The eukaryotic ribosome is a complex molecular machine responsible for the translation of mRNA to protein. Over the years we have gained detailed knowledge of ribosome structure, function, and biogenesis; however, a major unanswered question in the translation field is how cells monitor the integrity of the ribosome itself. Alterations in ribosome structure and function have been associated with diseases such as neurodegeneration, cancer, and ribosomopathies. Indeed, mutations, environmental stress, or mistakes during assembly can lead to malfunctioning ribosomes that need to be detected and marked for degradation to maintain translation fidelity. My group capitalizes on the recent advances in quantitative mass spectrometry, functional genomics methods, and CRISPR-mediated genome editing to study the molecular mechanisms of ribosome surveillance.
The assembly of the eukaryotic ribosome requires more than 200 accessory factors and numerous error-prone steps. Faulty intermediates resulting from mistakes during biogenesis are rapidly degraded, affirming the existence of a quality control pathway(s) monitoring ribosome assembly. In order to identify factors that differentiate between accurate and dead-end intermediates, we are developing novel systems to model ribosome biogenesis failure.
The ribosome is one of the most complex and long-lived machines in the cell and its many components are subject to both mechanical and chemical assaults. Accumulated damage can render the ribosome elongation incompetent, causing it to stall on messages. We are interested in identifying the molecular pathways that cope with such damaged ribosomes.
We want to build on our studies of ribosome surveillance and understand how the pathways identified in cultured cell lines contribute to the development of an intact organism. We use zebrafish as a model system to study how the integrity of the translation apparatus is monitored during development, and how dysregulation of the ribosome surveillance pathways leads to complex disease.
Ribosome-associated quality control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to degradation of faulty mRNA and partially synthesized polypeptides. Here we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific inhibition of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.
Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates.
Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. Here we show that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. We define amplification of MAPK signalling as a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. We propose that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. Our observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations.
The adenomatous polyposis coli (APC) gene product is mutated in the vast majority of human colorectal cancer. APC negatively regulates the WNT pathway by aiding in the degradation of β-Catenin, the transcription factor activated downstream of WNT signaling. APC mutations result in β-Catenin stabilization and constitutive WNT pathway activation leading to aberrant cellular proliferation. APC mutations associated with colorectal cancer commonly fall in a region of the gene termed the mutation cluster region and result in expression of an N-terminal fragment of the APC protein. Biochemical and molecular studies have revealed localization of APC/Apc to different sub-cellular compartments and various proteins outside of the WNT pathway that associate with truncated APC/Apc. These observations and genotypephenotype correlations have led to the suggestion that truncated APC bears neomorphic and/or dominant-negative function that support tumor development. To investigate this possibility, we have generated a novel allele of Apc in the mouse that yields complete loss of Apc protein. Our studies reveal that whole-gene deletion of Apc results in more rapid tumor development than the ApcMin truncation. Furthermore, we found that adenomas bearing truncated Apc had increased β-catenin activity compared to tumors lacking Apc protein, which could lead to context-dependent inhibition of tumorigenesis.