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Q&A: Max Planck's Gunter Meister Discusses How Importin 8 Influences microRNA Silencing

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meister.jpgName:
Gunter Meister

Position:
Head, RNA biology research group, Max Planck Institute for Biochemistry

Background:
• Postdoc, Rockefeller University — 2003-2004
• PhD, Max Planck Institute for Biochemistry — 2002
• BS, biology, University of Bayreuth — 1999

Gunter Meister and colleagues from Max Planck published data in Cell last month showing that the nuclear protein Importin 8 is a component of microRNA-guided regulatory pathways.

Specifically, the team found that Importin 8 “interacts with Argonaute proteins and localizes to cytoplasmic processing bodies, structures involved in RNA metabolism,” they wrote in the paper’s abstract.

“Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool … [and demonstrated] that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing.”

This week, RNAi News spoke with Meister about the findings.

Let’s start with an overview of your lab and your research focus.

I did a postdoc with Tom Tuschl [at Rockefeller University] and that’s how I became interested in working with small RNAs and RNAi. I am a biochemist and [at Tuschl’s lab] we were always interested in finding out what proteins are associated with small RNAs. We were also interested in the molecular mechanism behind Argonaute proteins, which are the binding partners of small RNAs.

Later, I started my own lab in Munich at the Max Planck Institute of Biochemistry and the main focus of our work is the function of human Argonaute proteins. We’ve focused on Argonaute 1, 2, 3, and 4 because these proteins are ubiquitously expressed. We started to produce monoclonal antibodies against these proteins and we’re interested in what complexes they form, what other proteins are associated with Argonaute, what small RNAs are associated with Argonaute proteins, [such as] different subsets of microRNAs, for example.

Finally, we are also interested what mRNAs are regulated by miRNAs and have developed biochemical methods to identify such targets.

The work described in Cell was related to your efforts to figure out how Argonaute proteins find the correct binding sites on mRNAs.

Exactly. It is quite well understood how microRNAs are processed, how they are transported from the nucleus to the cytoplasm and how they are incorporated into silencing complexes. But there is not much known about how these individual steps are regulated. How microRNAs find and bind [to the] correct mRNAs is still unclear.

There was a paper from [Reuven] Agami [at the Netherlands Cancer Institute] that presented a negative regulator, a protein that occupies a microRNA-binding site so that the microRNA cannot bind anymore. We now have identified a positive regulator: we need Importin 8 … so that Argonaute can bind to a specific mRNA.

This is the first positive regulator of Argonaute function.

Had researchers previously found Importin 8 to be involved with Argonaute function?

Importin 8 had been identified before because it is an import receptor [that] imports proteins from the cytoplasm to the nucleus after they have been synthesized in the cytoplasm. Importin 8 is one of these proteins, but not much was known about its specific function … and there was no link between Argonaute and Importin 8 before.

When we purified an Argonaute complex and did mass spectrometry, we found Importin 8 in this complex.

Were you able to find out exactly how Importin 8 does its job with Argonaute?

That is still not clear. We were able to figure out the exact step in the microRNA pathway were it functions — it’s downstream of Argonaute/microRNA associations, so the microRNAs are transcribed, are processed, and incorporated into silencing complexes, and Importin 8 is not required for this.

[It is required] at the step when Argonaute/microRNA complexes have to find the right mRNA targets. How exactly it works, we don’t know yet.

In your research, you found that Importin 8 is not required for RNAi, correct?

That’s right. We can simply recapitulate by generating a piece of RNA that has a perfect complementary target site. When we bring into the cell such an RNA … that is the perfect site for an endogenous microRNA, for example, this microRNA functions like an siRNA and guides cleavage of the RNA.

We have shown before that the endonuclease that is doing this cleavage is Argonaute 2, and in an in vitro system, you simply need Argonaute 2, the small RNA, and the target and you can get cleavage.

Those are kind of the minimal requirements for RNAi, and it’s not very likely that many more proteins are required for this cleavage event. This was more or less expected. When we take a perfectly complimentary, artificial target RNA, it gets cleaved by Argonaute 2 and Importin 8 is not required for this.

On the other hand, this is not a natural [situation] because there are almost no perfectly complementary target sites for microRNAs in the cell; 3’ UTRs typically contains binding sites [that] are only partially complimentary to microRNAs.

Using natural 3’ UTRs, exactly how they look in vivo without a perfect complementary site, we saw that we need Importin 8.

With these data in hand, is there work underway to find out how Importin 8 works with Argonaute?

The next step is clearly finding out what exactly Importin 8 is doing with Argonaute. Why do Argonaute proteins need Importin 8 to find the right target?

One scenario that one could imagine is, let’s say, that [in the] vicinity of a microRNA binding site, there could be sequence elements that proteins like Importin 8 could somehow detect or identify. [In this kind of situation], if you don’t have Importin 8 in the cell, Argonaute might not be able to find the right binding site anymore.

Maybe it could even influence the Argonaute structure, and there are data available now that have been published by Dinshaw Patel at Memorial Sloan-Kettering showing that when Argonaute is bound to an mRNA, it has a different conformation compared to Argonaute not bound to an mRNA. So one idea is that one conformation can bind Importin 8 and the other cannot, and that [the Importin 8-bound conformation] somehow helps [Argonaute] to find its target.

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