Novel functional small RNAs are selectively loaded onto mammalian Ago1

Abstract

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1–4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein–Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1–4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Interestingly, we identified F-actin as a key player in nuclear transportation of promoter-targeted siRNAs in mammalian cells, using the same siRNA constructs [205] . Results from this study are consistent with selective transport of promoter-targeted sncRNAs, which has also been shown for AGO-1 by other groups [135] , as mentioned earlier.…”

“…In this way, complexes could share a common core, but would vary in accessory proteins that modify their function to induce either TGS or PTGS. Consistent with this model, a recent study unveiled a sorting mechanism in humans, which directs differential loading of AGO-1 proteins for unique sncRNAs in the setting of a viral infection [135] . However the determinants of this selection remain unknown.…”

MicroRNA hsa-miR-29a-3p modulates CYP2C19 in human liver cells

“…Such a sorting mechanism for Ago family proteins in mammals was previously reported by Yamakawa et al [37], who found that some novel small RNAs were able to selectively distinguish and load onto human Ago1-RISC. Also, Turchinovich et al [38] observed that many miRNAs derived from antisense strands of pre-miRNAs (i.e., 3p-miRNAs) were Ago1-biased, while the counterparts derived from the sense strand (i.e., 5p-miRNAs) were Ago2-biased.…”

Regulation of LINE-1 in mammals

“…Finally, recent technical developments have enabled the identification of a multitude of novel types of small RNA molecules that do not fit into the well-established classes (35,98). Moreover, RNAi pathways expand beyond the post-transcriptional regulation, by having a role in the maintenance of cellular integrity (35) and by acting as an antiviral defense mechanism (99).…”