Gateway to Understanding Argonaute Loading of Single-Stranded RNAs: Preparation of Deep Sequencing Libraries with In Vitro Loading Samples

Abstract

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.

Hidden sequence specificity in loading of single-stranded RNAs ontoDrosophilaArgonautes

“…To further study the mechanism, we sought to find features including sequences and potential secondary structures of ssRNAs that determine their loading efficiency. To this end, we set up an in vitro assay system that allows profiling of loading efficiency of individual ssRNA species in a massively parallel manner using complex mixtures of RNA oligos with partially randomized sequences (Figure 3A) (22). The fly system was chosen as a model, taking advantage of the fact that there are two biochemically distinct Argonautes showing clearly different ssRNA loading preferences (Figure 1A and B).…”

“…We chose to use splinted-ligation to specifically add 3′ linker to the 3′ end of the introduced RNA oligo (40). By using this method, the ligation specificity and efficiency were high enough to construct small RNA libraries, and the majority of reads in the resulting libraries were derived from introduced oligos (Figure 3B and Supplementary Figure S4) (22). We applied the same experimental procedure to the ‘input’ RNA samples, which were prepared by mixing endogenous RNAs loaded to AGO1 or FLAG-AGO2 and randomized RNA oligos that were not subjected to the in vitro loading procedure (Figure 3A).…”

“…5′-5N HISSA libraries were constructed as previously described with some modifications (22). For 5′-8N HISSA libraries, we optimized the 3′ linker ligation condition and used a bridge DNA oligo that is complementary to 12nt of the 3′ end of mir-317 loop sequence and the 8nt of the 5′ end of the 3′-linker.…”

RNAs on the Go: Extracellular Transfer in Insects with Promising Prospects for Pest Management

“…First, several factors have been shown to influence the loading of sRNAs into Ago proteins, such as their sequence and structure [ 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 ]. In insects, generation of siRNAs with species-dependent length have been observed [ 265 ], and certain sRNA chemical modifications seem to vary among species.…”

The hidden rule in argonaute protein loading : sequence specificity

“…of labeled oligos was added to the lysate forming final reaction with RNaseOUT (Invitrogen, USA), 0.5mM ATP (Sigma-Aldrich, USA), 5mM DTT, 0.1M KOAc and water. The reactions were incubated at 25°C for 1 hour followed by the addition of PXL buffer (1xPBS (pH7.4), 0.1% SDS, 0.5% deoxycholate, 0.5% NP40) for quenching (Goh and Okamura, 2018). The original mir-34 loop sequence (1; blue) was shortened (2; blue) by bringing the last 7 nucleotides (underlined) forward while the original mir-317 loop sequence (3; green) was lengthened (4; green) by repeating the last 7 nucleotides at the 3' end.…”