In the roughly 10 years since lin-4 became the first microRNA discovered, hundreds more have been identified in mammals, invertebrates, and plants. And yet some of the most fundamental questions about miRNAs remain unanswered. With the help of a three-year grant from the National Science Foundation worth $420,000, Yale researcher Frank Slack aims to help remedy this situation.
Slack said that the NSF-funded project — which is set to begin on Jan. 15 this year and end on Jan. 31, 2007 — has two goals: The first is to “understand how… these microRNAs are expressed only at particular times during development — what are the transcriptional inputs to these microRNAs that turn them on only at a particular time?” The second is figure out just what these so-called temporal miRNAs are doing.
“My lab’s main interest is in the timing of development, and through that interest we stumbled upon … miRNAs, because two of them turn out to be very important for the timing of C. elegans development,” Slack told RNAi News. “They come on only at particular times during development — one’s called lin-4 and one’s called let-7, and they’re sort of the founding members of the microRNA family.”
Specifically, lin-4 acts as a timing switch to initiate a cascade of developmental changes in the somatic cells between the L1 and L4 stages, the NSF grant’s abstract explains, while let-7 turns on in the L4 and adult stages.
Studying these miRNAs “has become more relevant recently because lin-4 and let-7 have both been discovered to have human and fly and mouse … homologues,” Slack said. “And these RNAs are temporally regulated in those species,” as well. “What we’re hoping is that what we discover in C. elegans will also be true in those other organisms,” he added.
Slack noted that since the miRNA families appear to be regulating “important developmental steps, the timing of when a cell will differentiate for example, we predict that these RNAs are going to be doing a similar thing in humans.”
If true, he said, this means that “these genes are going to turn out to be oncogenes or tumor-suppressor genes, because they’re timing when cells stop dividing. There is an increasing number of papers where people have implicated microRNAs in cancer,” he added.
Among these, said Slack, is a paper in Molecular Cancer Research by Michael Michael and colleagues at Flinders University of South Australia, which identified miRNA sequences associated with colorectal cancer (See Molecular Cancer Research, October 2003; 1: 12, 882-891).
Slack also cited a paper in the Journal of Virology by Wayne Tam and colleagues from the Joan & Sanford Weill Graduate School of Medical Sciences of Cornell University, Memorial Sloan-Kettering Cancer Center, and the NCI Frederick Cancer Research and Development Center (See Journal of Virology, May 2002; 76: 9, 4275-4286); as well as a paper published in the Proceedings of the National Academy of Sciences by Carlo Croce from Thomas Jefferson University and colleagues from the National Institute for Research on Cancer, the University of Texas MD Anderson Cancer Center, the Long Island Jewish Medical Center, and the University of California at San Diego (See PNAS, Nov. 26, 2002; 99: 24, 15524-15529).
In terms of microRNAs linked to cancer, Slack said, “in some cases they’re mutated, and in others they are just poorly expressed. I would guess that many of these would turn out to be tumor suppressor genes,” he noted.
While in the future this kind of miRNA research may yield treatments for cancer, by delivering a specific miRNA to an oncogene for example, Slack pointed out that right now such things are “pies in the sky.
“The delivery process is just not there yet,” he said. “You’ve heard about the problems of getting siRNAs into animals — it’s even more difficult to get a microRNA in.”
First Things First
Slack said that his miRNA project has roots in work conducted by the Whitehead Institute’s Dave Bartel, who showed that a number of other miRNAs in C. elegans besides lin-4 and let-7 “also come on at particular times during development. [Bartel] found about 20 that are very robustly turned on at any one particular time during development,” he added.
We’re also interested in what those microRNAs might be doing and what their targets might be,” Slack said. And while he initially proposed that his lab study all of them under the project, “the reviewers thought that might be a little too ambitious, so we cut the number down to just the microRNAs that are in the let-7 and lin-4 families.” He noted that there are four mi- RNAs in C. elegans that are “very similar to let-7, and there are two that are similar to lin-4.”
According to the grant’s abstract, Slack and his colleagues will determine the expression pattern of the lin-4 and let-7 family miRNAs by developmental Northern blot and GFP fusion technology. At the same time, the abstract states, the project will also genetically test the developmental role of the mi-RNAs by examining the phenotype resulting from over-expression of let-7 and lin-4 homologs in a wild-type background.
It’s not exceedingly novel, but what we discover in terms of targets might be novel,” Slack said.
The basic thing that we’re learning from this is that all of these microRNAs come on at particular times and in a particular cell,” he added. “There is some overlap in when and where they’re expressed, but for the most part they seem to be non-redundant in terms of their expression pattern.”