Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans

Steiner, Florian; Hoogstrate, Suzanne W.; Okihara, Kristy L.; Thijssen, Karen L.; Ketting, René F.; Plasterk, Ronald H.A.; Sijen, Titia

doi:10.1038/nsmb1308

Cited by 87 publications

(86 citation statements)

References 47 publications

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“…These in vivo observations bolster recent biochemical work that has shown that depending on the complementarity of the hairpin precursor structure miRNA or siRNAs get sorted into distinct silencing complexes (Forstemann et al 2007;Steiner et al 2007;Tomari et al 2007). This sorting is not 100% discriminative as we observe that within a heterologous context (unc-54 39 UTR), three repeated, perfectly complementary lsy-6 sites confer a rde-1-dependent, though modest, down-regulation of the sensor in ASEL (L. Cochella and O.…”

Section: Lsy-6 Requires Imperfectly Complementary Target Sites To Medmentioning

confidence: 61%

Molecular architecture of a miRNA-regulated 3′ UTR

Didiano¹,

Hobert²

2008

RNA

163

124

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Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 39 UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 39 UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 39 UTR and in the context of heterologous 39 UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNAmediated 39 UTR regulation. The contextual features reside 39 of lsy-6 binding sites in the 39 UTR and act in a combinatorial manner; mutation of each results in limited defects in 39 UTR regulation, but a combinatorial deletion results in complete loss of 39 UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 39 UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 39 UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 39 UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.

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Section: Lsy-6 Requires Imperfectly Complementary Target Sites To Medmentioning

confidence: 61%

Molecular architecture of a miRNA-regulated 3′ UTR

Didiano¹,

Hobert²

2008

RNA

163

124

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“…Perfectly matched duplexes (siRNA-like) are preferentially incorporated into Ago2, and the loading is achieved by cleaving the passenger strand, whereas duplexes with central mismatched bulges (miRNA-like) are sorted to Ago1, and the unselected strand (asterisk strand) is eliminated through a "bypass" pathway, which is sensitive to the structure of the small RNA duplex and independent of the weak cleavage activity of Ago1 (12). A similar sorting mechanism exists in Caenorhabditis elegans, whereby small RNA duplexes with perfectly matched or bulged stems are channeled into RDE-1 and ALG-1, respectively (13). In plants, a different sorting mechanism has been reported.…”

mentioning

confidence: 96%

Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes

Jin

Zhang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage-and non-cleavage-based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1-Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila. Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.RNA interference | gene silencing M icroRNAs (miRNAs) and 21-to 23-nt siRNAs regulate gene expression in almost all eukaryotic organisms. Small RNA-mediated gene regulation is sequence-specific and believed to regulate at least one-half of all protein-encoding mRNAs (reviewed in refs. 1-4).Argonaute proteins (Agos), which directly associate with small RNAs, are the core of all known RNA-induced silencing complexes (RISCs). These proteins are functionally specialized into distinct RNA silencing pathways. In Drosophila, Ago2-RISC is involved in target mRNA cleavage, and Ago1-RISC mediates translational repression (5, 6). The human genome encodes four Ago-like subfamily proteins (Ago1-Ago4) that have redundant functions as negative regulators of gene expression, but only Ago2 has cleavage activity (7-9).In Drosophila, siRNAs and miRNAs are actively sorted into functionally distinct Ago-RISCs based on differences in structure rather than on their biogenesis (10, 11). Perfectly matched duplexes (siRNA-like) are preferentially incorporated into Ago2, and the loading is achieved by cleaving the passenger strand, whereas duplexes with central mismatched bulges (miRNA-like) are sorted to Ago1, and the unselected strand (asterisk strand) is eliminated through a "bypass" pathway, which is sensitive to the structure of the small RNA duplex and independent of the weak cleavage activity of Ago1 (12). A similar sorting mechanism exists in Caenorhabditis elegans, whereby small RNA duplexes with perfectly matched or bulged stems are channeled into RDE-1 and ALG-1, respectively (13). In plants, a different sorting mechanism has been reported. The composition and phosphorylation status of the 5′ nucleotide of small RNA duplexes influences their binding by the different Agos (14,15). In mammalian cells, several studies have shown that miRNAs are similarly associated with four human Agos (7,8,16), suggesting the abse...

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“…A x 2 test indicated that this periodicity was therefore enriched in a highly statistically significant manner in the pri-miRNA data set (p-value < 1.0 3 10 À10 ). The decrease in stability at positions 10 and 11 is known to be important for efficient loading of the miRNAmiRNA* duplex into the miRNA-specific Argonaute in C. elegans (Steiner et al 2007), but it is unclear if the other positions with decreased stability are also important for miRNA biogenesis or function. It is plausible that these positions are important for recognition of the pri-miRNA by Drosha.…”

Section: Thermodynamic Profile Of Pri-mirnas and Pre-mirnasmentioning

confidence: 99%

Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer

Warf

Johnson

Bass³

2011

RNA

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MicroRNAs (miRNAs) are small regulatory RNAs that are essential in all studied metazoans. Research has focused on the prediction and identification of novel miRNAs, while little has been done to validate, annotate, and characterize identified miRNAs. Using Illumina sequencing,~20 million small RNA sequences were obtained from Caenorhabditis elegans. Of the 175 miRNAs listed on the miRBase database, 106 were validated as deriving from a stem-loop precursor with hallmark characteristics of miRNAs. This result suggests that not all sequences identified as miRNAs belong in this category of small RNAs. Our large data set of validated miRNAs facilitated the determination of general sequence and structural characteristics of miRNAs and miRNA precursors. In contrast to previous observations, we did not observe a preference for the 59 nucleotide of the miRNA to be unpaired compared to the 59 nucleotide of the miRNA*, nor a preference for the miRNA to be on either the 59 or 39 arm of the miRNA precursor stem-loop. We observed that steady-state pools of miRNAs have fairly homogeneous termini, especially at their 59 end. Nearly all mature miRNA-miRNA* duplexes had two nucleotide 39 overhangs, and there was a preference for a uracil in the first and ninth position of the mature miRNA. Finally, we observed that specific nucleotides and structural distortions were overrepresented at certain positions adjacent to Drosha and Dicer cleavage sites. Our study offers a comprehensive data set of C. elegans miRNAs and their precursors that significantly decreases the uncertainty associated with the identity of these molecules in existing databases.

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Structural features of small RNA precursors determine Argonaute loading in Caenorhabditis elegans

Cited by 87 publications

References 47 publications

Molecular architecture of a miRNA-regulated 3′ UTR

Molecular architecture of a miRNA-regulated 3′ UTR

Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes

Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer

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