Biochemical and cell biological analysis of the mechanism of RNA interference in human cells

Abstract

RNA interference (RNAi) in human cells is efficiently triggered by short interfering RNA (siRNA) duplexes of 19-24 base-pairs (bp), which mimic the double-stranded processing intermediates of the endogenous small RNA species, microRNAs (miRNAs). Single-stranded siRNAs and miRNAs are incorporated into Argonaute protein-based RNA silencing complexes, to mediate inactivation of complementary mRNAs by cleavage or translational repression. There are eight Argonaute proteins expressed in humans, four Ago and four Piwi subfamily members, however only one of the Ago proteins is capable of the small RNA-guided cleavage of target mRNA. While all Agos can repress mRNA translation, the precise mechanism of this regulation is unknown. The role of mammalian Piwi proteins in RNA silencing processes has not been addressed. MiRNAs are predicted to regulate expression of one-third of the human genes. In light of the growing biological significance of RNA silencing processes, the current study addressed the mechanism of RNAi in human, focusing on two key components of RNA silencing complexes, small RNAs and the Argonaute proteins.To examine the fate of small RNAs in human cell lysate recapitulating target RNA cleavage activity, stability and processing of the radioactively labeled single- and double-stranded small RNAs was monitored in time-course experiments. The RNA duplexes were stable in the extract and their termini were converted to 5' phosphate and 3' hydroxyl groups, similar to the endogenous small RNA intermediates. Single-stranded siRNAs were rapidly degraded. The efficacy of 5'-phosphorylated single- and double-stranded siRNAs was tested in the cell culture. The single-stranded siRNAs were sufficient to trigger RNAi in the cultured cells. This suggests, that although siRNA duplexes are the preferred triggers of RNAi, single-stranded siRNAs can bypass the regular assembly pathway of the RNA silencing complex, especially while being 5'-phosphorylated.To examine the expression pattern of Argonautes in human cell lines, levels of the individual transcripts were quantified by qRT-PCR. Agos were found expressed in multiple human cell lines, while Piwis were detected only in the control samples from testis and ovary tissue. To understand the functional differences between the four Ago proteins, the association of Agos with the endogenous miRNAs was examined. The results suggest, that miRNAs are incorporated indiscriminately of their sequence into the different Ago-containing silencing complexes. The restricted expression of Piwis is in agreement with the reports on their germline expression in mouse, and implicates involvement of the RNA silencing in germline-specific processes in human. To check if the Piwis are capable of siRNA-guided target cleavage, the tagged Piwi proteins were ectopically expressed and purified. None of the Piwis was catalytically active in the tested conditions. To allow investigation of the role of Piwi proteins in the testis tissue, individual Piwi-specific antibodies were raised and/or characterized.