91影库

The plenary speaker for this year’s 91影库 and Molecular Biology annual meeting is of the University of California, San Francisco. An investigator with the Howard Hughes Medical Institute, Weissman is a protein biochemist whose laboratory focuses on protein folding, amyloid formation and next-generation genomic sequencing. ASBMB’s science writer, Rajendrani Mukhopadhyay, spoke with Weissman to learn more about his scientific interests and influences. The interview has been edited for length and clarity.

What does your research focus on?

We’re interested in how proteins fold in cells, especially how chaperones assist protein quality control and how a cell distinguishes between correct and incorrect proteins both in the cytosol and endoplasmic reticulum. We also have a broad interest in developing general approaches for querying biological systems, especially around next-generation sequencing. The most mature of those efforts is an approach we call ribosome profiling. It allows us to globally monitor translation in cells at single-nucleotide resolution.

This idea of using deep sequencing techniques to define the information encoded with these genomes and to watch it being expressed in space and time is really exciting. Ribosome profiling is one example of these techniques. We get to watch, with exquisite precision, the ribosome get in the act of converting an informational molecule, the RNA, into a functional molecule, the protein.

I’ll be talking about our ribosome-profiling work during my plenary lecture.

What was the genesis for ribosome profiling?

Our original interest was on the yeast prion phenomenon called [PSI⁺]. The phenotype of [PSI⁺] is that when the cell contains this prion, or self-propagating infectious protein, it increases the rate of translation read-through. We wanted to develop an assay that let us watch where ribosomes were reading through stop codons in vivo. But then, once we developed ribosome profiling, it was clear it was a much broader tool.

What are some of the technical challenges with ribosomal profiling?

The biggest challenge is interpretation and data analysis. We get huge amounts of data. It takes longer for us to analyze the data and ask the right questions than it does to actually collect the data. The other technical challenge is working with relatively small amounts of cells. We’d really like to optimize the amount of sample we use in our system.

How do you balance your scientific efforts between asking fundamental biological questions and developing techniques?

For my lab, it’s very important that the techniques we develop be driven by fundamental questions that we want to answer. My lab is very interested in seeing what happens to a protein as it’s synthesized and how it is recognized by the cell’s quality-control systems and doing this in an in vivo setting. Although the ribosome profiling is an -omics tool and it broadly looks at protein translation, I’m personally most excited about its value as a high-precision tool for looking at mechanisms of how proteins are synthesized and folded. I see the methods development as something that drives our ability to ask questions that we want to ask.

To put your work in a larger context, what do you think are some of the big-picture questions in biochemistry and molecular biology?

One of the things is: How do cells maintain integrity of their proteome and detect misfolded proteins? How do the different quality-control systems work together in a coherent way to allow cells to function, and when and why do they break down?

I think there’s another, broader question of how we define the function of a protein. What do we mean when we identify protein X to do job Y? A lot of this has been classically done, arguably, in an ad hoc way where you look for some activity in a cell and you try to find the proteins that are responsible for it. You do biochemical and genetic assays and say protein X is involved in DNA replication. Maybe you set up an in vitro system, solve the crystal structure (of protein X) and find out which side chains are catalyzing the addition of the next nucleotide.

This type of reductionist, top-down approach has been incredibly successful, but it has some inherent flaws and biases to it. We’re trying to develop more systematic and less ad hoc, more principled and faster approaches to finding gene function.

The other general area, which is related, is that we have built tools with which we can sequence any individual or organism’s genome rapidly and at low cost. There’s now a huge challenge to define the information encoded in genomes and how it’s expressed in time. Ribosomal profiling is a part of that effort. It lets you know what proteins are being encoded for in a cell in an objective way and see how much is made at a given time. If we really want to test our ideas and try to use this information to design new systems, we have to have much better ways of turning on and off genes and to engineer genomes. I’m very excited about approaches that we and others are working on using the CRISPR system.

Tell me more about the CRISPR system.

CRISPR is an adaptive-immunity system from bacteria that’s been known for some time. It’s turned out to be a very useful system for engineering genomes. The CRISPR system that has become particularly popular now is the CAS9-based system. In a nutshell, when a cell becomes infected with a phage, it grabs part of the phage’s DNA and inserts it into its own genome in these special cluster repeat regions. It then later transcribes that copy of the phage DNA as RNA and uses that RNA to guide an endonuclease (called) CAS9. Now, if the bacterium is ever infected with the same phage, the CAS9 plus this guide RNA will specifically cut up that phage DNA by hybridization.

What revolutionized things is that Emmanuelle Charpentier (at Molecular Infection Medicine Sweden) and Jennifer Doudna (at University of California, Berkeley) showed that a single