Novel Method Described for Blocking Action of Cancer-Promoting MicroRNA

MicroRNAs (miRNAs) are a small noncoding family of 19- to 25-nucleotide RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, inducing translational repression or mRNA degradation, depending on the degree of complementarity between miRNAs and their targets. Many miRNAs are conserved in sequence between distantly related organisms, suggesting that these molecules participate in essential processes. In fact, miRNAs have been shown to be involved in the regulation of gene expression during development, cell proliferation, apoptosis, glucose metabolism, stress resistance, and cancer.

In order to block the activity of the miRNA miR-21, which is present in high levels in many tumors where it increases the expression of cancer-promoting genes and decreases cancer suppressors, investigators at the University of Washington (Seattle, USA) manipulated the specificity of the RNA recognition motif (RRM) of the protein Rbfox2 (RNA binding protein, fox-1 homolog 2). They did this by engineering the conserved RRM of the Rbfox2 protein to specifically bind to the terminal loop of the microRNA precursor (pre-miR-21) with high affinity.

The modified pre-miR-21 could not be processed by the enzyme Dicer. Dicer, which is encoded by the DICER1 gene, trims double stranded RNA, to form small interfering RNA (siRNA) or microRNA (miRNA). These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation.

In a separate series of experiments the investigators attached the miR-21 binding regions of Rbfox2 onto the Dicer enzyme. This hybrid Rbfox2-Dicer protein sliced miR-21 into inactive fragments. Results published in the July 18, 2016, online edition of the journal Nature Chemical Biology revealed that the hybrid enzyme degraded pre-miR-21 specifically in vitro and suppressed mature miR-21 levels in cells, which resulted in increased expression of the tumor suppressor PDCD4 and significantly decreased viability for cancer cells.

"What we show here is a proving ground - a process to determine how to make the correct changes to proteins," said senior author Dr. Gabriele Varani, professor of chemistry at the University of Washington. "This method relies on knowledge of high-quality structures. That allowed us to see which alterations would change binding to the microRNA target."

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