microRNA-directed Cleavage Research Articles

MicroRNA-directed cleavage of targets: mechanism and experimental approaches.

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators, which are 21-24 nt in length and play a role in a wide variety of biological processes in eukaryotes. The past few years have seen rapid progress in our understanding of miRNA biogenesis and the mechanism of action, which commonly entails a combination of target degradation and translational repression. The target degradation mediated by Argonaute-catalyzed endonucleolytic cleavage exerts a significant repressive effect on target mRNA expression, particularly during rapid developmental transitions. This review outlines the current understanding of the mechanistic aspects of this important process and discusses several different experimental approaches to identify miRNA cleavage targets. [BMB Reports 2014; 47(8): 417-423]

Cascading cis-Cleavage on Transcript from trans-Acting siRNA-Producing Locus 3

The production of small RNAs (sRNAs) from phased positions set by microRNA-directed cleavage of trans-acting-siRNA-producing locus (TAS) transcript has been characterized extensively; however, the production of sRNAs from non-phased positions remains unknown. We report three cis-cleavages that occurred in TAS3 transcripts in Vitis vinifera, by combining high-throughput sRNA deep sequencing information with evolutional conservation and genome-wide RNA degradome analysis. The three cis-cleavages can be deciphered to generate an orderly cleavage cascade, and can also produce distinct phasing patterns. Each of the patterns, either upstream or downstream of the cis-cleaved position, had a set of sRNAs arranged in 21-nucleotide increments. Part of the cascading cis-cleavages was also conserved in Arabidopsis thaliana. Our results will enhance the understanding of the production of sRNAs from non-phased positions that are not set by microRNA-directed cleavage.

Akt-Mediated Phosphorylation of Argonaute 2 Downregulates Cleavage and Upregulates Translational Repression of MicroRNA Targets

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was usedto discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.

RNA Captor: A Tool for RNA Characterization

BackgroundIn the genome era, characterizing the structure and the function of RNA molecules remains a major challenge. Alternative transcripts and non-protein-coding genes are poorly recognized by the current genome-annotation algorithms and efficient tools are needed to isolate the less-abundant or stable RNAs.ResultsA universal RNA-tagging method using the T4 RNA ligase 2 and special adapters is reported. Based on this system, protocols for RACE PCR and full-length cDNA library construction have been developed. The RNA tagging conditions were thoroughly optimized and compared to previous methods by using a biochemical oligonucleotide tagging assay and RACE PCRs on a range of transcripts. In addition, two large-scale full-length cDNA inventories relying on this method are presented.ConclusionThe RNA Captor is a straightforward and accessible protocol. The sensitivity of this approach was shown to be higher compared to previous methods, and applicable on messenger RNAs, non-protein-coding RNAs, transcription-start sites and microRNA-directed cleavage sites of transcripts. This strategy could also be used to study other classes of RNA and in deep sequencing experiments.

MicroRNA-directed cleavage and translational repression of the copper chaperone for superoxide dismutase mRNA in Arabidopsis

microRNA398 (miR398) is a conserved miRNA of plants that targets two of the three copper/zinc superoxide dismutases (SOD) of Arabidopsis (CSD1 and CSD2) by triggering cleavage or inhibiting translation of their mRNAs. We analysed the transcriptomes of mutants impaired in miR398 production, and found that the mRNAs encoding the copper chaperone for superoxide dismutase (CCS1), which delivers copper to CSD1 and CSD2 apoproteins in different cellular compartments, are undiscovered targets of miR398. We identified the cleavage site in CCS1 mRNAs by 5'-RACE PCR. We further show that both CCS1 protein and mRNA levels are tightly linked to the quantities of miR398, which are themselves dependent on the copper content in the medium. We generated transgenic plants carrying a CCS1 mRNA version resistant to cleavage by miR398, and demonstrated that both CCS1 mRNAs and proteins accumulate in these plants when miR398 is abundant and copper limiting. Moreover, we show that one of the ten ARGONAUTE proteins of Arabidopsis (AGO10) is involved in miR398-directed translational inhibition of CCS1 mRNAs, as CCS1 protein, but not CCS1 mRNAs accumulates in ago10 (zll) mutants. Thus, miR398 mediates the cleavage and translational inhibition of mRNAs encoding CCS1, the chaperone protein that is essential for generating the mature copper/zinc SODs of Arabidopsis. Our results also imply that new targets that have not been identified by computing analyses have yet to be discovered, even for an extensively studied miRNA such as miR398.

RNA 1997–2007: A Remarkable Decade of Discovery

The past decade has witnessed spectacular progress in the field of RNA biology and biochemistry; old problems have been solved, and new ones have emerged. This perspective briefly reviews where we are today and where we might be 10 years hence.

