Tool dissects small RNA functions
attention paid to finding cellular factors that control gene
expression. Micro RNA (miRNA) - small sequences of 20-24
nucleotides that do not code for proteins - are thought to be
involved in this process, but it has been difficult to work out
exactly what they do in the cell.
In mammals there are often multiple versions of the same miRNA genes which makes them difficult to study by using gene deletion approaches, and conventional in situ hybridisation experiments are unreliable as miRNAs are too short to generate a signal. Now, researchers have developed a way of using antisense - oligonucleotides that bind to and block RNA - to knock out specific miRNA sequences.
The technique stops the miRNA fulfilling its function in the cell, and researchers can look at the effects of this to help identify its role.
And if it turns out that miRNA are indeed involved in the control of gene transcription - and studies suggest that the proportion of the genome made up by transcription factors is very close to that taken up by these sequences - then miRNA could emerge as a new target for interventions aimed at treating disease, or as a therapeutic in its own right.
The researchers have developed a way of modifying antisense sequences chemically so that they can effectively bind to miRNA. They created oligonucleotides that bind with the miRNA sequence and stabilised them using a 2'-O-methyl group side chain.
This prevents the antisense compound from breaking down, but does not interfere with its binding activity. The process can be used to knock out miRNA in cells, tissues and even in vivo, they report, allowing researchers an unprecedented glimpse at the regulatory roles and mechanisms that lie behind RNA's effects on gene function.
They first tested the approach in slightly longer RNA sequences, known as small interfering RNA (siRNA), that are already the subject of intensive research for their ability to inhibit gene expression. The results indicated that the inhibitor could block the activity of an siRNA sequence designed to switch off the gene for luciferase, a commonly-used marker gene that produces a fluorescent protein.
The acid test of the approach, however, came when they used the technique to knock out one of the handful of miRNAs whose function is already described.
They constructed an oligonucleotide inhibitor based on the sequence of a microRNA called let-7, which blocks the production of the protein Lin-41 and is important for proper developmental timing in roundworm larvae. Larvae injected with the oligonucleotide had the exact features of a let-7 deficient worm, showing that the inhibitor did indeed block this microRNA's function.
Other challenges remain before miRNAs can break out as a real prospect in drug discovery. For example, no-one has been able to isolate a complex between an miRNA and its target in order to explore the mechanism of gene repression further.
However, the research also uncovered clues into the mechanisms behind siRNA's ability to silence genes. Using different concentrations of siRNA and the antisense sequence, they established that a protein seems to be involved in the formation of the RNA-induced silencing complex (RISC) that inactivates genes. This protein acts as a rate-limiting step, so above a threshold level adding more siRNA does not lead to the formation of any more RISCs.
And this will have an impact on genetic studies that use RNA interference to uncover the function of sequenced, but unknown, genes; knowing the minimum required concentration of siRNA, researchers can avoid a buildup and any unwanted cell activity that goes along with it.
