Final Report Summary - CELLULAR IRESES (Mechanism of cellular IRESes translation)
This study sought to shed light on the largely unexplored molecular mechanism of internal initiation of translation via IRES employed by an extensive number of cellular mRNAs, for which deregulation has been implicated in the development and progression of tumour, and clearly links translation to cancer. Translation normally initiates with the recruitment of the initiation factors to the 5' cap of the mRNA for ribosome binding. Curiously, some normally capped cellular messages, which are involved in cell-cycle control, growth and apoptosis, rely alternatively on an Internal ribosome entry site (IRES) at their 5' end and the 3' poly(A) tail to support translation. The advantage conferred by the IRES is obvious during shutdown of cap-dependent translation: these mRNAs remain bound to the polysomes and maintain steady expression. The precise mechanism of translation via cellular IRES and its regulation, the structural/sequence consensus, and their importance in use remain largely unexplored. In this research project, we proposed to study the mechanism of poly(A) enhancement of cellular IRES function using the proto-oncogene c-myc IRES as a model. The c-myc is an oncogenic transcription factor, which controls expression of an array of genes involved in mitosis and apoptosis. Its translation is up regulated in a vast range of cancers, with a large contribution by the activation of its IRES. The specific goal of the research proposal was to determine the role of the 3'poly(A) tail and the possible involvement of distinct set of regulatory factors for the IRES activity, which does not require the canonical poly(A) tail partner, PABP. This research project used functional proteomic approaches and involved cross-disciplinary collaboration from bioinformatics, biochemical tools to expertise in mass spectrometry analysis.
Key from this research project was the establishment of proven experimental techniques, which are applicable beyond the field of translation and therefore contribute in transfer of knowledge.
1. We developed a solid functional complementation assay to identify the regulatory factor(s) responsible for the poly(A) tail stimulation of IRES activity. Based on the fact that the c-myc IRES driven translation is poly(A) tail dependent but PABP-independent, the assay consisted of depleting the PABP from the HeLa cell extract and then passing the PABP-depleted extract over a poly(A)-sepharose column. As anticipated, the depletion of PABP from the HeLa cell extract impaired cap-mediated translation but has no effect on c-myc IRES translation. The cmyc-IRES mediated translation was lost in the double depleted extract. Unlike the cap-driven translation, it couldn't be rescued with the addition of PABP. This functional complementation assay was then used to follow the activity of the poly(A)-enhancer factor(s) through the biochemical fractionations of the ribosome-bound factor complex. The approach was successful in the identification of six potential candidates by mass spec within the active fractions that were able to restore c-myc translation in the depleted extract. The assay has identified metabolic enzymes involved in the glycolysis pathways as specific cellular IRES-driven translation factors, with no ability to restore canonical cap-dependent translation in the depleted extract and with no role in viral IRES translation.
2. For the validation of the candidates, we have implemented a high-throughput small scale in vitro translation extract preparation from silenced HeLa cells for readable phenotype on translation. The in vitro translation assay has been presented as a robust approach for screening and published in the peer-reviewed Nature protocols journal. In complementation with the siRNA treatment assays, we have performed a battery of biochemical approaches, which take advantage of the double depleted extract, trans-inhibition assay, as well as pull-down assays. The approach helped to narrow the screen to one factor. As expected, the candidate specifically binds to poly(A). While the validation requires further studies, we can foresee that the acquired results will enable to gain insight on mechanistic details of translation control of cellular IRES.
While massive efforts have been provided to characterise the translation machinery, we have just touched the tip of the iceberg on understanding the basic regulation of translation. The central importance of translation control in cancer is just beginning to be fully appreciated. The current research contributed in unravelling a largely unexplored field of moonlight functions of metabolic enzymes in the aberrant regulation of translation of oncogenic mRNAs. A subset of metabolic enzymes has been reported to bind specific mRNAs by non-canonical RNA-binding domains. However, their regulatory mechanisms remain poorly understand and raise the question of a crosstalk between cell metabolism and the control of gene expression by RNA-binding enzymes. This research project has a wide-range implication beyond gene regulation with an undeniable socio-economical significance for human health. It opens new avenue of research on the molecular bases of disregulation of metabolic pathways reported in cancer cells. This extends to the potential development of novel diagnostic and therapeutic strategies based on this knowledge.
Key from this research project was the establishment of proven experimental techniques, which are applicable beyond the field of translation and therefore contribute in transfer of knowledge.
1. We developed a solid functional complementation assay to identify the regulatory factor(s) responsible for the poly(A) tail stimulation of IRES activity. Based on the fact that the c-myc IRES driven translation is poly(A) tail dependent but PABP-independent, the assay consisted of depleting the PABP from the HeLa cell extract and then passing the PABP-depleted extract over a poly(A)-sepharose column. As anticipated, the depletion of PABP from the HeLa cell extract impaired cap-mediated translation but has no effect on c-myc IRES translation. The cmyc-IRES mediated translation was lost in the double depleted extract. Unlike the cap-driven translation, it couldn't be rescued with the addition of PABP. This functional complementation assay was then used to follow the activity of the poly(A)-enhancer factor(s) through the biochemical fractionations of the ribosome-bound factor complex. The approach was successful in the identification of six potential candidates by mass spec within the active fractions that were able to restore c-myc translation in the depleted extract. The assay has identified metabolic enzymes involved in the glycolysis pathways as specific cellular IRES-driven translation factors, with no ability to restore canonical cap-dependent translation in the depleted extract and with no role in viral IRES translation.
2. For the validation of the candidates, we have implemented a high-throughput small scale in vitro translation extract preparation from silenced HeLa cells for readable phenotype on translation. The in vitro translation assay has been presented as a robust approach for screening and published in the peer-reviewed Nature protocols journal. In complementation with the siRNA treatment assays, we have performed a battery of biochemical approaches, which take advantage of the double depleted extract, trans-inhibition assay, as well as pull-down assays. The approach helped to narrow the screen to one factor. As expected, the candidate specifically binds to poly(A). While the validation requires further studies, we can foresee that the acquired results will enable to gain insight on mechanistic details of translation control of cellular IRES.
While massive efforts have been provided to characterise the translation machinery, we have just touched the tip of the iceberg on understanding the basic regulation of translation. The central importance of translation control in cancer is just beginning to be fully appreciated. The current research contributed in unravelling a largely unexplored field of moonlight functions of metabolic enzymes in the aberrant regulation of translation of oncogenic mRNAs. A subset of metabolic enzymes has been reported to bind specific mRNAs by non-canonical RNA-binding domains. However, their regulatory mechanisms remain poorly understand and raise the question of a crosstalk between cell metabolism and the control of gene expression by RNA-binding enzymes. This research project has a wide-range implication beyond gene regulation with an undeniable socio-economical significance for human health. It opens new avenue of research on the molecular bases of disregulation of metabolic pathways reported in cancer cells. This extends to the potential development of novel diagnostic and therapeutic strategies based on this knowledge.