Researchers from Mount Sinai School of Medicine this month published details on the development and application of two new tools for examining the function of microRNAs in the cell, reporting data that suggest that most miRNAs — even some that are highly expressed — may have far less functional activity than expected.
Specifically, the team found that nearly 60 percent of the miRNAs they detected via deep sequencing had “no discernible suppressive activity,” supporting the hypothesis that miRNAs expressed in low numbers in the cell have a small impact on target suppression, they wrote in Nature Methods.
In addition to indicating that a significant portion of the reported miRNome of a cell may have “little biological activity,” the findings highlight the need to consider both absolute expression and differential expression when examining miRNA signatures, they added.
According to Brian Brown, senior author of the paper, he and his colleagues have long been interested in the relationship between miRNA concentration and target concentration, and developed tools based on existing technologies to explore this issue.
The first tool is an miRNA decoy library that takes advantage of the so-called miRNA sponges developed several years ago by several labs working independently, including Phillip Sharp's at the Massachusetts Institute of Technology (GSN 8/31/2007).
“The principle of the technology is that if you over-express enough of the target of a microRNA, you end up saturating it — you [add] so much synthetic substrates that the microRNA can't regulate its natural target,” Brown explained to Gene Silencing News this week.
Although Sharp and others had previously created miRNA sponges, or decoys, against mammalian miRNAs, Brown's team built a library comprising 600 vectors against 300 different miRNAs, which they showed can be used in a pooled manner for loss-of-function studies.
More importantly, the Mount Sinai investigators built an miRNA sensor library that was used with a high-throughput assay dubbed Sensor-seq to quantify the activity of hundreds of miRNAs simultaneously, which revealed that “only the most abundant miRNAs in a cell mediate target suppression,” according to the paper.
To generate the sensor library, the researchers synthesized target sites for 291 miRNAs conserved between mice and humans “as five tandem copies with either perfect complementarity or mismatches at nucleotides 10 and 11 of the miRNA,” they wrote in Nature Methods.
The mismatched configuration, they noted, creates a bulge that prevents Argonaute 2-mediated slicing and results in the transcript being regulated in the more common non-slicing pathway.
Notably, “we made the sensors more sensitive than a natural target,” Brown said. “We souped up the system so it would be extremely responsive to the concentration of microRNAs in the cell, and the fact that we can look at 300 microRNAs simultaneously using this assay allowed us to … [examine] how many actual molecules of microRNA are needed to regulate even a very sensitive target.”
“An outstanding question in miRNA biology is how miRNA concentration relates to target suppression,” the team wrote. “Deep sequencing indicates that hundreds of miRNAs are expressed in a cell, but how many of these are functional is not known.”
To tackle this problem, Brown and the other researchers used the sensor library to study the expression of the monocyte miRNome via deep sequencing and quantitative PCR, and detected more than 310 miRNAs.
“Our library included sensors for 165 of these miRNAs (188 when considering families), but we detected the suppression of only 67 sensors,” they wrote. “Thus, 59 percent of the expressed miRNAs that we sampled did not have suppressive activity.”
For around 80 percent of the sensors that were significantly suppressed, the corresponding miRNA was expressed above 100 reads per million, they noted.
“The cognate miRNAs of some suppressed sensors were not highly expressed, but many of these were part of a miRNA family in which one of the family members was highly expressed, such as the miR-17 or let-7 family,” they added. “Because the targets of one miRNA are subject to regulation by all family members, we refined our analysis by considering the cumulative concentration of an entire miRNA family.”
They found that the majority of suppressed sensors corresponded to miRNAs or miRNA families that expressed above 1,000 reads per million.
“Although the observed activity threshold may reflect the sensitivity of Sensor-seq, by incorporating multiple, sometimes perfectly complementary, target sites, and by expressing the reporter at physiological levels, our sensors are more sensitive to regulation than a natural target,” the investigators wrote. “Thus, our results strongly suggest that miRNAs expressed below 100 to 1,000 [reads per million] could not themselves mediate substantial regulation of a natural target.”
To Brown, these findings confirmed his belief that miRNAs expressed below around 100 copies per cell have little regulatory capacity, and may help in overcoming the resistance to this notion that he has encountered.
Some in the field wonder, “'How can the microRNA be there if it's not doing anything?'” he said. “Part of the problem is that people profile microRNA and think about it like an RNA. I think you should think about a microRNA more like a protein. Essentially, a microRNA is the recognition domain of a protein — Argonaute.”
In Nature Methods, the researchers also suggested that one explanation as to why miRNA concentration must be high for target regulation is that a “high abundance is necessary to facilitate target interaction [since] ... miRNAs must locate their targets in a cell through diffusion and sampling. The rate of interaction between a low-abundance miRNA and an mRNA may be slower than the rate of mRNA production and natural decay, and thus have little effect on the mRNA’s expression level.”
As for why there are so many low-abundance miRNAs that are reliably detected in a cell but that have little capacity for target regulation, they said that the stability of miRNAs may be a root cause.
“Most of the genome is transcribed at low levels, but because mRNAs have a short half-life, an aberrantly transcribed gene may not reach a perceptible abundance,” they wrote. “However, because miRNAs generally have a long half-life, even low-level production can result in a consistent presence.”