Investigating Translational Repression by MicroRNAs in Arabidopsis

Regulation of gene expression by microRNAs (miRNAs) is an important facet of many developmental and physiological processes in both plants and animals. miRNAs are small (∼20 to 24 nucleotides) RNAs that recognize target genes based on sequence complementarity and typically act to repress expression of the target gene, either through cleavage of the target mRNA or translational repression independent of mRNA cleavage. Research to date suggests that most animal miRNAs function by translational repression, whereas most plant miRNAs trigger mRNA cleavage (e.g., Axtell and Bowman, 2008; Mallory and Bouché, 2008). A recent report provided evidence that translational inhibition by miRNAs and other small interfering RNAs in fact is widespread in plants (Brodersen et al., 2008). Now, Lanet et al. (pages 1762–1768) present more direct biochemical evidence for translational repression by miRNAs in Arabidopsis.

The authors assessed the subcellular distribution of several highly expressed miRNAs using polysome fractionation of cytoplasmic extracts from Arabidopsis seedlings. The results showed that miRNAs are associated with active polysomes via association with their target mRNA. miRNAs are formed by cleavage from longer double-stranded RNA precursor molecules by the action of Dicer-like RNases. Only one strand of the resulting miRNA duplex (termed miR/miR*) is the active, silencing strand, which forms a silencing effector complex with an Argonaute (AGO) protein. The miR* strand does not form part of this complex and does not have silencing activity. For one of the highly expressed miRNAs, miR168, Lanet et al. investigated subcellular localization of the nonactive miR168* strand. In contrast with miR168, miR168* was found only within untranslated cytoplasmic fractions, suggesting that only the active miRNA strand associates with polysomes (see figure). Further experiments suggested that miRNAs targeting translatable mRNAs are more likely to be associated with polysomes than miRNAs targeting noncoding mRNAs. In addition, the AGO1 protein, which associates with many miRNAs and has been implicated in both translational inhibition and mRNA cleavage, was also found to be associated with polysomal fractions.

Figure 1.

Functional microRNAs are associated with polysomes in Arabidopsis. At top, absorbance (OD) at 254 nm is from heavier (left) to lighter (right) fractions of a continuous sucrose gradient. Polysomes (Poly.) are fractions 1 to 3; monosomes (Mono.), fraction 4; and supernatant (SN), fractions 5 and 6. Bottom panels are RNA gel blots from total RNA isolated from each fraction showing that functional miR168 is associated with polysomes, whereas inactive miR168* is not.

The authors next investigated whether the association of miRNAs with polysomes is linked to translational repression, using mutant and transgenic plants with altered AGO1 activity: the ago1-25 mutant that has a point mutation in AGO1 and transgenic plants expressing Cucumber Mosaic Virus 2b protein that specifically inhibits mRNA cleavage activity of AGO1. In the 2b transgenic plants, miRNA-directed mRNA cleavage was lost but translational repression persisted, whereas both activities were lost in the ago1-25 mutant. miR168 association with polysomes was lost in ago1-25 mutants but persisted in 2b overexpressing plants, further indicating that the presence of miRNAs and AGO1 in polysomes is correlated with translational repression.

This work provides biochemical evidence for translational repression as a component in the plant miRNA pathway. As with the work of Brodersen et al. (2008), the results are certain to generate many intriguing questions about the nature of translational repression in plants. For example, what mechanisms discriminate between mRNA cleavage and translational repression, and what other components are involved in each process? What is the importance of translational repression relative to mRNA cleavage in plant development and physiology?