Rnai

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  • Topic: DNA, RNA, Polymerase chain reaction
  • Pages : 11 (3925 words )
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  • Published : May 1, 2013
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Methods to elucidate gene function

Historically, studies to identify genes that function in a particular process involve forward genetics. One way to mutate genes using this process is to expose organisms to a mutagen (typically either a chemical or gamma radiation), randomly mutating the genome in many animals. We then screen these animals for defects in the process we wish to study – essentially, looking for physical or behavioral changes in the organism. Mutagenesis (creating mutations) is very time-consuming and generally cannot target specific genes, making it very difficult to study the function of a particular gene if no mutation in the gene already exists. It is also a time-consuming process to map or figure out exactly where a mutation occurred and to make sure no additional mutations exist. A powerful alternative to forward genetics is to decrease the expression of genes with RNA interference (RNAi).

RNAi is an endogenous cellular mechanism that is present in some organisms, including plants and worms. Biologists have learned to use the RNAi mechanism to their advantage. By deliberately introducing defined sequences of dsRNA identical to the sequence of a gene, biologists can observe the physiological consequences of “silencing” that gene. Silencing genes in this way is also called reverse genetics because one can knock down the function of particular genes in a targeted way without mutating the gene. RNAi is a cellular mechanism thought to have evolved to protect organisms from infection by RNA viruses. When double-stranded RNA (dsRNA) is present in a cell, it is recognized by a protein named Dicer (Figure 1). Dicer is an RNase that cuts these dsRNA molecules into short pieces. These 21- to 25-base pair pieces, called small interfering RNAs (siRNAs), are bound by a protein complex called RISC (RNAi-induced silencing complex). One strand from the siRNA is destroyed after siRNA binding, leaving the other strand bound to Argonaute, the RISC complex RNase. The remaining strand acts as a guide that hybridizes to messenger RNAs (mRNAs) that are complementary to the guide strand. Once bound, Argonaute cleaves the target mRNAs within the complementary sequence, thereby inhibiting, or silencing, gene function. siRNA-bound RISC can then search for and destroy additional complementary target transcripts.

Figure 1: RNAi pathway (Alberts Essential Cell Biology 3rd edition) RNAi is a cellular mechanism thought to have evolved to protect organisms from infection by RNA viruses. When double-stranded RNA (dsRNA) is present in a cell, it is recognized by a protein named Dicer (Figure 1). Dicer is an RNase that cuts these dsRNA molecules into short pieces. These 21- to 25-base pair pieces, called small interfering RNAs (siRNAs), are bound by a protein complex called RISC (RNAi-induced silencing complex). One strand from the siRNA is destroyed after siRNA binding, leaving the other strand bound to Argonaute, the RISC complex RNase. The remaining strand acts as a guide that hybridizes to messenger RNAs (mRNAs) that are complementary to the guide strand. Once bound, Argonaute cleaves the target mRNAs within the complementary sequence, thereby inhibiting, or silencing, gene function. siRNA-bound RISC can then search for and destroy additional complementary target transcripts.

Figure 1: RNAi pathway (Alberts Essential Cell Biology 3rd edition)

RNAi in C. elegans
The nematode C. elegans is used as a model organism – one that is commonly studied by biological researchers. The RNAi mechanism was discovered using these organisms. Amazingly, RNAi can be activated in C. elegans by simply feeding worms bacteria expressing dsRNA corresponding to part of the gene to be silenced. The dsRNA enters cells through the intestine and is recognized by the RNAi machinery, leading to silencing of the specific gene through transcript (mRNA) degradation. In C. elegans the triggering of the RNAi mechanism can spread from...
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