penetrates nucleus membrane. Following polysome fractionation showed that CTIF-KASH1 mainly resides in the monosomal fraction while CTIF-F460A-KASH1 was evenly distributed from monosome to polysomal fraction, suggesting that the participation of CTIF in CT is majorly influenced by CTIF-DDX19B interaction itself. The effect of restricted participation of CTIF in CT was evaluated by NMD assay. Overexpression of CTIF-F460A enhanced NMD efficiency of exogenously introduced NMD reporter as well as endogenous NMD targets. Downregulation of DDX19B and further restoration of DDX19B-W6A/V10A enhanced NMD efficiency of exogenously introduced NMD reporter as well as endogenous NMD targets indicating that disruption of CTIF-DDX19B interaction let CT occurs indiscriminately.
more목차
Overview…………………………………………………………… 1
References………………………………………………………… 13
Abbreviations…………………………………………………… 16
Curriculum vitae…………………………………………………… 18
Chapter Ⅰ
Crosstalk between translation and the aggresome–autophagy pathway ……………………………………………………………………… 21
Chapter Ⅱ
Nonsense-mediated mRNA decay factor UPF1 promotes aggresome formation …………………………………………………………… 26
Chapter Ⅲ
The pioneer round of translation is regulated by CTIF-DDX19B interaction
Abstract ………………………………………………………… 63
Introduction …………………………………………………… 64
Materials and methods ………………………………………… 66
Results ………………………………………………………… 73
Discussion ……………………………………………………… 99
References …………………………………………………… 101
Summary in Korean……………………………………………… 106
Acknowledgements……………………………………………… 108
