Principles and Applications of RNA Interference Technology (Part 1)-RNAi

RNAi was first discovered by Fire et al. in C. elegans. They found that injecting dsRNA into the nematode can inhibit the expression of homologous genes, and confirmed that this inhibition mainly acts after transcription. Therefore, RNAi is also known as post transcriptional gene silencing (PTGS). Subsequently, RNAi was discovered in Drosophila, Trypanosomes, Planaria, Zebrafish, Arabidopsis thaliana, mice, rats, and humans. Genetic studies have shown that RNAi is a widely present and highly conserved mechanism in eukaryotes, closely related to many important biological processes in eukaryotic cells. Through genetic and biochemical research on RNAi, its mechanism of action has become increasingly clear (see Figure 1). Zamore et al. established an in vitro system using fruit fly embryo extracts and confirmed that RNAi is an ATP dependent process. During this process, dsRNA (exogenous or in vivo) is first degraded into small molecule double stranded RNA with a 5'2 monophosphate and a length of 21-23 bp. This RNA molecule is called small interfering RNA. siRNA recognizes mRNA with homologous sequences through base complementary pairing and mediates its degradation. Research has shown that siRNA has similar structural features in living organisms: it is a double stranded RNA approximately 21-23 bp in length, with a 5 'monophosphate and 3' hydroxyl terminus, and a 2-3 nt single stranded protrusion at the 3 'end of the complementary double stranded RNA. In the process of RNAi, a nuclease called Dicer is responsible for converting dsRNA into siRNA. It belongs to the RNase III family and has two catalytic domains, one helicase domain, and one PAZ (Piwi/Argonaute/Zwille) domain. Dicer appears in the form of a dimer during the catalytic process, and its catalytic domain is arranged antiparallel on dsRNA, forming four active sites. However, only two sites on both sides have endonuclease activity, which cleave dsRNA at a distance of about 22bp. Dicer structures vary slightly in various organisms, resulting in slight differences in siRNA length. After the formation of siRNA, it binds to a series of specific proteins to form siRNA induced interference complex (RISC), which recognizes target mRNA through base complementary pairing and degrades it, resulting in specific gene silencing. In RISC, the antisense strand of siRNA plays a role in target sequence recognition. Zamore et al. found that during RNAi, the first active precursor produced is RISC, with a molecular weight of approximately 250kD. When ATP is added, a 100kD active complex can be formed. The conversion from inactive precursors to active enzyme complexes is similar to the activation of proteasomes, which requires the cleavage of siRNA double strands bound to them. In the presence of ATP, ATP dependent helicases cleave the double stranded siRNA and replace its sense strand with the target mRNA. 

The mRNA replaces the sense strand and complements the antisense strand, and then the activated RISC cleaves the target mRNA sequence in the middle of the complementary region, about 12bp from the 3 'end of the siRNA antisense strand. The RNAi effect has two distinct characteristics, specificity and efficiency. The efficiency of interference suggests the presence of signal amplification steps in the mechanism. Fire et al. discovered as early as 1998 that a small amount of dsRNA could lead to the degradation of a large amount of target mRNA in nematodes, but the high efficiency cannot be explained solely by the degradation of a small amount of dsRNA into dozens of siRNA by Dicer. Many studies have shown that new dsRNA molecules are synthesized during RNAi. When the siRNA antisense strand recognizes and binds to the target mRNA, the siRNA antisense strand can be used as a primer to synthesize new dsRNA using the target mRNA as a template under the catalysis of RNA dependent RNA polymerase (RNA2depender RNA polymerase, RdRP). Then, Dicer cleaves to produce new siRNA, which recognizes a new set of mRNA and generates new siRNA. After several synthesis and cleavage cycles, the silencing signal will continue to amplify (Figure 1). It is this mechanism known as target2directed amplification that endows RNAi with high efficiency and persistence. 

 

In addition, many studies have shown that RNAi signals can cross the intercellular barrier and spread to other cells and tissues. In plants, RNAi signals can be transmitted between cells through two pathways: ① short distance transmission between adjacent cells. Plant cells have rich connections - intercellular filaments, and silencing signals (such as siRNA molecules) can be transmitted between cells through intercellular filaments; ② Silent signals can also be transmitted over long distances through the criss crossing vascular system in plants. In animals, the diffusion of RNAi signals requires the involvement of special proteins. Recently, Hunter et al. identified a protein related to the propagation of silencing signals in nematodes, which is a transmembrane protein encoded by the sid21 gene. It can form transmembrane channels on the membrane for silencing signals to pass through. SID21 protein homologs do not exist in fruit flies, but studies have shown that SID21 protein homologs exist in mammals.

