Hidden sequence specificity in loading of single-stranded RNAs onto Drosophila Argonautes.

Eling Goh,Katsutomo Okamura

doi:10.1093/nar/gky1300

Abstract

Argonaute proteins play important roles in gene regulation with small RNAs (sRNAs) serving as guides to targets. Argonautes are believed to bind sRNAs in a sequence non-specific manner. However, we recently discovered that Argonautes selectively load endogenous single-stranded (ss) RNAs, suggesting that Argonaute loading may conform to sequence specificity. To identify sequences preferred for Argonaute loading, we have developed HIgh-throughput Sequencing mediated Specificity Analysis (HISSA). HISSA allows massively parallel analysis of RNA binding efficiency by using randomized oligos in in vitro binding assays and quantifying RNAs by deep-sequencing. We chose Drosophila as a model system to take advantage of the presence of two biochemically distinct Argonautes, AGO1 and AGO2. Our results revealed AGO2 loading to be strongly favored by G-rich sequences. In contrast, AGO1 showed an enrichment of the ‘GAC’ motif in loaded species. Reanalysis of published sRNA sequencing data from fly tissues detected enrichment of the GAC motif in ssRNA-derived small RNAs in the immunopurified AGO1-complex under certain conditions, suggesting that the sequence preference of AGO1-loading may influence the repertoire of AGO1-bound endogenous sRNAs. Finally, we showed that human Ago2 also exhibited selectivity in loading ssRNAs in cell lysates. These findings may have implications for therapeutic ssRNA-mediated gene silencing.

Highlights

Argonaute and Cas (Clustered Regularly Interspaced Short Palindromic Repeats-associated system) effectors provide flexible platforms whose target specificities could be artificially programmed by guide DNAs or RNAs
RNA species loaded as guide molecules and those bound as target RNAs, our results suggested that AGO2 may have an intrinsic property in preferring G-rich sequences (Supplementary Figure S8A)
Because enrichment of G-rich and GAC-containing sequences were highly unexpected, we considered the possibility that the small RNAs enriched in the Argonaute HIgh-throughput Sequencing mediated Specificity Analysis (HISSA) libraries may represent RNA oligos that were recognized as targets by endogenous small RNA species in the Argonaute complexes

Summary

Introduction

Argonaute and Cas (Clustered Regularly Interspaced Short Palindromic Repeats-associated system) effectors provide flexible platforms whose target specificities could be artificially programmed by guide DNAs or RNAs. With little sequence specificity of these effector proteins for guide nucleic acids, these proteins can be programmed to target a broad range of sequences. This feature of the effectors must be important for their ancestral roles to serve as natural defense systems against foreign agents including viruses and transposable elements (TEs) with various sequences that rapidly evolve [3,4,5]. Understanding the sequence specificity of Argonaute or Cas effectors may be important for developing effective biotechnological methods, such as RNA interference (RNAi) and genome-editing

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Conclusion