BioWorld International Correspondent

LONDON - A tiny fragment of RNA has turned out to be a master regulator of the process of cell differentiation in a type of human blood cell.

A group of Italian researchers has elucidated both the role of the RNA fragment and the molecules that act as brake and accelerator to reduce or boost its levels. The discoveries could provide new targets for therapies to treat leukemia.

Irene Bozzoni, professor of molecular biology at the University of Rome, told BioWorld International, "We have worked out how this microscopic molecule - only 22 nucleotides long - provides the crucial means of telling a precursor blood cell what type of cell it has to become."

Bozzoni, together with collaborators also at the University of Rome, reported the study in the Dec. 1, 2005, issue of Cell in a paper titled: "A Minicircuitry Comprised of MicroRNA-2223 and Transcription Factors NFI-A and C/EBPa Regulates Human Granulopoiesis."

The RNA fragment that the group identified belongs to the micro-RNAs (miRNAs) class. The first one was discovered in 1993 in the worm, Caenorhabditis elegans. The second was not discovered until 2000, but also in C. elegans.

Since then, however, scientists have discovered miRNAs throughout the plant and animal kingdoms. Cloning techniques have identified almost 400 in mammalian genomes, and methods based on predicting miRNAs from DNA sequences suggest that there may be up to 800 miRNAs in mammalian genomes.

Several studies already have shown that miRNAs appear to play a key role in cellular differentiation. One group, for example, showed that a specific miRNA was expressed during hematopoietic differentiation in mice; inducing the expression of that miRNA in precursor cells resulted in the cells differentiating.

Bozzoni said, "We were inspired by the work, and we decided to investigate further, as we had cell lines of precursor hematopoietic cells that we could induce to differentiate in vitro into mature blood cells."

During hematopoiesis, some stem cells develop into precursor blood cells called promyelocytes, which contain a few granules. Eventually those cells develop into the class of heavily granulated white blood cells called granulocytes.

In people with the subtype of acute myeloid leukemia, called acute promyelocytic leukemia (APL), the promyelocytes accumulate. Those immature cells build up in the bone marrow and the blood, replacing normal blood cells.

The cell lines available to the Italian group were promyelocytic precursor cells that, when treated with retinoic acid, develop into mature granulocytes. The group's studies showed that adding retinoic acid to the cells caused them to make more of a specific miRNA, called microRNA-223 (miR-223). Cells from patients with APL showed the same change following retinoic acid treatment - up-regulation of miR-223.

Further investigations showed that the promoter of miR-223 contained the binding site for a transcription factor called C/EBPa. That molecule, the researchers knew, previously had been shown to be important for granulocyte differentiation and was known to be activated by retinoic acid.

"Even more interestingly," Bozzoni added, "we found that the binding site for the promoter also displayed sequence specificity for another factor, called NF1-A. That molecule is a transcriptional factor previously shown to control many genes involved in cell proliferation."

The paper explained how, before the precursor cells differentiate, NF1-A binds to the promoter of miR-223. After retinoic acid treatment, C/EBPa displaces NF1-A, and strongly activates transcription of the gene, leading to greater expression of miR-223.

MiR-223, like most miRNAs, controls hundreds of messenger RNAs, and by binding to the 3' region of the messenger RNA, prevents translation. Bozzoni and her colleagues established that one of the messenger RNAs that miR-223 controls is derived from the gene that encodes NF1-A.

Bozzoni explained: "This is a really interesting loop. When miR-223 represses NF1-A, this is a way of really changing the status of the cells. It is possible that the shut-down of NF1A is important for blocking cell proliferation and that this allows the cell to differentiate."

Working on the hypothesis that miR-223 may be able to promote differentiation in precursor promyelocytic cells, even in the absence of retinoic acid, Bozzoni concluded, it might be possible to develop new treatments for that type of leukemia.

"We may be able to do this by knocking down NF1-A or by getting progenitor cells to overexpress miR-223," she said.