A Tale of a Microprocessor, RISC and a Few Loops of miRNA

The word ‘microprocessor’ is generally used to designate VLSI and SLSI (Very/Super Large Scale Integrated circuits) devices which accept, decode and execute instructions presented in binary coded forms. They may be called the heart of the computer. RISC (Reduced Instruction Set Computer), on the other hand, is a type of microprocessor architecture that uses a simplified, yet highly-optimized set of instructions to deliver good performance. However, like ‘cell’ and ‘nucleus’, they too have been adopted in biology, and not without reason!

Proteins are essential for cells as they perform various functions as enzymes, ion channels, receptors and so on. They are manufactured in the ribosomes, organelles present in the cytoplasm, under the instruction of messenger RNA (mRNA). This instruction code is encoded in the sequence of nucleotides that make the mRNA molecule. However, the sequence of nucleotides in mRNA is dictated in turn by the DNA that is present in the nucleus. Messenger RNA carries this message from the nucleus into the protein production units. But what would happen if we interfered with the ‘message’?

RNA interference (RNAi) would occur affecting the regulation of gene expression. Micro RNAs (miRNA) are one of the small RNAs that regulate the expression of protein-encoding-genes, after the mRNA strand has formed. miRNAs have partly or fully complementary sequence to one or more mRNAs. This enables them to latch on to the mRNA molecule masking the ‘instruction codes’ in the mRNA strand, interfering with protein formation (translation). In other words, the gene has been silenced!

miRNAs are first transcribed from DNA by the enzyme RNA polymerase II into primary miRNA (pri-miRNA). pri miRNA is then cleaved by another enzyme, RNAse III, called Drosha, into precursor miRNA (pre miRNA) (see the picture on the left). However, Drosha (an RNAase III endonuclease) is assisted by Pasha (partner of Drosha), another enzyme, in this task. Later, it was found out that these two resided in a 500 kilo Dalton complex, called the microprocessor (micro RNA processor). So far, all these have been happening in the nucleus of the cell. The pre miRNA then moves into the cytoplasm through the exportin 5 pathway. Next, Dicer, another RNase III endonuclease, makes a mature miRNA duplex, which is then ‘uploaded’ into a complex called RISC (RNA induced silencing complex). RISC then prevents translation of the mRNA strand, as the ‘partially’ complementary miRNA strand interferes with the translation of the mRNA molecule into specified amino acid sequences can not occur. We can compare complementarity of nucleotide bases in terms of a pair of gloves and its corresponding fingers. The information of the gloves' coordinates gets obliterated by the occupying fingers. This RISC dependent mechanism occurs in parts of the cytoplasm, called P bodies (‘p’ for processing).

RNAi is very important for plants as they lack an immune system. Invading organisms can not dictate foreign protein formations as their RNAs are destroyed, not merely inhibited, as is usually seen in higher animals (animal miRNAs exhibit only imperfect homology to the mRNA in contrast to plants, and thus they only inhibit translation). Some of the tumor suppressor genes inhibit tumor formation by the action of miRNAs and not through protein formation. In humans, exploiting RNAi may be a useful tool in combating diseases such as cancer, AIDS etc. So it remains to be seen whether the microprocessor can bring a revolution in medicine and research as its counterpart in electronics did in the field of computing.

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Reference: Saumet, A., & Lecellier, C. (2006). Anti-viral RNA silencing: do we look like plants ? Retrovirology, 3 (1) DOI: 10.1186/1742-4690-3-3
Processing of primary microRNAs by the Microprocessor complex. doi:10.1038/nature03049
Wikipedia
The Macro World of MicroRNA (pdf)

Metallica Goes The Stem Cell Way

I had previously written a little about stem cells. While researchers still don’t yet know exactly how the four factors transform the fully differentiated fibroblast cells back into pluripotency, possible explanations are pouring in.

Pluripotency (by which the stem cell may become any tissue; muscle or nerve, for example) and “self renewal” (cells should not only differentiate, some ready stock of stem cells must be there for future need) are important determinants for stem cells.

According to Shinya Yamanaka, the steps could be somewhat like this: c-Myc first confers the open chromatin state and immortality to the skin fibroblasts. But it also induces apoptosis by acting on the p53, “the guardian of the genome”. Apoptosis or cellular senescence causes the cells to die. Klf4 inhibits p53 induced apoptosis. Again, if we added only Klf4 and c-Myc we would get tumor cells (both being oncogenes). Oct4 here acts and makes ES like cells (ES= Embryonic Stem) out of what was destined to be tumor cells. Sox2 confers pluripotency and you’ve got what you wanted.

Now, we just have to hand pick the right cells from the petridish. Scientists can do it either by looking for Fbx15 expression or the expression of nanog in the treated sample. Both Fbx15 and Nanog are targets of Oct3/4 and Sox2; but Nanog is found to be more closely associated with pluripotency, as is evidenced by adult chimera formation (chimera is a monstrous fire breathing creature like dragon of ancient mythology).

There have been some important modifications. Researchers have shown that one could still get human induced pluripotent stem cells (iPSC) without the need of the c-myc oncogene. The mode of delivery of these four factors could also be undertaken by plasmids, rather than the traditional retroviral vector approach. Retroviruses (like c-Myc) could potentially induce cancer. You may like to hear this Nature Podcast where both Yamanaka and Rudolph Jaenisch give a very good summary. As a bonus, you may also appreciate another way of creating iPSC. Replace the genome in “early embryonic cells” or zygotes (fertilized eggs) during cell division. During cell division, the nuclear membrane disappears and the factors are no longer in the nucleus. They are in the cytoplasm. Dieter Egli explains that if you replaced the genome of this zygote with another (genome) while the cell was still dividing, the new genome would adapt to the new cytosolic environment and get instructions from the factors in the cytosol. It will go ‘back in time’ and become a stem cell.

Now, a bit of refreshment. Watch this awe inspiring Metallica video called 'All nightmare long'. It portrays the Tunguska event, A-bomb, Soviet Revolution, American supremacy (?) and ‘revival of organisms’. Some key phrases are:

• “like a split worm, a part of the organism can reconstitute the whole”. Check about Planarians (flat worms, picture on the left), they not only reconstitute but also become separate individuals!
  
• “Instead of offspring, they become skin cells, nerves and muscle”- just as we described! Seems Metallica is well informed! Do see this wonderful video in YouTube.

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Reference: hyper-links and

Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells Nature, 448 (7151), 313-317 DOI: 10.1038/nature05934

Developmental reprogramming after chromosome transfer into mitotic mouse zygotes, doi:10.1038/nature05879