The recent papers on endo-siRNAs raise fundamental questions regarding the biogenesis of small RNAs. Some of the most important questions concern mechanistic aspects of small-RNA sorting pathways. For example, how does LOQS work with DCR2? And given that R2D2 is needed to load exosiRNAs into AGO2 (REFS 43,44
), to what extent do endo-siRNAs require R2D2 for loading? How are miRNA and hpRNA precursors distinguished? Some ‘long’ miRNAs and ‘short’ hpRNAs in D. melanogaster
are indistinguishable in size and structure10,42
. They are effectively sorted, however, as long miRNAs make only a single small-RNA duplex (as is typical for DCR1 substrates), whereas short hpRNAs produce multiple duplexes (as is typical for DCR2 substrates). How can the cell distinguish these hairpins?
The regulation of dsRNA formation is another mystery. For example, the cis
-NAT-siRNA pathway accepts many substrates — at least 17 in mouse oocytes6,7
and at least 140 in D. melanogaster8,9,12
. However, cis
-NAT-siRNA loci constitute only 25% of co-expressed cis
-NATs in D. melanogaster9
. Is there active selection for entry into the RNAi pathway, which could be mediated at the step of dsRNA formation? Conversely, how do co-expressed mammalian cis
-NATs, and co-expressed pseudogene-gene complementary pairs, avoid triggering an interferon response outside of oocytes? Finally, although it seems evident that cis
-NAT and trans
-NAT siRNAs are generated from processed transcripts, it is not known whether the dsRNA substrate forms in the nucleus or cytoplasm, nor is it clear where the dsRNA encounters Dicer.
Valuable lessons were taught by the length and structure of primary hpRNA transcripts. Their dsRNA character was recognized only after genomic fragments of sufficient length were examined, and consequently their siRNAs were prone to being misannotated as having derived from shorter, unstructured precursors8
. The stems of some plant miRNA hairpins are separated by long, unstructured terminal loops and even introns54
, and we now recognize the same to be true for several hpRNAs10,12
. It is therefore conceivable that the stems of some hpRNA precursors might be separated by kilobases or tens of kilobases. Do the structured precursors of any anonymous cloned small RNAs that are currently deposited in public databases await discovery?
Endogenous sources of mammalian dsRNA remain to be recognized outside of oocytes. As is the case with oocytes, introduction of long dsRNA into embryonic stem cells (ESCs) does not activate an interferon response55,56
. Might ESCs also harbour endo-siRNAs, the action of which is relevant for maintaining pluripotency? Although endo-siRNAs were not previously found in ESCs57
this possibility might deserve further study.
Finally, although small-RNA sorting pathways have received little attention in mammalian systems, there is growing recognition of their importance to siRNA and miRNA function in plants58,59
. As only one of the four mammalian AGO proteins (AGO2) has slicer activity16,17
, the directed sorting of mammalian siRNAs is presumably important for their ability to slice complementary targets63
. Consequently, the elucidation of mammalian siRNA sorting rules might have important implications for attempts to improve siRNA efficacy for experimental and therapeutic purposes.