This section will give an overview of the general mechanism by which RNAi is thought to work. It will also look briefly at non-silencing mutant plants.
The mechanism by which RNAi is thought to work is a very complicated one. The process by which RNAi occurs in different organisms varies slightly. This is usually because different proteins are used in the process of RNAi. As the emphasis of this website is on plant science, the model plant Arabidopsis thaliana will be looked at. Other organisms will be very briefly considered as well.
Basically, the process of RNAi is triggered off by dsRNA precursors. These dsRNA precursors are processed into small interfering RNAs (siRNAs) that vary in length from 21-23 nucleotides (nt). This processing requires the presence of ATP, which is mediated by different proteins in different organisms. Even though these proteins are different, they are all dsRNA specific RNase-III-type endonucleases. The siRNAs are subsequently incorporated into a multiprotein complex. This complex is known as the RNA-induced silencing complex (RISC). It is thought that RISC undergoes an ATP-dependent step that activates the unwinding of the double stranded siRNAs. This activated RISC complex is thought to use a single stranded siRNA, to aid in identification of RNAs that are complementary to this single stranded siRNA. An endoribonuclease then cleaves this RNA, which is then thought to be degraded by exoribonucleases (Meister, G. & Tuschl, T. 2004).
There are three different types of small RNA's that occur naturally. These are small interfering RNAs (siRNAs), microRNAs and repeat -associated short interfering RNAs (rasiRNAs). Natural production of dsRNA can occur through RNA-templated RNA polymerisation (e.g. from viruses), or through the hybridisation of overlapping transcripts (e.g. from repetitive sequences). These kind of dsRNAs result in rasiRNAs or siRNAs, which guide the degradation of mRNA. Also, endogenous transcripts containing 20 to 50 base pair inverted repeats that are complementary or near complementary form dsRNA hairpins. These dsRNAs form miRNAs that repress translation. Artificially, long dsRNAs or siRNAs have been introduced into organisms or cells to silence expression of genes (Meister, G. & Tuschl, T. 2004).
Basic steps involved in the mechanism of RNAi
(http://www.ambion.com/techlib/tn/101/7.html, Last Accessed: 9th April, 2005)
Now, major steps in this process will be looked at individually.
Processing the dsRNA precursors
Catalysis of the process of maturation of the small RNAs is carried out by dsRNA-specific RNase-III-type endonucleases, known as Drosha or Dicer. Drosha is needed to process miRNA precursors, but is not needed for long dsRNAs. Transcription of miRNAs as long primary transcripts, is first carried out in the nucleus by Drosha. Once Drosha has excised the miRNA precursor, a 2-nucleotide 3' overhang and a 5' phosphate are left over at the stem base. This miRNA precursor is then moved to the cytoplasm by a nuclear export receptor called exportin-5 (Meister, G. & Tuschl, T. 2004).
After the miRNA precursor makes it into the cytoplasm, it is processed yet further by a protein called Dicer. When dsRNAs are processed by Dicer, RNA duplexes with a length of about 21 nt are produced. These also have 2-nucleotide 3' overhangs and 5' phosphates. Of course, different organisms have different numbers of Dicer genes that process different sorts of dsRNAs (Meister, G. & Tuschl, T. 2004).
In this case, we will look at Arabidopsis thaliana. In this plant, four different Dicer-like (DCL) proteins have been found: DCL1 to DCL4. Three of these DCL proteins are known to be involved in the processing of the various dsRNAs. DCL1 is known to process miRNA precursors. However, it needs two other proteins to help it with this: HEN1 and HYL1. DCL2 is needed to produce siRNAs from various plant viruses. DCL3, in conjunction with HEN1, assists in the production of rasiRNAs. This is different to other organisms. For instance, C. elegans and mammals only have one Dicer gene, and Drosophila melanogaster has two Dicer genes (Meister, G. & Tuschl, T. 2004).
RNA silencing effector complex assembly
These miRNA and siRNA duplexes that contain ribonucleoprotein particles (RNPs) are now made into RNA-induced silencing complexes (RISC). Generally, effector complexes containing siRNAs are known as a RISC, while those containing miRNAs are known as miRNPs. In Arabidopsis thaliana the rasiRNA-containing effector complexes are known as RITSs. All RISCs or miRNPs have a member of the Argonaute (Ago) family of proteins attached to them. RISCs and miRNPs differ in size and composition, based on which organism they are in. During the effort to identify more specific and active siRNA duplexes for guidance of cleavage of mRNA, it was observed that the sequence of the siRNA duplex had a significant impact on the ratio of sense and antisense siRNAs that were entering the RISC complex. Also, miRNAs that occurred naturally tended to only accumulate single strands into the miRNPs (Meister, G. & Tuschl, T. 2004).
There have been different numbers of Ago proteins identified in different organisms. Arabidopsis thaliana has ten members, D. melanogaster has five members, and humans have eight members of the Ago protein family. Only a small number of these proteins have actually had their function characterised (Meister, G. & Tuschl, T. 2004).
mRNA cleavage and repression of translation
Once the RISC complex has been formed, the siRNAs in the RISC complex guide degradation that is sequence-specific, of the complementary or near complementary mRNAs. The RISC works by cleaving the mRNA in the middle of its complementary region. The cleavage does not require the presence of ATP, however multiple cleavages are more efficient in the presence of ATP. RISC and miRNP complexes work by catalysing hydrolysis of the phosphodiester linkage of the target RNA (Meister, G. & Tuschl, T. 2004).
The mechanism by which repression of translation guided by miRNA works, is not as well understood as the mechanism by which mRNA cleavage works. Evidence that this occurred, was first found in studies of C. elegans mutants, where specifically targeted miRNAs reduced synthesis of proteins without affecting the levels of mRNA. It has been suggested that miRNAs affect translation termination or elongation rather than actual initiation of the process. In addition to this, it has been found that miRNAs can act as siRNAs and vice versa. This has in the past been wrongly interpreted as a suggestion that the siRNA or miRNAs function is determined only by the complementarity shared by the small RNA and the target RNA. Perhaps, a more accurate explanation would be that small dsRNAs are made into complexes that undertake specific functions. It is thought that mRNA degradation and translational regulation guided by miRNAs could be used as simultaneous mechanisms for natural regulation (Meister, G. & Tuschl, T. 2004).
Even though there are a lot of organisms that can undergo gene silencing through the use of dsRNA, there are nevertheless some organisms that are mutants that can resist RNA interference. In the case of plants, the model species Arabidopsis thaliana will be considered. Of course, other organisms such as C. elegans and Neurospora crassa also have mutants that resist interference.
An example of an Arabidopsis plant that has undergone gene silencing
(http://www.cals.ncsu.edu/botany/Robertson%20Lab%20web/research.html, Last Accessed: 27th April, 2005)
There are essentially four different mutants of Arabidopsis thaliana that resist interference. These are the AGO-1 mutant, SGS-2/SDE-1 mutant, SGS-1 mutant and SGS-3 mutant. AGO stands for the Argonaute family of proteins, and SGS stands for suppressor of gene silencing. The AGO-1 mutant of A. thaliana has other phenotypic differences associated with it. It has leaf differentiation that differs from that of normal plants, fewer secondary meristems, and is also sterile because of defective flower development (Hammond, S.M., 2001).