The RNAi phenomenon in the process of life is regulated by multiple genes and enzymes. Through research on N. crassa, Dictoyostelium, fruit flies, nematodes, Arabidopsis, etc., people have gained a certain understanding of genes and enzymes related to RNAi. In the chain spore enzyme, the QDE21 protein encoded by the qde21 gene is a homolog of RdRP, while the qde22 gene encodes a member of the Argonaute family. Three RdRP homologs were found in the parasitic protozoa, two of which are encoded by rrpA and rrpB genes, but only the enzyme encoded by rrpA is involved in the RNAi process. At least six genes (rde21, rde22, rde23, rde24, mut27, ego21) have been identified in nematodes that are associated with the RNAi process. Mutations in these genes can lead to RNAi deficiency. Among them, RDE21 protein is homologous to QDE22 in the cellulase and AGO21 in plants, both belonging to the Argonaute family. Mut27 encodes a protein with 3 '→ 5' exonuclease activity, while the ego21 gene encodes a RdRP homolog. If a mutation occurs, it will cause the silencing of certain germline genes to be released, ultimately leading to developmental defects in the germline. This also indicates a certain relationship between RNAi mechanisms and development. 

In addition, researchers have found that the smg22, smg25, and smg26 genes are associated with the maintenance of RNAi effects. smg22 encodes an ATP dependent RNA helicase, which may be involved in the unwinding of siRNA double strands during RNAi processes. In Arabidopsis, the sde23 gene encodes an RNA helicase similar to the nematode SMG22, which participates in the PTGS process, while the sde21 encoded protein mediates the silencing of the transferred gene and is unrelated to PTGS. In addition to the aforementioned genes, there are also some important gene products involved in the RNAi process, such as the dcr21 gene encoding Dicer enzyme in nematodes. Dicer is responsible for cleaving long dsRNA into siRNA during the initiation stage of RNAi, which is crucial for the RNAi process. Although dozens of genes related to the RNAi mechanism have been discovered, RNAi is an extremely complex biological process, and more genes and enzymes involved in the RNAi process will be gradually discovered.

 

The RNAi mechanism relies on strict base pairing between siRNA antisense chains and target sequences, making it highly specific. Studies have shown that in addition to participating in post transcriptional stability regulation of mRNA (i.e. post transcriptional gene silencing), the RNAi mechanism is also related to other important biological processes.

 

 

Recently, Volpe et al. used S. pombe and found that transgenic expression integrated into the centromere region was always suppressed. They referred to this phenomenon as "centromere silencing" and subsequently discovered that centromere silencing was caused by chromatin condensation resulting from Lys29 methylation of histone H3 in this region. They also demonstrated that gene mutations related to RNAi can lead to the disappearance of histone H3 methylation and the elimination of centromere silencing. From this, it can be inferred that at least in yeast, the RNAi mechanism can cause gene silencing in the centromere region by altering chromatin structure through histone methylation. 

At the same time, Bartel et al. isolated a small RNA molecule homologous to the centromere sequence from yeast and named it heterochromatin siRNA. They speculated that "centromere silencing" was caused by histone methylation in the homologous region of the sequence caused by siRNA. That is, in the centromere region, one strand of DNA is continuously expressed while the other strand is intermittently expressed, and the transcripts of the two strands complement each other to form dsRNA. Then, siRNA is formed through RNAi mechanism, which guides methyltransferase to the centromere region homologous to the sequence, causing histone methylation and ultimately leading to changes in chromatin structure, thereby regulating gene activity.

 

 

Research on RNAi related gene mutants has shown that the RNAi mechanism is involved in regulating the normal transcription of cellular coding genes. Three gene silencing mechanisms in organisms - DNA methylation, co inhibition, and transposon silencing - are all related to RNAi. Wessenegger et al. found in 1994 that chromosomal DNA methylation in plants depends on RNA replication, i.e., the presence of double stranded RNA. Subsequently, Wessenegger and Pelissier confirmed that the double stranded RNA formed by virus replication in cells can methylate homologous sequences of about 30 bp on host chromosomes, and found that as long as there is sequence homology, double stranded RNA can methylate genes. After DNA methylation, genes lose their transcriptional activity and no longer initiate transcription, resulting in the silencing of specific genes. Moreover, this methylation state can be passed on to offspring, causing the expression of the gene to remain suppressed in the offspring. 

The phenomenon of co inhibition refers to the silencing of homologous genes in cells caused by the transfer of genes. PalBhadra et al. confirmed that co inhibition in fruit flies is due to the binding of Polycomb complexes to endogenous genes. Polycomb is guided by siRNA and binds to the vicinity of homologous sequences, making chromatin inactive and inhibiting its initial transcription. In addition, Sijen et al. found that in Petunia, if the dsRNA produced by gene transfer is homologous to the promoter of the target gene, it will lead to transcriptional gene silencing (TGS). If it is homologous to the coding region, it will lead to post transcriptional gene silencing (PTGS). They also found that small molecule RNA is produced during both TGS and PTGS processes, and both processes are accompanied by DNA methylation of the target sequence, ultimately leading to gene silencing.