Nicholas Ingolia Discusses Ribosome Profiling

Emerging Research FRonts Commentary, April 2011

Nicholas Ingolia

Article: Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

Authors: Ingolia, NT;Ghaemmaghami, S;Newman, JRS;Weissman, JS
Journal: SCIENCE, 324 (5924): 218-223, APR 10, 2009
Addresses: Univ Calif San Francisco, Howard Hughes Med Inst, Dept Cellular & Mol Pharmacol, San Francisco, CA 94158 USA.
Univ Calif San Francisco, Howard Hughes Med Inst, Dept Cellular & Mol Pharmacol, San Francisco, CA 94158 USA.
Calif Inst Quantitat Biosci, San Francisco, CA 94158 USA.

Nicholas Ingolia talks with and answers a few questions about this month's Emerging Research Front paper in the field of Multidisciplinary.

SW: Why do you think your paper is highly cited? Does it describe a new discovery, methodology, or synthesis of knowledge?

Our work presents a new technique for measuring gene expression at the level of translation. This technique, which we call ribosome profiling, provides a quantitative and comprehensive snapshot of protein synthesis. It also indicates the exact position of ribosomes on a transcript, allowing us to identify sites where ribosomes stall during translation. These precise measurements of ribosome positions can also reveal translation outside of conventional protein-coding genes.

The data from our study provide the first experimental measurement of the speed of translation on individual codons genome-wide. This data has allowed us, as well as others, to address basic questions about the process of protein synthesis that were previously intractable. For example, there is a great deal of interest in the role of codon usage in regulating the speed of protein synthesis and the overall yield of translation. Our data has enabled new analyses such as that of Tuller et al. (2010), who propose that the excess of ribosomes that we observed at the beginning of genes represents a metering signal to avoid ribosomal traffic jams.

More broadly, biologists want to understand the control of gene expression, and translation is a key step in this process that has historically been harder to measure than mRNA abundance. More quantitative measurements of translation enable studies to determine what features of transcripts control their translation. These studies have correlated the translational efficiency of transcripts with codon usage, as described above, as well as with the secondary structure of the transcript and the presence of binding sites for specific proteins.

"One of the great current challenges in biology is to better understand the functional significance of genome sequences."

I think that we will soon start to see new ribosome profiling experiments that directly measure changes in translation. For example, I collaborated on a study to monitor the effects of microRNAs on translation (Guo et al., 2010). Here, we confirmed that microRNAs generally decrease both translation and mRNA stability together, and found no evidence for a large class of transcripts controlled solely at the level of translation.

SW: Would you summarize the significance of your paper in layman's terms?

We measure gene expression by looking directly at the process of translation. That allows us to see the effects of certain kinds of regulation that are invisible to microarray or RNA-Seq measurements. We also look at which sequences within an mRNA are actually being translated. This can expose errors in our prediction of what part of a gene actually encodes a protein.

It also reveals short, upstream translated sequences that may play a role in regulating translation of the main, protein-coding gene. These upstream sequences often serve as decoys that inhibit translation of the main gene, but their effects are not fully understood.

SW: How did you become involved in this research, and how would you describe the particular challenges, setbacks, and successes that you've encountered along the way?

Ribosome profiling is really a combination of an old technique with new advances in high-throughput sequencing. Important early studies of translation used nuclease digestion to precisely determine the position of the ribosome, which protected a short stretch of mRNA from digestion, in vitro (Steitz, 1969). However, there was no way to analyze the whole mixture of ribosome footprints from in vivo translation before deep sequencing. We found that deep sequencing was a very powerful tool for measuring biological information—as well as experimental artifacts.

One major challenge in developing ribosome profiling was optimizing the conversion of the ribosome-protected mRNA fragments, which are just ~30 nucleotides long, into a dsDNA library for deep sequencing. Existing approaches had strong preferences for certain fragments over others based on their sequence or structure, and I did extensive work to minimize these biases.

SW: Where do you see your research leading in the future?

Ribosome profiling promises to reveal translational control of gene expression in many important biological processes. We know that translation is regulated during cellular stresses such as hypoxia. Translation is closely connected to cellular growth and proliferation, and disruptions of this connection can play a role in cancer.

One important link between growth and translation is the mammalian target of rapamycin (mTOR) pathway, which also plays a major role in nutrient sensing and aging. There is great interest in comprehensive and quantitative measurements of translation in these pathways. Furthermore, the full set of translationally regulated target mRNAs will help to understand how transcripts are differentially controlled.

SW: Do you foresee any social or political implications for your research?

One of the great current challenges in biology is to better understand the functional significance of genome sequences. Ribosome profiling provides a direct, experimental approach for annotating the protein-coding capacity of the genome.End

Nicholas Ingolia, Ph.D.
Staff Member (Principal Investigator)
Carnegie Institution, Department of Embryology
Baltimore, MD, USA



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