Efforts to uncover the mechanism through which microRNAs regulate gene expression have thus far resulted in a number of different, and sometimes conflicting, hypotheses.
But according to one researcher, the challenge of pinning down precisely how and to what extent these small RNAs act may be due in part to the type of cellular systems in which the experiments are conducted.
Speaking at this year’s American Society of Gene Therapy meeting in Boston last week, Timothy Nilson, director of the Center for RNA Molecular Biology at Case Western Reserve University, noted that the majority of research looking at miRNA regulation has been done in traditional cultured cells such as HeLa or Drosophila S2, which don’t necessarily reflect the real-life situations in which miRNAs function.
Instead, researchers may want to consider expanding their investigations into different cell types, he said, adding that “I think that, perhaps, it may be time for a fresh start.”
“In general, it’s been accepted that microRNAs down regulate gene expression … by binding to 3’ UTRs of target mRNAs and that this binding leads to a repression of protein synthesis from targeted mRNAs either by degradation or in some way interfering with translation,” Nilson said.
It is also generally believed that miRNAs are synthesized in the nucleus as long precursors called pri-miRNAs, processed into pre-miRNAs, then exported to the cytoplasm where they are cleaved by Dicer into mature miRNAs that are incorporated into so-called miRNA-containing ribonucleoproteincomplexes, or miRNPs.
In the end, though, “if you can think of a mechanism [by which miRNAs function], it’s been proposed, and actually there is good data for [them] all,” Nilson said. Overall, it is important to keep in mind that “the biogenesis of all microRNAs is thought to be the same, the assembly into RISC … is thought to be the same, [and] microRNAs in general, despite the differences in their sequence, are all physically the same.”
But despite these similarities, “the magnitude of regulation can vary dramatically, from less than two-fold to greater than 10-fold,” although most regulation appears to be “quite modest,” he said. Furthermore, “the levels of regulation … are not strictly correlated with the number of target sites or the degree of pairing” of the microRNAs.
“We’ve been unable to reproduce most of the data that has been published by other labs. [As such], we decided to take a different path, which is to look at a differentiation system … [which] throws a new wrinkle into mechanisms of microRNP regulation.”
“There are examples … where there is one site that can give very dramatic regulation [and] there are other examples where four or five sites give one-and-a half-fold type regulation,” Nilson said.
Further complicating the issue is the recent finding that certain miRNAs can trigger up-regulation of gene expression, he added, although “it’s not clear at the moment whether this … phenomenon” can be generalized across all miRNAs.
With all this as a backdrop, Nilson said that he and his colleagues at the Center for RNA Molecular Biology set out to determine what factors are involved in determining the magnitude of gene regulation observed with miRNAs.
“We’ve been unable to reproduce most of the data that has been published by other labs,” he noted. As such, “we decided to take a different path, which is to look at a differentiation system … [that] throws a new wrinkle into mechanisms of microRNP regulation.”
Nilson said that his team aimed to follow up on findings from a key paper published in Nature in 2005 that suggested tissue-specific miRNAs could down-regulate a greater number of targets than previously believed.
Specifically, that paper shows that that delivering miR-124 into HeLa cells caused their mRNA expression profile to “shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed.”
Nilson noted that the levels of regulation detailed in that paper were “very small … and we wanted to see how [the mRNA] would behave in a real muscle cell.”
To do so, Nilson’s team used C2C12 skeletal muscle cells, which, when deprived of serum, will differentiate into developing muscle fibers called myotubes. Importantly, it has been shown that this differentiation process induces three highly conserved, muscle-specific miRNAs: miR-133, miR-206, and miR-1.
“We did the HeLa cell experiments [described in Nature] as well and [saw] about two-fold regulation of these constructs,” Nilson said at ASGT. However, in differentiated C2C12 cells “the magnitude of regulation goes up significantly,” by approximately 12-fold.
“What is going on? There are several possibilities,” he said. “It could be that the cell state in the differentiated myotube in some way promotes the ability of the microRNP to act. We think it is more likely that there is a muscle-specific factor that interacts with [proteins that make up RISC] such that the level of regulation is much higher in the differentiated cells.”
Nilson said that his lab is currently making stable C2C12 cell lines in order to identify the mechanism of the miRNA action in the cells and is purifying miR-1 from the differentiated cells to locate the factors that might be at work.
As it stands now, though, he said it seems likely that specific miRNAs interact with specific factors that affect levels of gene-expression regulation, and that these factors vary in different cell types.
“Most, if not all, of what has been done … in terms of determining the mechanism of regulation and magnitude of regulation … [has been done] in normal tissue culture cells,” Nilson said. “We would suggest that there is something quite different in a differentiated muscle cell.
“I would be very cautious of the mechanisms that have been worked out in rapidly proliferating cells like HeLa cells, which have been constantly growing since 1948 … [and] may have adapted to avoid microRNA regulation,” he added.