1. More specifically: what loads the siRNA onto RISC when Dicer is not present? I do know siRNA works quite well without Dicer; I just can't find any information on RISC loading in systems where Dicer is missing.
2. It's clear that only Ago2 can cleave mRNA; and that a complete match of the guide strand with the target will cause mRNA degradation as opposed to suppression. But herein lies the question... How does the small RNA "know" which RISC to be loaded onto? We only have one Dicer, so Dicer homologues can't determine the loading. I'm not sure if Dicer "remembers" what kind of RNA it processed into small RNA. Do other, RNA specific proteins "decide" which RISC complex gets loaded? If so, how can miRNAs induce cleavage if they happen to be perfectly complementary to their targets if they are loaded onto the "wrong" (non Ago2 containing) RISC?
Dicer is not part of RISC complex. Dicer process siRNA from dsRNA in order to activate RISC complex. Processed smRNAs methylated in order to prevent degradation by RNses. Therefore endogenously added or expressed siRNAs can work in dicer mutant cell. RISC is multiprotein and AGO2 might recognize siRNAs by methylated or in sequence specific manner. So in plants if add mRNA specific siRNA does not trigger PTGS but if design siRNA which is functional then it does.
To address your second question, I don't think that there is any evidence that smRNAs are selectively loaded onto certain RISCs. It is likely that siRNAs are loaded onto all 4 Agos (same goes for miRNAs), but that you will only detect repression by siRNAs that are loaded into Ago2-containing RISCs because they bind only a single site, usually in the ORF and efficient repression requires cleavage of the target. It is additionally possible that cleavage of the passenger strand is required to unwind a perfectly complementary siRNA duplex and, since Ago2 is the only one capable of this, siRNAs are only "active" (i.e. unwound) when in complex with Ago2.
Dear Tohir, thank you for the answer. I do know that Dicer is upstream of RISC in the RNAi process, however, Dicer is important in RISC loading; at least this is what most papers reviewing the process say. Hence the lack of Dicer must be impacting RISC loading somehow. (Nevertheless my Dicer -/- cells are happily processing siRNA.)
Dear Justin, Thank you for your answer as well. It does make sense; however, does it mean that imperfectly paired dsRNA does not need to be cleaved to get rid of the passenger strand?
The issue I'm having is: how separate are the miRNA/siRNA pathways in mammals? Is it possible to inhibit/suppress one (siRNA) but not the other (miRNA). One way I can imagine is the selective suppression of Ago2...
Thank you,
Andras
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