How The Endogenous Gene Of C. Elegans Could Be Interfered By The Interfering Rna
Introduction
RNA interference was useful in changing the gene expression of an organism, especially in Caenorhabditis elegans (C. elegans), researchers studied about this biological mechanism and questioned from their finding. They found two features were hard to understand. First, the sufficiency of each sense and antisense RNA preparations to cause interference. Second, despite the rapid degradation of many endogenous RNA transcripts in the early embryo, effects of interference could persist to the offspring. With these questions, the research aimed to find out how the endogenous gene of C. elegans could be interfered by the interfering RNA, of which the requirements for structure and delivery were examined.
Methods
RNA was synthesized with the use of T3 and T7 polymerase and purified by gel electrophoresis. Injection buffer was used for sense/antisense annealing and purified. For the microinjection to C. elegans, equal masses of sense, antisense and double-stranded RNA were used so as to compare their activities. In order to study what were required for the structure and delivery of interfering RNA, analysis of phenotypes of injected C. elegans targeted on several segments of different genes was done. Genes unc-22, fem-1, unc-54, hlh-1 and myo-3 driven GFP transgenes were chosen to test the RNA interference on the offspring of injected C. elegans. C. elegans strain PD425, which produces GFP over the body muscles, was used since it had three plasmids important for the study, including pSAK4, pSAK2 and dpy-20 subclone. Fluorescence micrographs were taken to see the interference effects of double-stranded(ds)-gfpG and ds-lac ZL RNA at a cellular level. Gonadal and body-cavity injections were done with needle insertion to investigate the effect of site of injection in the animals and their offspring. Semisynchronous cohorts were yielded after recovery of injected C. elegans, the phenotypes were then identified from them. Mex-3 RNA was used to test the interference on endogenous mRNA.
Results
For the effect of sense, antisense and mixed RNAs on offspring of injected C. elegans, most of the sense-antisense mixtures were much more effective than either form of the single strands. The amount of injected mixtures required to make interference was also much less than the amount of single strands used, only a few molecules were needed. The sense-antisense mixture was found predominantly double-stranded which indicated the potent interfering activity could be due to ds-RNA but not the single strand itself. There was one exception on the F1 phenotype, dsRNA interference on gene unc54C did not show the expected null-mutant phenotype. Another important note was that there was no obvious interference of dsRNA on intron and promoter sequences.
The C. elegans with injected ds-gfpG and ds-lacZL RNA showed decreased fluorescent cells and absence of reporter proteins. The non-striated vulval muscles that were born in late larval development were resistant to any concentrations of injected dsRNA while in embryonic and postembryonic development, higher concentration had greater effect. The amount of endogenous mRNA transcript decreased with the dsRNA injection. For the experiment on the effect of site of injection, it showed the dsRNA interference could cross cellular boundaries, which produced a strong and specific interference in offspring’s gene expression. Discussion The study suggested that the injected single strands might be degraded or became inaccessible since their interfering activity was extremely low in the absence of opposite strand. The exceptional result on unc54C might be interfered with other myosin heavy-chain genes. However, clear explanation was not found from the study. The finding of the effectiveness of dsRNA injection could be used for other researchers to study on many other coding regions even with no specific function defined. Nevertheless, there are limitations, for instance, interference between closely related genes of the shared sequence and resistance on RNA interference of a low level expression for some or all genes. The potential organisms to be examined with the use of dsRNA interference can be nematodes, invertebrates, vertebrates and plants. In their findings, it was important for a deeper understanding on gene regulation and RNA interference. The discovery on dsRNA injection to be significant in gene expression was very useful for other researchers to study the gene function and possibly led to the new development in medical field.
Conclusion
It was concluded that dsRNA was more effective for the production of interference than individual strand. Also, the interference affected both the injected animals and their offspring. Lastly, in the interference process, a catalytic or amplification component could be presented. This study suggests that RNA interference may be involved directly at the chromatin structure or transcription. Furthermore, organism may use dsRNA to silence physiological gene for a biological purpose and C. elegans may have an efficacious mechanism on the transportation of RNA. The mechanism of RNA-mediated interference in C. elegans was not known and so there can be more researches on the possible targets and mechanisms.
Comment
The arguments of the articles are well supported by the data and experiments conducted and the results are supportive of the authors’ earlier work. This is why the main authors got Nobel prize with this study. The data were presented in details and well organized. They had justified their data and provided suggestions in their conclusion. The paper format was not presented in a clear way compared to the recent journal articles. There is discussion mixed in the methods and results. In addition, not all methods were included with respect to their results, which made the readers a bit confusing when they did not know the topic well.