A Two-Hit Trigger for siRNA Biogenesis in Plants

In Arabidopsis, microRNA-directed cleavage can define one end of RNAs that then generate phased siRNAs. However, most miRNA-targeted RNAs do not spawn siRNAs, suggesting the existence of additional determinants within those that do. We find that in moss, phased siRNAs arise from regions flanked by dual miR390 cleavage sites. AtTAS3, an siRNA locus important for development and conserved among higher plants, also has dual miR390 complementary sites. Both sites bind miR390 in vitro and are functionally required in Arabidopsis, but cleavage is undetectable at the 5' site--demonstrating that noncleavable sites can be functional in plants. Phased siRNAs also emanate from the bounded regions of every Arabidopsis gene with two known microRNA/siRNA complementary sites, but only rarely from genes with single sites. Therefore, two "hits,"--often, but not always, two cleavage events--constitute a conserved trigger for siRNA biogenesis, a finding with implications for recognition and silencing of aberrant RNA.

Recapitulation of Short RNA-Directed Translational Gene Silencing In Vitro

microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.

MicroRNA-directed cleavage of ATHB15 mRNA regulates vascular development in Arabidopsis inflorescence stems.

Class III homeodomain-leucine zipper proteins regulate critical aspects of plant development, including lateral organ polarity, apical and lateral meristem formation, and vascular development. ATHB15, a member of this transcription factor family, is exclusively expressed in vascular tissues. Recently, a microRNA (miRNA) binding sequence has been identified in ATHB15 mRNA, suggesting that a molecular mechanism governed by miRNA binding may direct vascular development through ATHB15. Here, we show that miR166-mediated ATHB15 mRNA cleavage is a principal mechanism for the regulation of vascular development. In a gain-of-function MIR166a mutant, the decreased transcript level of ATHB15 was accompanied by an altered vascular system with expanded xylem tissue and interfascicular region, indicative of accelerated vascular cell differentiation from cambial/procambial cells. A similar phenotype was observed in Arabidopsis plants with reduced ATHB15 expression but reversed in transgenic plants overexpressing an miR166-resistant ATHB15. ATHB15 mRNA cleavage occurred in standard wheat germ extracts and in Arabidopsis and was mediated by miR166 in Nicotiana benthamiana cells. miR166-assisted ATHB15 repression is likely to be a conserved mechanism that regulates vascular development in all vascular plants.

Uridine Addition After MicroRNA-Directed Cleavage

One of the important roles of microRNA (miRNA) is to direct the cleavage of messenger RNA (mRNA). However, the mechanisms of decay of the cleaved mRNA products is not well understood. We show that miRNA-directed cleavage products in organisms as diverse as Arabidopsis, mouse, and Epstein-Barr virus have at their 3' ends a stretch (1 to 24 nucleotides) of oligouridine posttranscriptionally added downstream of the cleavage site. This 3' uridine addition, as shown for Arabidopsis, is correlated with decapping and 5' shortening of the cleaved products, suggesting a mechanistic step in the miRNA-directed mRNA decay mechanism.

MicroRNA-directed cleavage of Nicotiana sylvestris PHAVOLUTA mRNA regulates the vascular cambium and structure of apical meristems.

Leaf initiation in the peripheral zone of the shoot apical meristem involves a transition to determinate cell fate, but indeterminacy is maintained in the vascular cambium, a tissue critical to the continuous growth of vascular tissue in leaves and stems. We show that the orientation of cambial growth is regulated by microRNA (miRNA)-directed cleavage of mRNA from the Nicotiana sylvestris ortholog of PHAVOLUTA (NsPHAV). Loss of miRNA regulation in semidominant phv1 mutants misdirects lateral growth of leaf midveins and stem vasculature away from the shoot, disrupting vascular connections in stem nodes. The phv1 mutation also expands the central zone in vegetative and inflorescence meristems, implicating miRNA and NsPHAV in regulation of meristem structure. In flowers, phv1 causes reiteration of carpel initiation, a phenocopy for loss of CARPEL FACTORY/DICER LIKE1, indicating that miRNA is critical to the termination of indeterminacy in floral meristems. Results point to a common role for miRNA in spatial and temporal restriction of HD-ZIPIII mediated indeterminacy in apical and vascular meristems.

MicroRNA in HOX

Homeobox (HOX) genes are highly conserved transcriptional regulators that play a central role in development and pattern formation of higher eukaryotes. Genes encoding microRNAs, tiny 22-nucleotide RNAs involved in gene silencing, are found in almost all eukaryotes and have been implicated in a number of important cell and developmental pathways. Yekta et al. now show that an evolutionarily conserved and essentially complementary binding site for the miRNA miR-196 is found in the HOX gene HOX8B . miR-196 directs the cleavage of HOX8B messenger RNA during mouse development, and other evidence suggests that miR-196 can regulate the translation of other HOX genes in the mouse HOX cluster. S. Yekta, I. Shih, D. P. Bartel, MicroRNA-directed cleavage of HOXB8 mRNA. Science 304 , 594-596 (2004). [Abstract] [Full Text]

MicroRNA-directed cleavage of HOXB8 mRNA.

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, some of which are known to play important regulatory roles in animals by targeting the messages of protein-coding genes for translational repression. We find that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments diagnostic of miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting productive translation.