BIOCHEMISTRY - DR. JAKUBOWSKI
Last Updated: 03/30/16
Learning Goals/Objectives for Chapter 5D: After class and this reading, students will be able to
What accounts for the increased complexity of organisms like humans? As was discussed in the DNA chapter, it is not the number of chromosomes or even the number of possible genes in an organism. One big difference between bacterial and human cells, for example, is the percentage of DNA coding for proteins. In bacteria, most of the DNA codes for proteins, but in human eukaryotic cells, most of the DNA (up to 98%) is "junk" in that it does not code for proteins. The DNA consists of intervening sequences within DNA coding for a given protein, and sequences between genes. Up to 98 % of the RNA transcribed in human cells is derived from this "junk" DNA. What function does this RNA serve? New evidence shows that this transcribed RNA binds to other RNA molecules like mRNA (to inhibit its translation), to DNA (to control gene transcription) or to proteins (to alter gene transcription as well). These process are called RNA interference (RNAi).
An understanding of RNAi really began in 1998 with the study of RNAi in round worms (described below) by Fire and Mellon, who were awarded the Nobel Prize in Physiology and Medicine in 2006. The Nobel Foundation stated that "the discovery of RNAi has already had an immense impact on biomedical research and will most likely lead to novel medical applications in the future".
The terminology used to describe the RNA species involved in RNAi is often confusing (especially to a protein chemist). In part it depends on the sources of the RNA. The RNA can derive from the cells own endogenous RNA (transcribed from genes of the cell) or by exogenous RNA entering the cell from the outside. This can happen by infection by an RNA virus, a DNA virus (which forms RNA in the cell after transcription), by plasmids containing DNA that will be transcribed to RNA in the cell, or by synthetic RNA. These RNAs generally inhibit translation of mRNA for a given protein and are described below in more detail.
RNAi from enodgenous RNA
RNAi from exogenous RNA (or DNA)
RNAi pathways probably evolved from or with pathways for cellular resistance to viruses. Viruses often produced dsRNA during their life cycle. Some viruses like the HIV virus have a ssRNA genome. One method of host defense against viral infection is fomation of short interfering RNAs that could inhibit transcription of viral proteins from viral mRNA.
Jmol: dsRNA (still figuring out to convert to JSMol)
One of the first amazing demonstrations of the power of RNAi to module gene expression at the translational level was done in the nematode worm, C. elegans. This organism has about 20,000 genes which code for proteins. Kamathk et. al. fed these worms E. Coli transformed with plasmid DNA designed to produced dsRNA upon transcription, one strand of which was complementary to mRNA sequences in the worm. Plasmids containing almost 17,000 different dsRNA encoding genes were constructed and used to knockout gene expression by forming dsRNA complexes of the mRNA with the RNAi. Phenotypic changes in the organism were studied. About 1700 of the dsRNA experiments led to observable (phenotypical) changes in the organism. Genes whose inactivation was lethal (and hence were essential for survival) were generally those that had counterparts in all other organism, while those associated with nonlethal changes were more likely to be homologous to genes in higher organisms and more recently evolved. They also selectively looked at which genes influenced lipid metabolism by incorporating a fluorescent tag which bound to lipid deposits in the organism. Around 300 genes were found to influence fluorescence and hence regulate fat deposition in the organism.
RNAi is the basis of an new emerging industry. Many companies offer kits and free software that make RNAi studies simple. Invitrogen is one such company.
Two groups have deleted miRNA-155 and looked at effects on immune cells in mice. Immune cell function in B, T, and dendritic cells was affected, leading to animal death when exposed to salmonella after they were immunized. Animals in sterile environments showed no effect. In contrast to knockouts of protein-coding genes, these knockouts affect transcription of multiple genes. Knockout of miR-208 caused heart problems in mice placed in a stressful environment. These experiments indicated that some genetic diseases might arise from mutations in non-protein coding regions of the genome.
Recently, a new mechanism in control of gene expression has been offered which involves regulation of translation of a mRNA. mRNA must have a sequence, the Shine-Delgarno sequence, which allows it to bind to ribosomes. If a ligand binds to this site, mRNA could not bind to the ribosome and translation would be inhibited. Such is the case in the mRNA encoding proteins involved in the transport and synthesis of vitamins B1 (thiamine) and B12 (adenosyl cobalamin). Thiamin and thiamine pyrophosphate were shown to bind to the leader sequence of an E. Coli mRNA involved in thiamine biosynthesis and inhibit the translation of the mRNA. This allosteric mechanism for inhibition makes physiological sense since the presence of high levels of cellular B1 would obviate the need for its synthesis or transport.
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