Gateway to Understanding Argonaute Loading of Single-Stranded RNAs: Preparation of Deep Sequencing Libraries with In Vitro Loading Samples
- 1.2k Downloads
Identification of sequences preferred by individual RNA-binding proteins (RBPs) has been accelerated by recent advances in the quantitative analysis of protein–RNA interactions on a massive scale, and such experiments have even revealed hidden sequence specificity of RBPs that were assumed to be non-specific. Argonaute (AGO) proteins bind diverse guide small RNAs and were believed to have no sequence specificity besides the preference for particular bases at the 5′ nucleotide. However, we recently showed that short single-stranded RNAs (ssRNAs) are loaded to AGOs in vivo and in cell extracts with detectable sequence preferences. To study the sequence specificity, we established a protocol for preparing the oligo-specific deep-sequencing library. The protocol includes in vitro loading assay that uses RNA oligos containing randomized nucleotides at the first five positions and also splinted-ligation that specifically amplifies the introduced oligo RNA species from a complex mixture of endogenous small RNAs and exogenously introduced RNA oligos. With the current sequencing depth, this procedure will allow quantitative profiling of interactions between the AGO and ~1000 ssRNA species with different sequences. The method would aid in studying the mechanism behind the selective loading of ssRNAs to AGOs and may potentially be applied to study interactions between RNA and other RNA-binding proteins.
Key wordsArgonaute Single-stranded RNA High-throughput sequencing In-vitro
We thank Gregory Hannon for sharing the tagged AGO2 plasmid. Research in K.O.’s group was supported by the National Research Foundation, Prime Minister’s Office, Singapore under its NRF Fellowship Programme (NRF2011NRF-NRFF001-042).
- 9.Liu J, Yu D, Aiba Y, Pendergraff H, Swayze EE, Lima WF, Hu J, Prakash TP, Corey DR (2013) ss-siRNAs allele selectively inhibit ataxin-3 expression: multiple mechanisms for an alternative gene silencing strategy. Nucleic Acids Res 41(20):9570–9583. doi: 10.1093/nar/gkt693 CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Hu J, Liu J, Narayanannair KJ, Lackey JG, Kuchimanchi S, Rajeev KG, Manoharan M, Swayze EE, Lima WF, Prakash TP, Xiang Q, Martinez C, Corey DR (2014) Allele-selective inhibition of mutant atrophin-1 expression by duplex and single-stranded RNAs. Biochemistry 53(28):4510–4518. doi: 10.1021/bi500610r CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Matsui M, Corey DR (2016) Non-coding RNAs as drug targets. Nat Rev Drug Discov. doi: 10.1038/nrd.2016.117