Origin of RNAi-RNAi

The first clue that dsRNA can lead to gene silencing came from the study of Caenorhabditis elegans, a nematode. >In 1995, Dr. Su Guo and Kemphue from Cornell University discovered an unexpected phenomenon while attempting to block the par-1 gene in C. elegans. They originally used antisense RNA technology to specifically block the expression of the above-mentioned genes, while injecting sense RNA into nematodes in control experiments in order to observe an increase in gene expression. But the result obtained was that both equally cut off the expression pathway of the par-1 gene. This is exactly opposite to the traditional explanation of antisense RNA technology. The research team has been unable to provide a reasonable explanation for this accident.

This strange phenomenon was not resolved until three years later. In February 1998, Andrew Fire from Carnegie Institution in Washington and Craig Mello from the University of Massachusetts Cancer Center first uncovered this suspenseful mystery. Through extensive and arduous work, they confirmed that the phenomenon of suppression of gene expression by sense RNA encountered by Dr. Su Guo, as well as the blockade of gene expression by past antisense RNA techniques, were caused by contamination of trace double stranded RNA in the RNA obtained from in vitro transcription. When they purified the single stranded RNA obtained from in vitro transcription and injected it into nematodes, they found that the gene inhibition effect became very weak, while the purified double stranded RNA was exactly the opposite, capable of efficiently and specifically blocking the expression of the corresponding gene. In fact, a few molecules of double stranded RNA per cell are sufficient to completely block the expression of homologous genes. Subsequent experiments have shown that injecting double stranded RNA into nematodes not only blocks the expression of homologous genes throughout the nematode, but also leads to the silencing of homologous genes in its first generation offspring. This phenomenon is referred to as RNA interference (RNAi) by the team.

The potential role of RNAi prompted Fire and Timmons to continue their experiments. They fed nematodes with genetically engineered bacteria capable of expressing double stranded RNA homologous to the+C. elegans unc-22 gene, and the nematodes exhibited a phenotype similar to unc-22 deficiency. Subsequent experiments have shown that immersing nematodes in double stranded RNA can also induce gene silencing - this technique makes it possible to screen for functional loss mutants induced by nematode RNA i on a large scale, and has sparked extensive research on gene knockout in this model organism.

In the following year, the phenomenon of RNAi was discovered in eukaryotes such as fruit flies, cone worms, fungi, rotifers, plants, and zebrafish, indicating that RNAi may have occurred in the early stages of life evolution. Before the mechanism of RNAi was truly understood, naturally occurring RNAi phenomena were described in these organisms as follows:

1) The 'qelling' effect of the fungus genus Mycelium.

2) Posttranscriptional gene silencing (PTCS) and co suppression in plants, as well as RNA mediated antiviral effects. This is a protective mechanism against frequent viral infections.

3) The RNAi phenomenon in animals, including hydroids, rotifers, fruit flies, and nematodes, has the function of inhibiting the spread of transposons.

4) RNAi phenomenon in zebrafish and mice in vertebrates.

Due to the presence of a common mediator siRNA in these phenomena, as well as the homology of genes involved in different species, researchers believe that all of these silencing phenomena are based on the same core mechanism, and protective resistance against viruses and transposons may be the core function of the RNAi pathway [9].

More than 20 years ago, a strange discovery was made in the study of Petunias: Rich Jorgens and colleagues placed a gene that produces pigments in a strong promoter and introduced it into Petunias, attempting to deepen the purple color of the flowers. However, they did not see the expected deep purple flowers, and most of the flowers became spotted or even white. Jorgensen named this phenomenon cosuppression because both the introduced gene and its similar endogenous genes are simultaneously suppressed. At first, this was considered a peculiar phenomenon unique to dwarf morning glories, but later it was discovered that similar phenomena exist in many other plants, even in fungi, particularly in Neurospora+, where detailed research was conducted (referred to as the quelling phenomenon here).

However, what causes this phenomenon of gene silencing? Although for some plants, gene silencing caused by transgenes may be due to gene specific methylation, known as transcriptional gene silencing (TGS), there are indeed some plants where gene silencing occurs after transcription, known as post transcriptional gene silencing (PTGS). The nuclear transfer experiment showed that homologous transcripts did appear, but were quickly degraded in the cytoplasm without accumulation. Gene silencing can be transmitted between nuclei of heterokaryotes. Later, it was further confirmed that in plants, grafting genetically modified plants resulting in gene silencing onto another plant without gene silencing can also induce PTGS. In later experiments on nematodes and fruit flies, we learned that the trans acting factor causing PTGS in plants is double stranded RNA.

Transgenic organisms can trigger PTGS, but gene silencing may also be induced by some viruses. Once triggered, PTGS is mediated by a diffusible trans acting molecule. This was first confirmed in Neurospora: Cogoni and his colleagues found that gene silencing can be transmitted between nuclei of heterokaryotes. Later, Palauqui and colleagues further confirmed that in plants, grafting plants with genetically modified genes causing gene silencing onto another plant without gene silencing can also induce PTGS. In later experiments on nematodes and fruit flies, we learned that the trans acting factor causing PTGS in plants is double stranded RNA.

RNAi has also been discovered in the study of fruit flies. Although the experiment of feeding fruit flies with yeast capable of producing dsRNA ended in failure, gene silencing can be triggered by microinjection or gene gun injection of dsRNA into fruit fly embryos, or introduction of DNA with reverse repetitive sequences into fruit flies. In the past few years, RNAi technology has become a reverse genetic tool in fruit fly research for identifying functional loss phenotypes.

How does injected RNAi induce gene silencing? Multiple research groups have made unremitting efforts in the past few years. Baul Combe and Hamilton first identified approximately 25 base sized RNAs in plants undergoing co inhibition that do not exist in plants without gene silencing. These RNAs complement the sense and antisense strands of silenced genes, respectively. This becomes the first key clue to reveal the secrets of RNAi.

Later experiments in fruit fly cells further revealed this secret. In a series of famous experiments, Zamore and colleagues found that dsRNA injected into fruit fly cells was cleaved into RNA fragments of 21-23 nucleotides in length. They also discovered that mRNA of endogenous genes homologous to dsRNA was only cleaved into 21-23 nucleotide fragments at the corresponding sites to dsRNA. Soon, the mechanism of RNAi became increasingly clear

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Origin of RNAi-RNAi

Origin of RNAi-RNAi