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Harvard’s Carl Novina Discusses Function Of microRNA and Their Role in Cancer

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Name: Carl Novina
 
Position: Assistant professor, Dana-Farber Cancer Institute & Harvard Medical School
 
Background:
Postdoc, Center for Cancer Research, MIT — 2000-2004
MD, Columbia University College of Physicians and Surgeons — 2000
PhD, immunology, Tufts University — 1998
BS, biology/BA, English, Rutgers University — 1990
 
After completing his postdoc research in 2004 under RNAi pioneer Phillip Sharp, Carl Novina formed his own lab to continue investigating small RNAs.
 
That work primarily focuses on microRNAs, and Novina and colleagues recently published a paper in the Proceedings of the National Academy of Sciences detailing their findings regarding the mechanism of miRNA-mediated gene silencing.
 
This week, RNAi News spoke with Novina about his lab, the PNAS paper, and the direction of his research.
 

 
When we spoke last in 2004, you were just beginning to assemble your lab (see RNAi News, 10/22/2004). So let’s start with an overview of the work you do these days.
 
We’re looking at the mechanisms by which microRNAs repress genes, focusing particularly on the molecular interactions — RNA/RNA and RNA/protein interactions — that lead to translational repression.
The other part of my laboratory is looking at the role of microRNAs in tumor formation and progression using cell-based assays, as well as in vivo modeling for microRNA function in cancer.
 
You recently published a paper in PNAS [describing work] that falls under that first category. Can you talk a bit about the study and the findings?
 
We developed a cell-free assay for microRNA function back in 2006. The paper that was just published in PNAS is a follow-up to those early studies demonstrating that we can repeat the major biochemical hallmarks of microRNA activity first discovered in cell-based assays using our in vitro system.
 
There have been some observations that microRNAs repress translation during the initiation stage of translation. Our data goes into some molecular detail about how microRNAs repress translation initiation.
 
The early observations in worms suggested that microRNAs repress translation at post-initiation, [while] the early observations in mammals suggested that microRNAs work in a similar way as they do in worms. But more recent data suggests that microRNAs may actually block translation at the initiation step also. There are several lines of evidence suggesting this mechanism.
 
The principal observation that links many of these notions together is that microRNA repression requires a 7-methyl-G cap — a physiologic cap — on target mRNAs to mediate the repression. We demonstrated this property in our Molecular Cell paper in 2006. Several subsequent observations have confirmed that observation, and many groups now agree that microRNAs can repress translation at initiation, but the molecular details differ.
 
Some groups, for example one group [at McGill University led] by Nahum Sonenberg, suggest that microRNAs block translation initiation at the level of cap recognition so that it prevents the ribosome from ever joining the target mRNA. Another group, [Zissimos] Mourelatos’ group [at the University of Pennsylvania that published a relevant paper in] Cell, suggested that the critical microRNA protein Argonaute may even bind to the cap of target mRNAs and thus mediate the cap-dependent repression.

 

Our data suggests that there is an initiation block, but the point of repression is a little later during the initiation cycle. Our data suggests that microRNAs block not at the level of 40S ribosome subunit recruitment, but that the point of repression is at the level of 60S subunit joining.
 
These data are consistent with some other observations [including] one publication [that is the] combined work of [the University of California, San Diego’s Amy] Pasquinelli and [the Wistar Institute’s] Ramin Shiekhattar, suggesting that eIF6 is involved in microRNA repression. The role of eIF6 is to prevent 60S subunit joining, so our data mechanistically agrees with that observation.
  
It also agrees with another in vitro system developed by Matthias Hentze’s group [at EMBL], which suggests that in Drosophila, microRNA-targeted mRNAs lack 60S subunits.
 
So all these data suggest an initiation block, but the exact details of the block to initiation are different.
 
Are there plans to follow up on this work?
 
We are following up in several different ways looking at the exact mechanism of repression. You have to understand that repression the way we’re describing it is unusual. The majority of translational control occurs more like the way Nahum Sonenberg suggests, and that is at the level of cap recognition and subunit joining — that’s the majority of translational control. And there are other mechanisms of translational control, but blocking an initiation step the way we’re describing it — where the 40S binds to the mRNA, scans across the 5’ UTR, sits at the AUG waiting for the 60S to join, and blocks the microRNA there — is a very unusual mechanism of translational control.
 
But if you consider that microRNAs may target as many as a third to half of human genes, it suggests that what was previously considered to be a rather unusual mechanism of repression may be more ubiquitous. So we are interested in getting at a molecular understanding of the exact mechanism of this type of repression.
 
