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Small RNAs with a big effect

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by Carla B. Mellough

MicroRNAs (miRNAs) are endogenous RNAs that direct the repression of protein coding genes. The way in which miRNAs elicit their effects on protein production has been an issue of great debate, whether they act to decrease translational efficiency, decrease messenger RNA (mRNA) levels or both. Recent results published in Nature from the laboratory of David Bartel at the Whitehead and Howard Hughes Institutes, Massachusetts, provides an insight into the molecular consequences of these small regulatory RNAs1.

Initially thought to have no influence on mRNA levels and repressing protein output only (the ‘translational-repression’ model), subsequent work demonstrated miRNAs to decrease target mRNA levels by increasing the rapidity of mRNA degradation via the normal pathways for this process. This was complicated by the fact that repression mediated by heightened mRNA degradation was found to occur at modest levels only, favouring the translational-repression model. Various experiments utilising high throughput analysis however indicated that although miRNA-mediated mRNA destabilisation (the ‘mRNA destabilisation’ model) is modest, this represents the majority of miRNA-mediated repression.

To clarify this Guo et al.1 use ribosome profiling to assess the molecular impacts of miRNA expression on protein production and mRNA levels in mouse and human cells. Ribosome profiling generates data on the cell at a particular point in time (preventing distortion of detected mRNA destabilisation and protein decreases often associated with proteomics approaches) and allows detection of more modestly expressed mRNAs or proteins that often go undetected. This method also maps, at sub-codon resolution, the position of translating ribosomes in cells. To allow comparison between the methods adopted in the current study and others previously reported using more general methods, the authors examined the effects of introducing foreign expression of miR-1 or miR-155 into HeLa cells and knocking-out mir-223 in mouse neutrophils, on which the results of previous experiments examining the effect of these on thousands of proteins had already been reported. In order to assess whether their results supported the translation-repression scenario (repression of translation without a decrease in mRNA levels), Guo et al. removed the component of ribosome protected mRNA fragment (RPF) change attributed to miRNA-dependent changes in poly(A) mRNA, with that left over representing the change in ribosome density and thus a truer calculation of translational efficiency. Their results revealed that across all three experiments, a decrease in translational efficiency was observed in miRNA target sites resulting in fewer ribosomes on target mRNA sites, but that this effect was only a modest decrease amounting to a 7% reduction in translational efficiency. The conclusion here is that only a small proportion of observable repression (11-16%) is attributable to reduced translational efficiency for both ectopic and endogenous regulatory mechanisms. A great proportion of the repression observed (84%) was attributable instead to decreased mRNA levels.

For highly expressed genes targeted in their 3’UTRs (mapped to 3’-untranslated regions) downregulation of mRNA and ribosome drop-off was observed across the length of composite open reading frames (ORFs). But what about more modestly expressed genes? When miR-155 target sites were compared to genes without targets, fewer mRNA-Seq tags were observed along the ORF with a reduction in RPFs, but with a uniform reduction in translation.

The results of this study support the mRNA-destabilisation scenario for most targets, but do not rule out the possibility that in a subset of targets the translational-repression scenario might apply. Notwithstanding, analysis of the results of this study led the authors to conclude that miRNAs predominantly act to decrease mRNA levels in nearly all targets. They provide conclusive evidence that miRNA regulation occurs predominantly via mRNA destabilisation, as first suggested from proteomic analysis2 but in contrast to the translational repression dogma.

Clearly much remains to be determined regarding which targets are outliers to this trend and how repression mechanisms differ depending on the message and targets, but one thing is now clear: that these small, endogenous RNAs have a big effect on decreasing target mRNA levels.

 

References

1. Guo et al. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature. 466(7308):835-40.

2. Baek et al. (2008) The impact of microRNAs on protein output. Nature 455(7209):64-71.