An Arizona State University team has used a unique high-throughput screening system to complete the largest-ever analysis of microRNAs (miRNA), the puzzling little cousins of RNA that help regulate gene expression.
“People normally do this for one or two genes because it takes a lot of time to perform these experiments. But we have optimized this to do it in high-throughput,” said Marco Mangone of ASU’s Biodesign Institute, an author on the study.
Their findings, published in the online advance edition of Genome Research, also offer a new explanation for why groups of similar miRNA sequences, called miRNA families, are so plentiful in higher species of animals, including humans.
Measuring only around 22 nucleotides long, miRNAs cannot code for proteins. Instead, like a volume knob, they can mute gene expression, binding to and destroying messenger RNA, or let it go full blast — which is often the difference between health and disease.
The high-throughput system sequenced 2,000 miRNA targets, or 10 percent of the human total. Analyzing the results has provided a clearer picture of which genes certain miRNA target and repress, said Mangone.
“So you can say, ‘This microRNA can target gene Y, X and Z.’ And then you look in a specific cancer or in a specific disease, that microRNA is either too much abundant or not there at all. So you know already that, if this microRNA is there, and these genes are there as RNA, they’re going to be repressed.”
Discovered in 1993 in the nematode C. elegans, miRNAs were once considered the measly runt leftovers of a time when RNA, not DNA, dominated life processes on Earth.
But as time passed, scientist uncovered miRNA throughout higher life forms, including humans, in vast numbers and with sprawling variety. A new idea gradually took hold - that the small regulatory molecules had an outsize role to play in the degree to which genes are expressed.
“It turns out that if you could sequence the RNA in the cell — all the RNA present in the cell – and all the protein present in the cell, you never get a 100 percent correlation, one-to-one. And the reason is because you have these reactive little RNA, these microRNA, that bind on messenger RNA and repress the expression of this messenger RNA to protein,” said Mangone.
The study bolstered the idea that miRNA don’t glom onto genes randomly; rather, they target biological pathways — groups of closely related processes that alter a cell or make a product.
This capacity to mute gene expression helps to explain why miRNA is useful, but it only partly answers a fundamental question in the field: Why do they occur in the thousands in higher animals, including humans, when worms only have a relative few?
“What adds to the mystery is that the number of genes that humans and worms have, in terms of genes that they both have, it’s very similar — it’s between 20-25,000 protein-coding genes. So the question is, ‘How come the worms can develop and live only with few microRNA, and humans, having the same number of genes, instead, they need a lot more?’” said Mangone.
It also makes miRNA, when something goes wrong, prime suspects in a large number of diseases, said Mangone.
“I mean, at this point, you just name the disease you want — Alzheimer’s, Parkinson’s, autism … these microRNAs are misregulated, either over-expressed or not present anymore.”
The ASU lab developed a high-throughput system called “3’ LIFE”, Luminescent Identification of Functional Elements in 3’ UTRs (untranslated regions), to study how miRNAs bind to 2,000 human genes. Unlike indirect, predictive systems, 3’ LIFE actually clones the binding sites of genes and uses a luminescence test to physically check whether a given miRNA can target a given gene. This functional assay approach has the additional advantage that it indicates the strength of the binding.
“People normally do this for one or two genes, because it takes a lot of time to perform these experiments. But we have optimized this to do it in high-throughput,” said Mangone.
Mangone said the goal was to learn “the language” that miRNA use to target specific genes, “and eventually use this language in our own advantage and develop treatment that can allow us to control their expression, regulate their expression in cells, in order to restore normal state, that normally are not present anymore in normal diseases.”