Molecular and pharmacological studies of eIF1, eIF4G1 and eIF1A
Addressing mechanistic aspects of scanning dependent and independent translation revealed dynamic and mutually exclusive interaction of eIF4G1 with eIF4E and eIF1 (1). To examine further the interplay between them, we used HTS and identified i14G1s, the first eIF4G1-eIF1 inhibitors. The i14G1s established the importance of eIF4G1 cooperation with eIF1 in scanning, cap-proximal leaky scanning and stringency of AUG selection. Strikingly, the eIF1–eIF4G1 inhibitors uncovered a mechanism of translational control of stress-response via modulation of eIF1–eIF4G1 interaction (2).
By utilizing translation complex profiling (TCP-seq) and Selective-TCP-seq, we investigated 48S organization and the impact eIF4G1-eIF1 inhibition by i14G1-12. Our data unveil the landscapes and regulation of scanning, leaky scanning, and 48S initiation complexes provides a general overview of scanning and AUG selection and evidence for conformational rearrangements in vivo (3). The potential of these compounds as anti-tumor and anti-pathogens is currently investigated.
We also applied high-throughput drug screens to identify translation initiation inhibitors targeting eIF1A, a regulator of scanning and AUG selection. These inhibitors were found to be excellent research tools and potential therapy against cancer and SARS-CoV-2.
Dissection of ribosome intrinsic properties driving specialization
The ribosome is central to the entire translation cycle, interacting dynamically with stage-specific regulatory factors. Despite its fundamental importance, its inherent regulatory functions are often underestimated. Evidence across species suggests that heterologous ribosome composition and modifications are involved in the translation of specific mRNAs. We are studying an unexplored potential mechanism of specialization that is intrinsic to the ribosome and involves the ability of certain ribosomal proteins (RPs) to interact with the mRNA. RPs situated along the mRNA path of the 40S small ribosomal subunit may preferentially recognize specific mRNA sequences, enabling these interactions to impact the efficiency of translation of the target mRNAs. Additionally, RPs mRNA binding activities may be a target for regulation.
To address this hypothesis we used site-specific genome editing in mammalian cells to mutate endogenous RPS3 and RPS26 residues located at the entry or exit channel of the 40S, respectively, impairing their mRNA binding ability. Our findings revealed that these mutants display differential effects on cap-proximal initiation, leaky scanning and start codon fidelity (4,5). We demonstrated that RPS3 mRNA binding is a major target for regulation by SARS-CoV-2 encoded NSP1 and uncovered a central role of RPS26 mRNA binding in energy metabolism and the AMPK-mTOR signaling pathway. Collectively, these initial genetic and molecular studies validate the feasibility of our approach for elucidating the critical role of ribosome-mRNA interaction in selective translational control of cellular and viral mRNAs bearing defined features.
To further establish this concept, our current goal is to perform a systematic examination of the regulatory landscape and the functional significance of 40S RPs' interaction with mRNA.
References
1. Haimov, O., Sehrawat, U., Tamarkin-Ben Harush, A., Bahat, A., Uzonyi, A., Will, A., Hiraishi, H., Asano, K. and Dikstein, R. (2018) Dynamic interactions of eIF4G1 with eIF4E and eIF1 underlie scanning dependent and independent translation. Mol Cell Biol.
2. Sehrawat, U., Haimov, O., Weiss, B., Tamarkin-Ben Harush, A., Ashkenazi, S., Plotnikov, A., Noiman, T., Leshkowitz, D., Stelzer, G. and Dikstein, R. (2022) Inhibitors of eIF4G1-eIF1 uncover its regulatory role of ER/UPR stress-response genes independent of eIF2-alpha-phosphorylation. Proceedings of the National Academy of Sciences, 119, e2120339119.
3. Weiss, B. and Dikstein, R. (2024) Unraveling the landscapes and regulation of scanning, leaky scanning, and 48S initiation complex conformations. Cell reports, 43, 114126.
4. Havkin-Solomon, T., Fraticelli, D., Bahat, A., Hayat, D., Reuven, N., Shaul, Y. and Dikstein, R. (2023) Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling. Nucleic acids research, 51, 4415-4428.
5. Havkin-Solomon, T., Itzhaki, E., Joffe, N., Reuven, N., Shaul, Y. and Dikstein, R. (2023) Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding. Nucleic acids research, 51, 4208-4222.