In the following video we are introduced to the concept of RNA interference (RNAi), one of the most important discoveries made in the field of cell biology in the last two decades. It is so important that Andrew Fire and Craig Mello were awarded the 2006 Nobel Prize in medicine for their discovery of RNAi. Prior to this discovery, the common belief was that the main role of RNA in the cell was in its messenger form (mRNA), which is an intermediate step between a gene and its proteins. The discovery of RNAi demonstrated that RNA also possesses important regulatory functions in the cell.

Initially, the mechanisms of RNAi were studied in the worm Caenorhabditis elegans and in plants. Today it is known that these processes occur in many organisms, ranging from the unicellular yeast to the highest organisms, including humans. Further studies in plants and fruit flies (Drosophila melanogaster) have shown that the RNAi system is also active in defense against viruses by degradation of the viral RNA. Today researchers are investigating whether RNAi can be exploited in medicine, for example by introducing RNAi molecules that can silence specific genes that are necessary for tumor growth.

RNA interference is a type of post-transcriptional regulation, since it controls the level of expression of specific genes either by degrading (destroying) their mRNA transcripts or by hindering the translation of the mRNA into its protein product. The video describes the mechanism behind the RNAi system and how its molecular components are produced inside the cell. In addition, the video depicts how RNAi activity is amplified in plants, worms, mammals and other organisms by generation of a multitude of siRNA molecules. If you are not familiar with the concept of gene regulation and the process of protein synthesis, I would advise to watch the video The protein production process - the secret of life before reading the rest of the article.

 

The video was produced by Arkitek.
Special thanks to Beth Anderson for her great assistance.

 

As mentioned, the RNAi system regulates gene expression by either degradation of mRNA or by repression of its translation into a protein. The system is composed of two main participants: (a) short RNA molecules, about 20 nucleotides in length (compared to the >1000 nucleotides making up an average mRNA). These molecules are complementary to a segment on their target mRNA molecule and are thus able to repress it in a specific manner; (b) RISC – RNA-Induced Silencing Complex. As the "executor" of the system, this protein complex performs the actual chemical degradation of the target mRNA or, alternatively, acts as a translation repressor.

Short RNA molecules with repressive function exist in two main forms that differ in their origin. Short interfering RNA (siRNA) molecules are derived from double strand RNA which often originates from repetitive sequences in the genome. This type of molecules can be introduced into the cell from its surrounding, either by artificial means or as a result of natural transport processes between neighboring cells (in plants, for example). MicroRNAs originate from sequences that lie in between protein-encoding genes, or within gene introns (the non-coding sequences in a gene). Their distinct feature is the hairpin structure they adopt following their transcription (see diagram below). These molecules mature into active MicroRNA molecules in a process that includes their digestion by two different enzymes, the first digestion taking place already inside the cell nucleus and the second in the cytoplasm. The end result of this process is a double strand RNA molecule about 20 nucleotides long, where one of these strands is recruited into the RISC complex (the last step is also true for siRNA molecules).


The hairpin structure characteristic of immature microRNA molecules

In humans, as in all mammals, microRNAs are more common than siRNA. The microRNA molecule is not fully complementary to the mRNA that it regulates, and this is the reason that the target RNA is not digested at the center of the complementary region. The recognition element lies between nucleotides 2-8 of the microRNA molecule (longer recognition elements are sometimes observed), which are complementary to a segment on the target mRNA. As mentioned, this recognition leads to silencing of the mRNA either by translation repression or by enhanced degradation (but not through direct digestion). These processes have not been fully deciphered to date. Contrary to this, regulation by siRNA and by microRNAs in plants is accomplished by digestion of the target mRNA at the center of the recognition element (between nucleotides 10-11 of the microRNA or siRNA molecules).


Biosynthesis and maturation of a microRNA molecule (adopted from Wikipedia)

So far we have discussed the mechanisms by which short RNA molecules are produced and how they silence gene expression. But what is their physiological role and how important are they for our development?

More than 400 different microRNA molecules have been identified in humans to date. Computational studies show that these molecules regulate about one-third (!) of the gene repertoire.This implies that each microRNA molecule can regulate several genes by virtue of the short recognition element it employs in order to bind target mRNA molecules (as few as 6 nucleotides). It should be mentioned that while siRNA molecules can virtually shut down a gene's expression by digesting its mRNA transcript, regulation by microRNA in mammals is much more moderate and usually entails inhibition of up to 60%. Nonetheless, since each microRNA molecule can regulate several genes (dozens and more) its effect is actually very wide, and on top of that each mRNA can be regulated by more than one microRNA (either similar or different).

Regulation by microRNA in mammals occurs in most tissues and cell types. The significance of this system is manifested by the fact that a mouse embryo lacking microRNA regulation cannot develop and dies before birth. In addition to embryonic development, the role of microRNAs has been investigated extensively in several processes, including cell cycle and cell division, metabolism, programmed cell death (apoptosis) and many more. MicroRNAs also play a role in cancer development, as evident from the observation that different tumors are characterized by abnormal microRNA levels. In this context, tumor-promoting genes can be over-expressed due to low levels of a specific microRNA, or vice-versa, tumor-suppressing genes can be under-expressed due to abnormally high levels of a specific microRNA. In fact, nowadays it is possible to identify many types of cancers according to their abnormal microRNA profiles.

Ongoing studies are focused on attempts to employ synthetic siRNA molecules as potential therapy in a number of diseases, including various cancers. These will be discussed in another article.

Haggai Kaspi
Department of Biological Chemistry
Weizmann Institute of Science
 
A note to the reader
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2 comments

  • Sunil G babu

    Biotechnology

    The lecture is lucid and very good.
    can you suggest any other promising roles of miRNA other than diagnostic, markers in tumors?

  • חגי כספי

    answer

    Hi,

    Physiologically, microRNAs have important roles in regulating numerous processes in the human body during development and homeostasis.

    In medicine (in addition to diagnostics) a number of companies are developing siRNA based drugs. These drugs are designed to use the microRNA/RISC machinery in order to target and repress a specific mRNA. Till now, more than 20 siRNA based drugs have been developed and are subjected to clinical trials. These drugs are designed to treat more than a dozen different diseases including a number of cancers, macular edema, glaucoma and even asthma.

    Hope I've been helpful,
    Haggai