Molecular Cell biology

Introduction

Human gene expression is exquisitely regulated at several points of between transcription and protein synthesis to ensure fidelity in the conversion of genetic information into biological functions.
Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance pathway responsible for the recognition and degradation of mRNAs containing premature termination codons (PTCs).
NMD protects cells from nonfunctional, potentially harmful, polypeptides encoded by mutated mRNAs.
On the other hand, NMD degrade frameshift or miss-spliced mRNAs encoding cancer antigen.
Hence, manipulation of NMD activity will benefit to improve anti-cancer immune response.
We are challenging this fundamental post-transcriptional gene expression regulation pathway and developing novel drugs for cancer and rear genetic diseases.
In addition to NMD, we are analyzing disease related post-transcriptional event of gene expression mechanisms using our original technique, which developed during basic analysis of NMD.
The goal of the research is to find the critical factors of diseases and develop/perform the primary compound screening targeting these factors.

Research Project

1） Investigation of regulation mechanisms of mRNA surveillance.

SMG1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system known as nonsense-mediated mRNA decay (NMD).
NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins.
SMG1 directly phosphorylates Upf1 helicase, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation.
Phosphorylated-Upf1 recruits the SMG5:SMG7 complex to phospho-S1096 to induce ribosome dissociation and induce decapping mediated PTC-mRNA decay.
Phospho-Upf1 also recruits SMG6 endonuclease to phospho-T28 which might be involved in PTC-mRNA end-cleavage.
Upf1 ATPase/helicase activity are likely required for the activation of mRNA decay enzymes and mRNP dissociation to complete NMD.
We will reveal the physiological significance of NMD, and the application of NMD mechanisms will open the new strategy for treatment of genetic diseases and cancer.

2） Development of new drugs targeting for nonsense-mRNA and post-transcriptional mRNA regulation.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that eliminates aberrant mRNAs carrying premature termination codons (PTCs).
Up to 30% of all known mutations causing human diseases generate aberrant mRNAs that are degraded by NMD.
NMD degrades not only proteins that show dominant-negative function but also aberrant proteins that retain at least some aspect of their normal cell function.
If mutant proteins are still functional, the selective inhibition of NMD provides a strategy to ameliorate disease phenotypes in patients with PTC-related conditions.
Another strategy to inhibit NMD for the rescue of intractable diseases involves drugs causing translational readthrough as NMD inhibition expected in a synergistic effect on read-through efficiency.
Importantly, suppression of NMD causes an enhancement of tumor immunity; suppression of NMD causes induction of tumor specific neoantigen that induce tumor immunity in mice model.
The bottleneck of these strategy for the treatment of intractable diseases is a “pure” NMD inhibitor.
Although recent studies have reported the identification of novel NMD inhibitors, for most of them, NMD inhibitor could not be confirmed by other laboratories.
Hence, the identification of a potent “pure” NMD inhibitor is desired.

In this project, we will demonstrate NMD suppression at mouse level using our novel NMD inhibitor (P-DIRECT, patent) and several existing drugs that induce NMD inhibition (unpublished), which were discovered using our NMD evaluation system (patent).
By using these NMD inhibitors, we will establish a method for the treatment of cancer immunity and genetic diseases containing nonsense-mRNA.
In parallel, to obtain more effective compounds, we will conduct screening using the Okinawa natural compound library of the University of the Ryukyus and the compound library of the Drug Discovery Initiative using our NMD or read-through evaluation systems.
As a cancer model, we will use a transplantation model using mouse melanoma cells.
As a model for the treatment of genetic diseases, we will use cystic fibrosis nonsense mutant mice that we established.
Through these analyses, we will obtain proof of concept at the in vivo level.

The goal of the study is to strengthen the rationality of the strategy by obtaining the proof of concept for the induction of nonsense-mRNA via inhibition of NMD against intractable cancers and rear genetic disease.

3） Analysis of post-transcriptional gene expression regulation for diverse biological processes and diseases

Translational regulation of mRNA is an immediate and precise mechanism to control gene expression in various biological processes, including development, differentiation, and responses to extracellular stress.
Global quantification analysis indicates that the cellular abundance of proteins in mammals is predominantly controlled at the level of translation.
In vivo, mRNAs do not exist as bare mRNA molecules but as mRNA-protein complexes with RNA-binding proteins (RBPs).
More than one thousand RBPs have been identified, and they bind to specific cis-acting elements, consisting of sequence elements, stem-loop structures and/or modified nucleotides.
For many genes, alternative poly (A) addition and alternative splicing give rise to 3’-UTR variants.
These variants are controlled by specific post-transcriptional regulation.
The cap-dependent mRNA translational process is divided into three major steps: initiation, elongation, and termination.
Each step is elaborately regulated by multiple mRNA 3’-UTR binding proteins in a cell type- and species-specific manner.
We are challenging this new frontier of RNA based biology by combining biochemistry, molecular biology, and cellular biology and mice.