by Monya Baker
A new screening technique identifies molecules that control the cell cycle
Short stretches of RNA molecules known as microRNAs (miRNAs) coordinate gene expression. Although their importance as gene regulators is now accepted, it can be hard to figure out the functions of particular miRNAs. An individual miRNA molecule can interact with multiple gene transcripts, and multiple miRNAs often serve similar functions and so mask each others' effects.
Using a new screening technique, Robert Blelloch and colleagues at the University of California, San Francisco, have identified a collection of miRNAs that allow embryonic stem (ES) cells to divide rapidly1. The team had previously engineered a line of mouse ES cells that cannot process miRNA because they lack a crucial gene called Dgcr8. Unlike normal ES cells, these knockout ES cells do not proliferate rapidly. By introducing commercially available miRNA mimics, however, the researchers were able to get the cells to start dividing more quickly. After screening nearly 300 mouse miRNAs, the researchers found 14 that greatly improved proliferation of the Dgcr8-knockout ES cells but had no effect on wild-type ES cells. The researchers then concentrated their analysis on five of the miRNAs that are also highly expressed in human ES cells.
As cells divide, they go through a set series of steps collectively known as the cell cycle; the knockout ES cells get stuck in what is called the G1–S transition, the stage immediately before cells start replicating their DNA. Therefore Blelloch and colleagues looked for interactions between the identified miRNAs and proteins that regulate this part of the cell cycle. Computer analysis showed that all five of the identified miRNAs could potentially silence a gene that encodes a protein called P21, which inhibits one of the cyclin-dependent protein kinases, enzyme complexes that help cells move through the cell cycle; specifically, it inhibits cyclinE–Cdk2. At the G1–S transition, levels of the P21 protein are low in wild-type ES cells. But when the P21 gene was overexpressed, the wild-type ES cells behaved in ways similar to the Dgcr8-knockout cells.
The actions of the miRNAs seem more complex than a single miRNA preventing expression of a single protein. Eleven of the 14 miRNAs share a similar 'seed sequence', the sequence by which they are targeted to a messenger RNA, and Blelloch says he was surprised both by the level of redundancy the miRNAs have with each other and by the number of genes a single miRNA can affect. "It's not entering the pathway but entering the pathway at multiple points," he says. "I think we'll see that paradigm over and over."
Besides being less able to proliferate, Dgcr8-knockout ES cells are also less able to differentiate, and Blelloch is looking for another suite of miRNAs to explain this effect. In fact, he says, miRNA's roles in controlling the cell cycle and differentiation might be intertwined, as halting a cell's progression through the cell cycle gives it more time to respond to cues about how it should differentiate.
"This is important work," says Jeanne Loring at the Scripps Research Institute in San Diego, California, who is studying the regulation of pluripotency in human cells. "We've had to look at miRNAs at a group, and they've been able to look at them one at a time." She says the redundancy and multifold effects of miRNAs observed by Blelloch and colleagues is "good news" for researchers who hope to use miRNAs to manipulate the fate of pluripotent stem cells.
Source: Nature