Gene Inhibition Therapy

Endogenous RNAi Pathway

The endogenous RNAi pathway can be triggered by different types of double-stranded RNAs.

In this video, we will be reviewing the endogenous or naturally occurring RNA interference (or RNAi) pathway.
The endogenous RNAi pathway can be triggered by different types of double-stranded RNAs. One of these triggers is microRNA18. Endogenous microRNAs play important roles in the biological processes of cell development, differentiation, apoptosis, and proliferation22–25.

A non-coding microRNA gene is usually transcribed by RNA polymerase II to form primary microRNA or “pri-microRNA”18,21. Pri-microRNA is a double-stranded, stem-loop hairpin structure that is cleaved to form precursor microRNA or “pre-microRNA” of approximately 70 to 100 nucleotides, which is then transported into the cytoplasm21,22. Once in the cytoplasm, pre-microRNA loses the stem loop and is further cleaved into shorter duplexes of approximately 22 nucleotides and two 3-prime nucleotide overhangs to form microRNA18,22,26.

MicroRNA then associates with the RNA-induced silencing complex, otherwise known as RISC18,21. Within the microRNA–RISC complex, the microRNA is unwound, releasing the “passenger strand”, while the guide strand—the one that is complementary to the messenger RNA (or mRNA) that is being targeted—remains associated with RISC. This leads RISC to bind to the target mRNA by partial complementarity18,21,27,28.

If the guide strand is only partially complementary to the target mRNA, it can bind to multiple complementary sites or multiple mRNAs, and cause translational repression21,28-30.

Less frequently, when the guide strand is fully or almost fully complementary to the target mRNA, cleavage of the target mRNA occurs21,30,31.

RNAi can also be triggered by small pieces of double-stranded RNA called small interfering RNA, or siRNA21. Although not completely understood, endogenous siRNAs may have roles in repression of transposable elements, chromatin organization, and gene regulation at the transcriptional and post-transcriptional level32,33.

A long double-stranded RNA is first transcribed from cellular genes or infecting pathogens. This RNA is cleaved in the cytoplasm into a smaller double-stranded RNA molecule called siRNA that is 21 to 23 nucleotides long21. siRNA then associates with RISC, and within the siRNA–RISC complex, the siRNA is unwound, releasing and degrading a “passenger strand”. The guide strand, however, remains associated with RISC and leads RISC to bind to the target mRNA18,21,27,28.

To induce gene inhibition, the guide strand must be fully complementary to its target mRNA, which results in gene inhibition via cleavage of the target mRNA21. This summarizes the endogenous RNAi pathway. The next section will discuss key therapeutic triggers of RNAi and how these have been harnessed as therapeutic strategies.

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