Faculty Research Page
Associate Professor of Biochemistry, Biophysics and Structural BiologyLab Homepage: http://www.ingolia-lab.org/
We seek to learn how cells control the translation and stability of mRNA transcripts and understand the role of this dynamic regulation in maintaining homeostasis and adapting to changing environments. Cells tightly control which genes they express as proteins, and the amount of each protein they make, and post-transcriptional regulation can modulate gene expression based on cellular needs and extracellular conditions. Indeed, rapid reprogramming of protein synthesis in dynamic environments often depends on modulating the translation and stability of existing transcripts. This regulation depends on mRNA-binding proteins, which are as numerous as sequence-specific transcription factors, suggesting a pervasive role for post- transcriptional regulation in cell physiology. We are thus very interested in a genetic and molecular understanding of these RNA-binding proteins, and much of our work focuses on developing comprehensive and high-throughput techniques for elucidating post-transcriptional regulatory networks.
RNA-binding proteins as trans-acting regulators of translation. Recent studies in yeast and in mammalian cells have emphasized that hundreds of proteins bind to specific subsets of mRNAs. However, we do not know the impact of these proteins on the transcripts they bind. It seems likely that many of these RNA-binding proteins act, directly or indirectly, to alter the translation and stability of bound transcripts. The ways in which they affect their targets are not well understood, nor is it known how cell signaling pathways impinge on these regulators. We are interested in comprehensively characterizing RNA regulators through functional genomics.
Comprehensive dissection of gene expression regulation. We are developing approaches to survey comprehensively how genetic perturbations, especially CRISPR/Cas9-based transcriptional interference (CRISPRi) and activation (CRISPRa) impact quantitative molecular phenotypes including transcription, translation, and mRNA decay. Our approaches circumvent many of the limitations arising from the use of flow sorting and single-cell based approaches to provide precise and quantitative phenotypic measurements from pooled screens.
Start site selection. Ribosome profiling revealed an unexpected degree of variability in translation initiation in mammalian cells, including substantial use of non-AUG codons. These diverse start sites led to the translation of alternate protein isoforms as well as short reading frames that likely served principally as decoys repressing translation of a protein-coding gene. Alternate initiation has been recognized as a mechanism for controlling protein expression level and isoform choice for a few specific genes. Our finding that alternate initiation was pervasive posed questions about the ways in which start site selection is controlled and its impact on expression genome-wide. We are using ribosome profiling to study the effects of modulating the factors that control start site usage. We are also pursuing one specific, interesting mode of alternate protein production: the regulated expression of truncated dominant negative isoforms from the same transcript that encodes a full-length protein. This mode of regulation may have unique properties that we can assess by quantitative, single-cell analysis.
Padron A, Iwasaki S, Ingolia NT. Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules. Mol Cell, accepted (2019).
McGeachy AM, Meacham ZA, Ingolia NT. An Accessible Continuous-Culture Turbidostat for Pooled Analysis of Complex Libraries. ACS Syntho Biol 8: 844 (2019).
Iwasaki S, Iwasaki W, Takahashi M, Sakamoto A, Watanabe C, Shichino Y, Floor SN, Fujiwara K, Mito M, Dodo K, Sodeoka M, Imataka H, Honma T, Fukuzawa K, Ito T, Ingolia NT. The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA. Mol Cell 73: 738 (2019).
Mills EW, Green R, Ingolia NT. Slowed decay of mRNAs enhances platelet specific translation. Blood 129: e38 (2017).
Mills EW, Wangen J, Green R, Ingolia NT. Dynamic Regulation of a Ribosome Rescue Pathway in Erythroid Cells and Platelets. Cell Rep 17: 1 (2016).
Iwasaki S, Floor SN, Ingolia NT. Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor. Nature 534: 558 (2016).
Ingolia NT, Brar GA, Stern-Ginossar N, Harris MS, Talhouarne GJ, Jackson SE, Wills MR, Weissman JS. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes. Cell Rep 8: 1365 (2014).
Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nature Protocols 7: 1534 (2012).
Ingolia NT, Lareau LF, Weissman JS. Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes. Cell 147: 789 (2011).
Gracheva EO*, Ingolia NT*, Kelly YM, Cordero-Morales JF, Hollopeter G, Chesler AT, Sánchez EE, Perez JC, Weissman JS, Julius D. Molecular basis of infrared detection by snakes. Nature 464: 1006 (2010).
Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324: 218 (2009).
Ingolia NT, Murray AW. Positive-feedback loops as a flexible biological module. Curr Biol 17: 668 (2007).
Ingolia NT. Topology and robustness in the Drosophila segment polarity network. PLoS Biol. 2: e123 (2004).
* denotes equal contribution
Photo credit: Mark Hanson at Mark Joseph Studios.
Last Updated 2019-07-24