RNA interference has been one of the most significant and exciting discoveries in recent history. The impact of this discovery is enormous with applications ranging from knockdown and loss-of-function studies to the generation of better animal models with conditional knockdown of desired gene(s) to large-scale RNAi-based screens to aid drug discovery.
History of discovery
The discovery of the gene silencing phenomenon dated back as early as the 1990s with Napoli and Jorgensen demonstrating the down-regulation of chalcone synthase following introduction of exogenous transgene in plants . Similar suppression was subsequently observed in other systems [10, 22]. In another set unrelated work at the time, Lee et al. identified in a genetic screen that endogenous lin-4 expressed a non-protein- coding product that is complementary to the lin-14 gene and controlled the timing of larval development (from first to second larval state) in C. elegans . We now know this as the first miRNA to be discovered. In 2000, another miRNA, let-7, was discovered in the same organism and was found to be involved in promoting the late-larval to adult transition . The seminal work by Mello and Fire in 1998 (for which was awarded the Nobel Prize in 2006) demonstrated that the introduction of exogenous dsRNA in C. elegans specifically silenced genes via RNA interference, explaining the prior suppression phenomenon observed in plants . Subsequent studies found the conversion of dsRNA into siRNA in the RNAi pathway. In 2001, the term miRNA and the link between miRNA and RNAi was described in three papers in Science . With this, we have come to realize the gene regulatory machinery was composed of predominately of two classes small RNAs, with miRNA involved in the regulation of endogenous genes and siRNA involved in defense in response to viral nucleic acids, transposons, and transgenes . Later works revealed downstream effectors: Dicers (for excision of precursor species) and Argonaute proteins (part of the RNA-induced silencing complex to perform the actual silencing effects), completing our current understanding of the RNA silencing pathways. The details of the mechanism and the differences among the species are further discussed below.
There is a common theme involved for both siRNA-mediated and miRNA-mediated silencing. In the biogenesis of both siRNA and miRNA, the double-stranded precursors are cleaved by a RNase into short ∼22 nt fragments. One of the strands (the guide strand) is loaded into an Argonaute protein, a central component of the larger ribonucleoprotien complex RISC that facilitates target RNA recognition and silencing. The mechanism of silencing are either cleaveage of the target mRNA or translation repression.
Aside from this common theme, the proteins involved in these processes differ among species and there exists additional steps in miRNA processing prior to its maturation and incorporation into RISC (Figure 13.1). For the biogenesis of siRNA, the precursors are dsRNAs, oftentimes from exogenous sources such as viruses or transposons. However, recent studies have also found endogenous siRNAs . Regardless of the source, these dsRNAs are processed by the RNase III endonuclease, Dicer, into ∼22 nt siRNAs. This RNase III-catalyzed cleavage leaves the characteristic 5’phosphates and 2 nt 3’ overhangs . It is worth noting that different species have evolved with different number of paralogs. This becomes important as, to be discussed later, the miRNA biogenesis pathway also utilizes Dicer for the processing of miRNA precursors (more specifically pre-miRNAs). For species such as D. melanogaster, there are two distinct Dicer proteins and as a result there is typically a preferential processing of the precursors (e.g. Dicer-1 for miRNA cleavage and Dicer-2 for siRNA cleavage) . In contrast, mammals and nematodes only have a single Dicer protein and as such both biogenesis pathways converge to the same processing step . In subsequent steps of the siRNA biogenesis pathway, one of the strands in the siRNA duplex is loaded into RISC to silence target RNAs (Figure 13.1C).
In the miRNA biogenesis pathway, majority of the precursors are pol II transcripts of the intron regions, some of which encode multiple miRNAs in clusters. These precursors, in the form of a stem-loop structure, are named pri-miRNAs. The pri-miRNAs are first cleaved in the nucleus by a RNase III endonuclease (Drosha in animals and Dcl1 in plants) into ∼60-70 nt stem loop intermediates, termed pre-miRNAs . In animals, the pre-miRNA is then exported into the cytoplasm by Exportin-5. This is followed by the cleavage of pre-miRNA intermediate by Dicer to remove the stem loop. One of the strands in the resulting mature miRNA duplex is loaded to RISC, similar to that described for siRNA biogenesis Figure 13.1B. Interestingly, in plants, the pri-miRNA is processed into mature miRNA through two cleavages by the same enzyme, Dcl1, in the nucleus before export into the cytoplasm for loading (Figure 13.1A).
Functions and silencing mechanism
The classical view of miRNA function based on the early discoveries of miRNA has been analogous to a binary switch whereby miRNA represses translation of a few key mRNA targets to initiate a developmental transition. However, subsequent studies have greatly broaden this definition. In plants, most miRNAs bind to the coding region of the mRNA with near-perfect complementarity. On the other hand, animal miRNAs bind with partial complementarity (except for a seed region, residues 2-8) to the 3’ UTR regions of mRNA. As such, there are potentially hundreds targets by a single miRNA in animals rather than just a few . In addition, in mammals, only a few portion of the predicted targets are involved in development, with the rest predicted to cover a wide range of molecular and biological processes . Lastly, miRNA silencing acts through both translation repression and mRNA cleavage (and also destabilization as discussed below)(as shown for example showed by Bartel and coworkers on the miR-196-directed cleavage of HOXB6 ). Taken together, the modern view of miRNA function has been that miRNA dampens expression of many mRNA targets to optimize expression, reinforce cell identity, and sharpen transitions.
The mechanism for which miRNA mediates the silencing of target mRNA is still an area of active research. As previously discussed, RNA silencing can take the form of either cleavage, destabilization (leading to subsequent degradation of the mRNA), or translation repression. In plants, it has been found that the predominate mode of RNA silencing is through Argonaute-catalyzed cleavage. However, the contribution of these different modes of silencing has been less clear in animals. Recent global analyses from the Bartel group in collaboration with Gygi and Ingolia and Weissman shed light on this question. In a 2008 study, Bartel and Gygi groups examined the global changes in protein level using mass spectrometry following miRNA introduction or deletion . Their results revealed the repression of hundreds of genes by individual miRNAs, and more importantly mRNA destabilization accounts for majority of the highly repressed targets (Figure 13.2).
This is further supported by a subsequent study using both RNA-seq and a novel ribosome-profiling first demonstrated by Inoglia and Weissman 2009 that enables the interrogation of global translation activities with sub-codon resolution . The results showed destabilization of target mRNA is the predominate mechanism through which miRNA reduces the protein output.