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MIT’s Chris Burge Discusses How 3’ UTR Shortening Affects microRNAs

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NAME: Chris Burge
 
POSITION: Associate professor, biology, Massachusetts Institute of Technology
 
BACKGROUND:
Whitehead career development associate professor, MIT — 2004-2006
Assistant professor, MIT — 2002-2004
Bioinformatics fellow, MIT — 1999-2002
PhD, computational biology, Stanford University — 1997
BS, biological sciences, Stanford University — 1990
 
Last week, Massachusetts Institute of Technology researcher Chris Burge and colleagues published a paper in Science showing how proliferating cells express mRNAs with shorter 3’ UTRs, which cuts the number of microRNA binding sites, compared with non-proliferating cells.
 
“Our data indicate that gene expression is coordinately regulated, such that states of increased proliferation are associated with widespread reductions in the 3’ UTR-based regulatory capacity of mRNAs,” the investigators wrote.
This week, RNAi News spoke with Burge about his findings and their implications.
 

 
Let’s start with some background on your lab.
 
My lab is in the biology department. We study RNA processing and microRNA regulation, and do both computational and experimental work. But a main focus of the lab is alternative splicing; to understand the rules for splicing in different tissues and under different conditions.
 
We’ve collaborated a lot with both the [David] Bartel lab and the [Phil] Sharp lab, [both of which are at MIT], on microRNA regulation. There, the questions have mostly been about the rules for target determination.
  
More recently, we’ve been getting into studying the roles of microRNAs in development and differentiation.
 
And that’s what led you to this work detailed in Science?
 
Yeah.
 
Can you touch on the findings in that paper?
 
A lot of work has been done on T cell activation; it’s a very classical system, but most of those have focused on regulation at the transcriptional level or … signaling — kinase cascades and things like that.
 
Less is known about alternative RNA processing, so we used a relatively new array platform that has probes in every exon so we could see what was happening with both alternative splicing and alternative cleavage and polyadenylation.
 
It’s widely recognized that most human genes are alternatively spliced; people have known this for several years now. But it’s less recognized that at least a third, and probably half, of all genes have alternative cleavage and polyadenylation sites. What we noticed was that there are a number of genes that change the expression of their alternative polyA isoforms — isoforms differing in their 3’ UTR regions.
 
There was a very strong trend of the genes that change following activation changing toward increased expression of the shorter 3’ UTR isoform. That was sort of surprising; the conventional view is that the alternative RNA processing of each gene might be independent of other genes, it might be regulated just to fit the needs of that individual gene, and the widespread trend of a whole bunch of genes changing in the same direction hasn’t been observed.
 
The potential connection to microRNAs would be that since microRNAs primarily target 3’ UTR regions, the genes would be effectively reducing the impact of microRNA regulation. On average, [in] the genes that have multiple polyA sites, the two UTR isoforms differ by about a factor of two in length. So when you go from the long form to the short form … you also reduce the number of microRNA target sites by a factor of two.
 
So this is a fairly large change.
 
And this occurs during the cellular proliferation process?
 
Originally, we observed it in T cell activation. Forty-eight hours after primary mouse T cells are activated, we see this strong trend. To ask whether this is conserved, we looked at publicly available array data for human T cells and we see the same phenomenon. We also saw it in other immune cell types; B cells and monocytes also [display] a trend toward increased expression of the short 3’ UTR isoform.
 
Whenever immune cells are activated, they generally will go from a quiescent state to a more proliferative state, so we wondered whether this was something specific to the immune system or a signature of proliferation generally.
We looked then at essentially all available array data. To assess proliferation, we developed a gene expression-based measure called the proliferation index. Essentially, you look at the relative expression of a set of genes that are observed to increase whenever cells are more rapidly proliferating. So we developed it based on known examples. … What we could then do is estimate the proliferation status of a variety of different cells and tissues, and look at what was happening with the 3’ UTRs.
 
We observed a strong correlation where those cells and tissues that are more proliferative preferentially express the shorter 3’ UTR isoforms, just as we saw with T cells.
 
Do you have any theories on what this is all about? Is this a natural mechanism the body uses to turn off microRNAs that inhibit proliferation during normal processes?
 
That’s certainly one possibility. Essentially, the cell will be going from a state where the UTRs tend to be long to one where they tend to be shorter. So, the impact of any type of regulation that involves the 3’ UTR would potentially be reduced in proliferating cells.
 
[Aside from miRNA targeting], there are other roles for 3’ UTRs in terms of subcellular localization of proteins and things like that, so those would also be reduced. It’s as if the role of the 3’ UTR is more important in quiescent or slowly dividing cells [as opposed to] proliferating [ones].
We focused on microRNAs because they are very widespread and important … and in one case we saw conserved seed matches to microRNAs that we knew were expressed in activated T cells. In that case, we were able to reverse the repression that was associated with the expression of the long 3’ UTR isoform.
 
The experiment is a luciferase assay where the expression of either the long UTR isoform or the short isoform was forced. In all the examples tested, expression of the long UTR isoform represses translation, [a finding that is] consistent with the extended region — the region that is unique to the long form — containing negative regulatory elements such as microRNA target sites.
 
In one example, we were able to mutate microRNA seed matches in that region and reverse the repression.
 
What are the implications of these findings?
 
If you are designing small RNAs for therapeutic purposes, you shouldn’t think of the target gene as being a static entity with a defined 3’ UTR. It very well may be that the target gene changes its 3’ UTR under different circumstances. For example, if you want to target the gene under all circumstances, you’ll want to target the region upstream of the proximal polyadenylation site if there are multiple polyA sites. Conversely, you could preferentially target a gene in quiescent cells by targeting the region that is unique to the long form of that gene.
 
Also, a lot of the experiments that have been done studying microRNA-targeting mechanisms with transfection of microRNAs into cultured cells have been done in cells like HeLa cells, which are very proliferative and therefore may be expressing mostly the shorter 3’ UTR isoforms. That impacts how you interpret those experiments, [which] emphasizes the need to not just define the 3’ UTRs of genes but to define the 3’ UTR isoforms that are being expressed in any cell or tissue where microRNA targeting is occurring.
 
Another thing to think about is that there must be some mechanism that causes genes to increase expression of the short 3’ UTR isoform, and that’s something that will need to be figured out. It could be that the cleavage and polyadenylation machinery is up-regulated when cells proliferate and that helps them preferentially recognize upstream polyA sites. That’s one possible model … but there is clearly something going on.
 
[Also], since inhibition of microRNA processing is associated with tumorigenesis, and [since] cancer cells have reduced levels of microRNAs, this suggests that microRNAs, on balance, are anti-proliferative. So the shortening of 3’ UTRs may be a [cancer’s] mechanism to escape the antiproliferative effects of microRNAs, and if one could reverse this shortening, that might be a way to bring cells back under the control of microRNAs.

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