RNA Silencing Process

RNA silencing is a complicated process that involves many different enzymes and molecules.  RNA silencing is carried out in a number of ways for different reasons, but ultimately the process serves the same function: to prevent mutation in an organism.  One such way is simply to maintain the regulation of gene expression, which occurs constantly throughout the translational process through the use of miRNA.  Another way is through the use of dsRNA, which is used to create siRNA.  In these two cases, RNA silencing is being utilized to prevent translation and is referred to as post-transcriptional RNA silencing.  In another case, however, RNA silencing can be utilized to prevent mRNA from even forming, which is pre-transcriptional RNA silencing and it called transcriptional RNA silencing.  The latter of these two methods is not well understood in the scientific community, but some information has been found on what may be occurring during this process.

One method of RNA silencing is carried out through the use or micro RNA, which is considered a regulatory RNA.  miRNA can carry out the RNA silencing process in multiple ways, but its main target is the same: mRNA.  miRNA is created in the nucleus of a cell, but before becoming miRNA it is primary miRNA or pri-miRNA.  Pri-miRNA must undergo post-transcriptional modification before becoming miRNA and each pri-miRNA may contain the precursor for up to six miRNA.  This allows amplification of the RNA silencing process if needed. Once miRNA is mature, it can become part of a RNA-Induced Silencing Complex (RISC).  The RISC is composed of miRNA, a Dicer, and many different associated proteins.  Primarily, members of the argonaute protein family are critical to the RISC function, because they bind to the miRNA and orient it for interaction with the target mRNA.  Also, it is important to note that a RISC may also be referred to as miRISC when it has miRNA as a component (this will make more sense later on when the other method of RNA silencing is described).  The following is an illustration of this process so far.

Once miRISC is formed, the silencing process may take place through a number of ways.  Typically miRISC can target many different mRNA molecules due to having an incomplete base pairing, which allows it to target multiple mRNA codons.  Therefore, one miRISC may regulate the expression of many different genes.  The miRISC can do this by locating its target mRNA and then base pairing with it.  After this occurs, a number of things may take place.  One event that may occur is simply that the miRISC will remain on the mRNA and block translation from occurring by blocking the pathway of tRNA so that it can not read the genetic code in order to create a polypeptide.  Another event that may occur is the cleavage of the mRNA into two mRNA sections.  One section may still be translated while the other is broken down and recycled by the cell.  This creates specificity and control of the polypeptides being synthesized.  The decay of the mRNA is sped up through the deadenylation of it by the miRNA, which is when an anion is dissociated from a nucleotide in the RNA.  The following is an illustration summarizing the above information.

Another post-transcriptional RNA silencing process involves dsRNA.  dsRNA can either be synthesized in an organism or it can be introduced into an organism (such as a virus) in order for the RNA silencing process to occur.  No matter where the dsRNA originated, the same RNA silencing process takes place.  Endogenous dsRNA, which is dsRNA that originated in the organism or cell, initiates the RNA silencing process by first activating a ribonuclease protein Dicer.  The Dicer binds to the dsRNA and then cleaves it into two double-stranded fragments with twenty to twenty-five base pairs and a two nucleotide overhang at the three prime (3′) end.  These fragments are called single interfering RNA or siRNA duplexes, which are then broken down into single stranded siRNA after becoming part of the RISC complex.   Exogenous dsRNA (dsRNA that originated outside of the cell or organism, like a virus) initiates the RNA silencing process by first being detected by an effector protein.  The protein stimulates Dicer activity, the protein binds to the dsRNA, and then cleaves it into two fragments.  These fragments are also considered siRNA duplexes and are transformed into single stranded siRNA like in the endogenous dsRNA process. From this point forward, both processes occur in the same manner.

Once single stranded siRNA is formed, it is part of the RISC complex, which has many of the same components as miRISC.  Those components include a siRNA (in place of miRNA), a Dicer, and multiple proteins (again, primarily proteins from the argonaute protein family).  Once the complex is formed, it then targets its specific mRNA depending on the base pairing present.  One of the main differences between the siRNA RISC complex and the miRISC complex, is that the siRNA RISC complex does not target a variety of mRNA, but instead has the base pairing code for only one mRNA while one miRISC can target a variety of mRNA.  Once the RISC complex finds its target mRNA, it binds to it, and then cleaves it into two fragments.  These two fragments are then recycled by the cell, and the RNA silencing process has been completed.  The following is an illustration of the dsRNA silencing process.

Transcriptional RNA silencing is another RNA silencing process, but it is not very well understood.  What is understood about it is that it involves a complex called the RITS complex, which functions to down-regulate genes pre-transcriptionally through the modification of histones and the induction of heterochromatin formation.  Modifying the histones serves to regulate the transcription of genes into pre-mRNA, because histones are a protein component of chromatin, which is what DNA structure depends upon.  If the DNA structure changes, then how it can be transcribed or what part of it (which genes) can be transcribed also changes.  Heterochromatin also influences the transcription of genes, because it is a tightly packed form of DNA, and when DNA is too tightly packed it cannot be transcribed.  Therefore, gene regulation is again induced.  The following is a chart that shows how similar transcriptional RNA silencing and post-transcriptional RNA silencing are relative to the beginning of their processes, but also how their differing complexes that are formed carry out different functions.

Overall, the RNA silencing process may be carried out in different ways, but every way serves the same purpose: to control gene expression and prevent and protect from mutation in an organism.

The following video is a great example of the RNA silencing process.

The following is a great animation of the RNA silencing process when the RISC complex acts to block or prevent transcription by bonding to mRNA or when it cleaves mRNA, respectively.  Although it can be a little confusing to understand, since none of the molecules are labeled, after reading this section and the previous sections, it should be quite simple to identify which molecules are which based on the function that they are carrying out.

Sources:

“MicroRNA (miRNA) Resource.” Ambion, Inc. – The RNA Company. Web. 10 Nov. 2010.

<http://www.ambion.com/techlib/resources/miRNA/index.html&gt;.

Pfeffer, Sébastien, Gunter Meister, Markus Landthaler, and Thomas Tuschl, comps. “RNA Silencing.” B.I.F. FUTURA 20 (2005):             83-91. The Rockefeller University. Web. 10 Nov. 2010. <http://www.rockefeller.edu/labheads/tuschl/Pfeffer,BImeeting05.pdf&gt;.