Far from being junk, these short stretches of the genome exert wide-ranging influence in the cell Thursday, 31 July 2008 MicroRNAs (miRNAs) subtly influence a vast number of proteins involved in most key biological processes, according to the first large-scale analyses of how these small pieces of non-coding RNAs affect proteins. The work is presented in two articles in the latest issue of the journal Nature. These studies could help answer long-standing questions about how miRNAs work. These tiny snippets of genome, just 21 to 25 nucleotides long, were once thought to be “junk” material because they are not translated into protein. In fact, in animals they control protein levels through two mechanisms: by breaking down the messenger RNA “read-out” from a gene; or by stopping the messenger RNA from being translated into protein. But previous studies had taken only a global look at how miRNAs affect messenger RNA, because of the technical difficulties in examining how miRNAs affect thousands of proteins at once. Two teams of scientists have now tackled this problem using a state-of-the-art version of mass spectrometry. The technique, called SILAC (stable isotope labelling with amino acids in cell culture), relies on the use of heavy isotopes to label proteins as they are being produced by cells. The researchers used the isotopes to label thousands of proteins being made in cells that had been forced to over-express or down-regulate certain miRNAs. They then compared labelled protein levels in these cells with levels of corresponding proteins in control cells. One group of researchers come from the Max Delbrück Center for Molecular Medicine (MDC) in Berlin-Buch, Germany. Two teams lead by Matthias Selbach (Proteomics) and Nikolaus Rajewsky (Systems Biology) have shown that a single miRNA can directly regulate synthesis of hundreds of different proteins. In this way, miRNAs can program the way human cells act, they report. All body cells contain the same genes, may they be muscle-, brain-, blood-, or liver cells. Therefore, all have the same blue prints for the production of proteins. However, different cells produce different proteins at different times – a prerequisite for the body to develop normally and stay healthy. For this to happen, genes must be regulated differently in different cell types – that is, turned on and off at the right time. Only a few years ago, researchers discovered that miRNAs play an important role in gene regulation and, thus, help determine which proteins are produced by which cells. Proteins are the building and operating materials of life and are required for the structure, function, and regulation of the body's cells, tissues, and organs. Diseases can result if protein production goes awry. Worldwide, scientists are seeking to develop methods to detect which miRNAs are active in tissue samples and which proteins are regulated by them. To date, researchers have identified a few hundred human miRNAs but it is not clear which proteins they regulate. A further complication is that miRNAs are known to regulate synthesis of proteins, which is difficult to measure. Using a novel experimental approach carried out by PhD students Björn Schwanhäusser and Nadine Thierfelder, the MDC researchers for the first time were able to quantify protein synthesis for thousands of different human proteins. Together with extensive computational analyses, they could further identify and quantify the direct impact of specific miRNAs on target protein synthesis. Changing the fate of a cell The MDC researchers could demonstrate that the regulation of protein synthesis typically is mild, with a number of interesting exceptions. "MicroRNAs screw many switches, but most of them only slightly", Matthias Selbach explains. "Thus the system is robust and flexible. A single miRNA can have profound impact on the fate of a cell. miRNAs active in cancer cells, for example, are different from those active in normal cells." Using a trick, the MDC researchers were for the first time able to measure changes in protein production after artificially changing the activity of specific miRNAs. They labelled amino acids (the building blocks of proteins) with a stable, non-radioactive isotope and put it together with miRNA in cell culture. This allowed them to distinguish labelled proteins in a mass spectrometer. They could show that only newly produced proteins were heavier. A single miRNA can tune the protein levels of thousands of genes The MDC researchers also compared the impact on protein production when artificially boosting or repressing the activity of an individual miRNA. They found that this impact is largely inverse for thousands of different proteins. Thus, “it is as if a single miRNA can alter a large fraction of the entire protein production program of a human cell in a reversible fashion”, comments Nikolaus Rajewsky. The findings of the two Berlin research teams in collaboration with Raya Khanin from Glasgow University (UK) are anticipated to have a big impact in the future, as miRNAs are considered to be promising diagnostic and therapeutic candidates for the treatment of human diseases. The second group obtaining similar results was led by Steven Gygi, a protein chemist at Harvard Medical School in Boston, Massachusetts, and David Bartel, a molecular biologist and Howard Hughes investigator at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts. The most intriguing finding from these studies is that the effects of miRNAs on proteins are usually quite modest, changing their expression levels by less than twofold. Although most miRNA effects may be small, they can still be powerful in the cell. The data paint a picture of miRNAs as regulators that fine-tune protein expression in complex and overlapping patterns. That could complicate the picture for drug developers, because most miRNAs have so many different targets. But this is not necessarily a bad thing, because some of the most effective drugs hit multiple molecular targets. References: Widespread changes in protein synthesis induced by microRNAs Matthias Selbach, Björn Schwanhäusser, Nadine Thierfelder, Zhuo Fang, Raya Khanin & Nikolaus Rajewsky Nature advance online publication 30 July 2008, doi:10.1038/nature07228 The impact of microRNAs on protein output Daehyun Baek, Judit Villén, Chanseok Shin, Fernando D. Camargo, Steven P. Gygi & David P. Bartel Nature advance online publication 30 July 2008, doi:10.1038/nature07242 ......... ZenMaster
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