What are the primary research efforts you have ongoing in regards to your work investigating microRNAs and tumors?
 
We are looking at a couple of different leukemias, chronic lymphocytic leukemia and chronic myeloid leukemia … [as well as] a solid tumor, melanoma. We are asking different questions using these cancers; with the leukemias we are, more or less, looking at the microRNA expression profiles and seeing what they tell us about these cancers and whether we can intervene in the pathobiology of these leukemias by manipulating microRNA expression as a way to affect the progression of the disease or change its drug sensitivity.
 
In the case of melanoma, it is an unusual cancer in that you can harvest melanoma cells from patients, drop those cells in culture, and the cells will actually grow without doing any other manipulation to the cells.
 
We are asking a different question in the melanoma project. MicroRNA expression is altered in very characteristic ways specific for cancers [and] altered expression of microRNAs in cancers is not random. What we’re asking with melanoma is, “if microRNA expression can be used to classify cancer … what are the mechanisms that lead to this characteristic expression pattern in cancer?” Melanoma … is very convenient for interrogating that issue.
 
At this point, do you have target microRNAs that you’ve already implicated in the [cancers] or are you still defining those?
 
We’re still defining them. In the case of chronic lymphocytic leukemia, this is a cancer that has been studied very carefully by several groups, and the interesting thing is that the microRNA expression profiles described by several different … groups [researching the issue] are not identical. That goes against the notion … that microRNA expression profiling can be used to classify cancers. It seems like there are several microRNAs that are similar amongst the groups, but none of them are identical in their signature. We’re not exactly sure why. It may have to do with the different patient populations or the different ways of testing microRNA expression, so there may be biological, as well as experimental, differences to account for the different profiles from the different groups.
 
We have our own profile, and our profile is different from any of the other published profiles, which differ from each other. I guess what I’m saying here is that we do have some ideas, but we haven’t exactly honed in on one microRNA that can be used to account for this cancer formation or that cancer’s response and sensitivity to drugs.
 
So when papers come out linking a particular microRNA to a particular cancer, we should keep in mind that these signatures can vary and it’s not clear cut?
 
That’s right. What’s interesting about the microRNA field is that not only can the microRNA expression tell you about the identity of the cancer, but it can also give you some insight into the identity of the cell that was transformed to lead to the cancer.
 
We’re working on those ideas, too. The [hypothesis] is there are some microRNAs that are specific to certain cells whereas others are more ubiquitous. So if you can detect certain microRNAs or combinations of microRNAs that are unique to a certain cell, you can conceivably identify the cell that was transformed leading to the cancer. And just that amount of information may be helpful in treating patients and, one day, manipulating microRNA expression to alter the sensitivity of that cancer cell to certain drugs.
 
There is quite a bit of information that can come out of this [work], and there are some microRNAs that may vary naturally between cancer patients, but one would hope there are certain common microRNAs in a particular population that can be used to identify the cell of origin.
 
I’d like to say, too, that mRNA expression profiling has been very poor at defining cell types, normally or in cancer. It may be that there are so many mRNAs expressed in a particular cell that it becomes hard to discern the specific signals from the noise. It may be that microRNAs are very good at this either because they are very persistent and long-lived, or maybe that we’re getting better at detecting them. Or it might be that if you looked at a subset of mRNAs, like transcription factors, this class of mRNAs might be able to provide that kind of detailed information about a particular cell’s identity like microRNAs do.
 
So it seems microRNAs have become a large focus of the lab. Are you still working with the more traditional side of RNAi?
 
That is pretty much secondary to the microRNA work. A lot of people come to me asking for help using RNAi as a tool. And I still do that, but I’m not really focused on the molecular details of siRNA-mediated cleavage of mRNA, per se. Although I do use it and do study it, it is not the focus like microRNAs.
 
There are companies interested in using microRNAs therapeutically and diagnostically. Are you collaborating with any of them?
 
Yeah. I’m looking at a few different collaborations. We’re in the process of setting up collaborations with Santaris [Pharma], which is a company that makes locked nucleic acids to inhibit microRNA function. We’re also looking at collaborating with Nastech [Pharmaceutical], another company that is interested in looking at microRNAs for therapy.
 
You’re on [Nastech’s] scientific advisory board, right?
 
Correct. I’m also on the SAB of Qiagen, a company that uses microRNAs as … tools. … My list of industry collaborators is short, but my list of academic collaborators is long.

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