RNAi and the Dicer protein

Introduction to RNAi and the Dicer protein

RNA interference (RNAi) is a post transcriptional gene regulatory mechanism which acts via inhibition (by blocking) or degradation of the transcribed mRNA. It then blocks corresponding proteins to be expressed within the cell at the end. Two general types of noncoding RNAs, either ‘short interfering RNA’ (siRNA) or ‘microRNA’ (miRNA), can induce RNAi in cells by binding to their complementary mRNA which it downregulates.

The key difference between miRNA and siRNA is that miRNAs are natural products encoded from the organisms’ genome, whereas siRNAs are primarily obtained from the extracellular (exogenous) environment, which are usually viral (nature) or artificial (laboratory).

Both dsRNA, that has been transported into the cells, and pre-miRNA, that has been made in the nucleus then transported to the cytoplasm, must be modified in order to become siRNA and miRNA. Protein Dicer, endoribonuclease, plays the main role of this modification. Modified siRNA or miRNA are both recognized in the cytoplasm, and then they go through other sets of modifications and form complex with other proteins to downregulate a gene.

Real World Applications of RNAi

RNAi plays its role in several major fields including medicine and novel therapeutics in our current world. RNAi can selectively, and with high specificity, target and downregulate or knock-out disease causing genes (e.g. oncogenes). Main problems with treating cancer is in its specificity, and RNAi provides an approach that is highly specific. However, RNAi may not alone provide an effective treatment by itself. It should be working in parallel with current chemotherapy (Nandakumar, 2016).

RNAi is also a foundation of the pest control researches. Pests are bringing loss of billions of dollars’ worth of crop yield per year. In past decades, the main solution of dealing with the pests were chemical pesticides, which has many flaws. One of the main flaws that is frequently brought up is a harm to an environment and ecosystem (e.g. DDT and its bioaccumulation), and an application of RNAi can resolve these issues.

Environment is barely harmed (however can depend on the nanoparticle that will be used to deliver dsRNA to the pests) because dsRNA is biodegradable, highly species-specific, and non-toxic to non-target species. Much research has been done and are one going in order to confirm RNAi as an effective pest control method, and many of the researches suggested it to be a valuable pesticide.

There had been a demonstration of a foliar application of RNAi against Colorado potato beetle actin gene, which showed a high effectiveness for the pest population control. Moreover, it was shown that RNAi protected potato plants for at least 28 days under greenhouse conditions (San Miguel & Scott, 2016).

As described, RNAi has a wide range of implications. However, although significant researches had been done in order to figure out the structures of the biomolecules involved in RNAi and how the mechanism works, yet we still do not know everything about the RNAi. As its applications involve medical treatments and our ecosystem, we have to be very careful to not cause irreversible side effects, and this is prevented by making sure that we know everything involved in the mechanism.

RNAi Mechanism

As described in Figure 1, most parts of the gene silencing mechanism by miRNA and siRNA has already been determined. miRNA is first transcribed in the nucleus of the cell as a form of Pri-miRNA. Pri-miRNA then gets modified into pre-miRNA after removal of its single stranded RNA flanking regions by Drosha (RNAse III).

Pre-miRNA is then imported into the cytoplasm by Exportin 5 protein located on the nuclear membrane. Pre-miRNA or exogenous dsRNA, when recognized in the cytoplasm by Dicer protein, are cleaved into 18 to 25 nucleotides of miRNA and siRNA respectively.

These RNAs are then bound to the Argonaute 2 (AGO2) protein attached to the RISC complex which discards or cleaves the passenger strand of miRNA or siRNA respectively. Such activated RISC complexes then are guided by the guide strand to target mRNA which either cleave mRNA (siRNA induced RNAi), or inhibits mRNA via translational repression, cleavage, or degradation (miRNA induced RNAi).

Protein Dicer

Dicer enzymes are important proteins in all living organisms in order to initiate RNAi as described (cleaving pre-miRNA or dsRNA), and therefore they are highly conserved throughout many organisms. Normally, they are comprised with domains of: helicase on N-terminal, DUF283 (Domain of Unknown Function), PAZ, and two RNAaseIII (MacRae and Doudna, 2007).

Some structural research found that some of the Dicers lack some domains (called Dicer like proteins), but Dicer protein have never been shown to have sequence cleavage preferences. However, Hoehener, Hug, and Nowacki showed that Dicer like enzymes in Paramecium demonstrates cleavage preferences.

There are total of three types of recognized Dicer proteins (Dcr 1-3), and five types of Dicer-like proteins (Dcl 1-5) in Paramecium, which previous gene knock-down studies indirectly showed that they induce different RNAi’s within Paramecium (1, 2).

Hoehener, Hug, and Nowacki were interested in testing whether specific targeting of miRNA or siRNA (collectively small RNA) for specific to-be-inhibited gene (because the cut small RNA must be complementary with the portion of the to-be-inhibited gene) was due to a specific Dicer cleavage by different types of Dicers and Dicer like enzymes, or just due to selections done by Argonaute (AGO2) proteins to form RISC complexes as already presented by other studies using different organisms.

References 

San Miguel, K., & Scott, J. (2016). The next generation of insecticides: DsRNA is stable as a foliar‐applied insecticide. Pest Management Science, 72(4), 801-809.

Nandakumar, G. (2016). Gene Silencing as a Therapy for Cancer. Young Scientists Journal,(18), 29-32.

Macrae, I, & Doudna, J. (2007). Ribonuclease revisited: structural insights into ribonuclease III family enzymes. Current Opinion in Structural Biology, 17, 138-145.

1. G. Lepère, M. Nowacki, V.Serrano, J.-F. Gout, G. Guglielmi, S. Duharcourt, E. MeyerSilencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia

Nucleic Acids Res., 37 (2009), pp. 903-915

2. P.Y. Sandoval, E.C. Swart, M.Arambasic, M. NowackiFunctional diversification of Dicer-like proteins and small RNAs required for genome sculpting

Dev. Cell, 28 (2014), pp. 174-188