Hidden sequence specificity in loading of single-stranded RNAs ontoDrosophilaArgonautes

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.

Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression

“…It is possible that Dcr2 recognises the stem–loop structures of the guide RNA backbone and directs these to the Dcr2 small interfering RNA pathway. However, as Dcr2 knockout AF319 cells also showed some limited Cas13b-independent silencing, other alternatives should be considered, such as a direct loading of single-stranded RNA guides onto Ago1 and Ago2, the catalytic components of RNA-induced silencing complexes (RISC) 34 – 37 . It may be that the short, highly structured RNAs that are recognised by Cas13b are loaded efficiently onto the RISC complexes even in the absence of Dcr2 in insect cells.…”

Endogenous RNAi silences a burgeoning sex chromosome arms race

“…In Drosophila , components of the dedicated RNAi pathway (i.e., Dcr-2 and AGO2) are similarly required for antiviral defense (Mondotte et al, 2018; Wang et al, 2006), and ongoing research has focused on mechanisms of siRNA biogenesis (Goh and Okamura, 2019; Lee et al, 2018; Sinha et al, 2018; Tsuboyama et al, 2018) and/or new regulatory arenas such as transcriptional silencing and dosage compensation (Deshpande and Meller, 2018; Lee et al ., 2018; Nazer et al, 2018).…”

Identification of oligo-adenylated small RNAs in the parasite Entamoeba and a potential role for small RNA control

“…5 ). Differential loading of sRNA populations into different Agos has been reported in several model systems [ 41 , 42 ]. As demonstrated in Drosophila and Arabidopsis , miRNAs and siRNAs are sorted into specific Ago proteins [ 43 ]; the first nucleotide at the 5′-end of the sRNA is used as selective identity for AGO loading: AGO1 has a preference for 5′-uridine, whereas AGO2 and AGO4 prefer 5′-adenosine [ 44 ].…”