Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

Abstract

Translation initiation in eukaryotes is an intricate process that requires specific interactions between multiple eukaryotic initiation factors (eIFs) and the mRNA to be translated. Primarily, a 43S pre-initiation complex is formed from the association of the 40S small subunit of the ribosome with protein factors eIF1, eIF1A, eIF3, eIF5 and the ternary complex (TC) of eIF2-guanosine 5 ́- triphosphate (GTP)-methionine initiator transfer RNA (tRNAiMet). On the other hand, the mRNA that needs to be translated would be preactivated by association with eIF4F, which is a protein complex consisting of DEAD-box RNA helicase (eIF4A), Cap-binding protein (eIF4E) and a large scaffold protein (eIF4G). Another factor participating in translation initiation is DEAD box RNA helicase DDX3X, which has been proposed to resolve 5 ́ UTR secondary structures to promote mRNA translation. Interestingly, DDX3X has been implicated in several cancers such as hepatocellular carcinoma, breast cancer and medulloblastoma carcinoma. DDX3X is a stress response factor and shows localization in intracellular membraneless compartments known as stress granules during stress. However, the function of DDX3X during normal cell cycle and integrated stress response is still not well understood. Our proteomic data from DDX3X immunoprecipitates suggest that DDX3X associates with the eIF3 complex. Therefore, we tested the relationship between eIF3D and DDX3X and how both bind to transcripts during translation initiation. We employed CLIP-Seq to identify the exact locations on mRNAs that these proteins are found. eIF3D mainly binds to the transcript in the 5 ́ UTR which is in concordance with the proposed role of eIF3 in the 43S pre-initiation complex, while DDX3X binds mainly at and downstream of the start codon, suggesting a role of DDX3X beyond 5 ́ UTR secondary structure resolution. To uncover the role of DDX3X in translation initiation during stress, we performed CLIP-Seq, polysome profiling and RNA-seq in two different states, the first one during rest

condition and the second one is after treatment with Thapsigargin, to induce endoplasmic reticulum stress. Our data showed that DDX3 binds specifically on and after the start codon of most genes, the main peak of binding is immediately after the start codon with a decrease in the binding in the same location in stress condition. Interestingly, we noticed that DDX3X binds transcripts whose translation doesn’t change during stress, which lead us to seek a deeper understanding of CLIP-Seq data. Moreover, we serendipitously noticed an enrichment of cytidine on DDX3X mRNA binding sites, which lead us to investigate the role for acetylation of cytidine in the function of DDX3X during stress. Our data reveal new important features of mRNA translation regulation by DDX3X in rest and stress as we move closer to an understanding of how different RNA helicases select and act on their mRNA targets.

Date

1-18-2024

Committee Chair

Anastasios Vourekas

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Available for download on Saturday, January 25, 2031

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