Associate professor, cell and developmental biology, University of North Carolina, Chapel Hill
• Assistant professor, cell and developmental biology, UNC — 2002-2008
• Postdoc, Cold Spring Harbor Laboratory — 2002
• Postdoc, Genetica — 1999-2001
• PhD, molecular/cellular pharmacology, State University of New York at Stony Brook — 1999
• BS, chemistry, San Jose State University — 1993
Researchers from the University of North Carolina, Chapel Hill, and Massachusetts General Hospital this month published a paper in RNA showing a mechanism of microRNA production control, the addition of an oligo-uridine tail to the 3' end of the miRNA's precursor, is far more widespread than previously reported.
The modification, the paper notes, had previously been observed in the let-7 family in embryonic cells. In this work, however, the investigators found that “non-templated addition is a widespread phenomenon occurring in many miRNA families.”
This week, Gene Silencing News spoke with the paper's senior author, UNC's Scott Hammond, about the findings.
Let's start with a little background on your lab.
We're very interested in studying microRNAs, and our major research focus is on the biochemical pathways involved in producing microRNAs and how their production is controlled. Our interest [is driven] by the fact that microRNA expression is so altered in many diseases including cancer.
Our current focus is trying to understand how these widespread alterations in microRNA expression take place and the biochemical mechanism for that, [with the goal of] better understanding diseases like cancer.
Can you talk about the oligo-uridine tails that were the focus of the RNA paper?
Several years back, several labs including our own demonstrated that the microRNA let-7, which is very important in development and is also a tumor suppressor … is regulated after transcription during biogenesis by the protein lin-28, which could bind to let-7 and control its processing into its mature form.
The Narry Kim lab [at Seoul National University] showed, interestingly, that lin-28 can also trigger an oligo-uridylation process where you get a U tail added to the let-7 precursor. That helps promote degradation of the precursor thereby preventing production of the mature let-7.
Her work was focused on let-7, and as a kind of follow up, we wanted to ask if other microRNAs are regulated by this same kind of uridylation process because there really wasn't any information in the literature. When people have sequenced mature microRNAs, they have noticed non-templated additions to the ends [although] it's not very common. So the question was, “Are these vestiges of modifications to the precursor or are the mature [miRNAs] themselves being modified?”
What we wanted to do, then, was systematically look at precursors through sequencing to determine whether or not they, across many microRNA families, have these non-templated additions. We reported that this is very widespread — it's not just let-7, it's not just in conjunction with lin-28 regulation, but it's widespread in many tissues for many microRNAs.
For the sequencing method you developed to conduct this research, did you just need to tweak an existing approach or was it a matter of coming up with something specific?
It was definitely very specific. People have been using Illumina-based deep sequencing to look at many different types of RNA species and it has become a very popular tool to get very large numbers of sequence reads. The problem was that we were interested in looking specifically at precursors for microRNAs, and with any kind of method where you non-specifically sequence RNAs, you'll have a hard time finding precursors because they are the same size as tRNAs. [On top of this], tRNAs are probably the most abundant small RNA and microRNA precursors are the rarest.
We developed a method using specific primers to amplify only precursors and combined it with deep sequencing. It's kind of a novel idea; in the past, deep sequencing was always non-specfically sequencing everything in the population. We're combining specific PCR primers, but in a heavily multiplexed reaction, with sequencing to get just information about the RNAs that we're interested in.
It turned out to work well; over half the sequence reads we got mapped to a precursor instead of other stuff.
In the end, you found that these sorts of modifications are far more widespread than with the let-7 family?
Right. Also, the work with let-7 from Kim's group was all focused on oligo-uridylation in coordination with lin-28, and that only happens in embryonic cells. We looked at non-embryonic tissues and still see extensive uridylation. Not only do we see it in non-let-7 microRNAs, but we see it outside of the embryonic compartment, which is where it was expected to be found.
It was actually quite surprising to find it so widespread and it really suggests that the role of uridylation is not quite so simple. It doesn't just cause degradation because a lot of these microRNAs that are uridylated are still highly processed into the mature form in some cell types. In some cases, it's not just a signal for degradation, it must be something else. That is certainly where we want to go now, to understand the biology of these events and what they mean to the production of microRNAs and their function.
Uridylation wasn't the only modification you observed, right?
It wasn't the only one, but it was the most common. We did see some adenylation, and also some cytidine addition, which is less common although some microRNAs prefer different kinds of additions. There is some underlying biology there; we just need to try to understand what it is.
At this point, is it at all clear why some modified precursors are degraded and some are not?
That is something we really don't have any information on at all. You'd expect that there is a second signal of some kind that would trigger degradation for the microRNAs that are meant to be degraded, but we have no real understand of what that is.
And the process of degradation is still a bit of a mystery, as well?
There are several major RNA turnover pathways in the cell. What is interesting is that histone mRNAs are oligo-uridylated in a sort of similar fashion as a method to promote degradation of the mRNA. In that case, it has been shown that there are several RNA degradation pathways involved.
What's cool is that two totally different types of RNAs have been found to undergo the same oligo-uridine addition, and maybe that gives some idea of how microRNAs might be treated, as well.