August 31, 2007
Researchers Discover Emergency Protein Synthesis Pathway
Researchers have identified an “emergency” mechanism that yeast cells use to switch on protein synthesis in times of stress. The studies by Howard Hughes Medical Institute researchers
use the fact that when yeast cells are starved of nutrients, they are transformed into invasive foragers. Although numerous proteins are required for this developmental switch, overall protein synthesis - which is responsible for maintaining basic cellular processes - shuts down. The backup system appears to play a crucial role in carrying yeast through lean times.
The scientists speculate that cells in higher animals, including humans, may well have a similar backup plan for regulating protein synthesis when cells are stressed.
“An important take-home message from this work is that IRESs actually have a physiological function -- in this case helping yeast respond to starvation by entering a foraging pathway.”
Jennifer A. Doudna
The researchers, led by Howard Hughes Medical Institute investigator Jennifer A. Doudna, published their findings in the August 31, 2007, issue of the journal Science. Wendy Gilbert in the Doudna Laboratory at the University of California, Berkeley, was the lead author on the paper. Other co-authors were research specialist Kaihong Zhou, and Tamira Butler, who performed the research as part of HHMI's Exceptional Research Opportunities Program for undergraduates. EXROP provides talented undergraduates from disadvantaged backgrounds with summer research experiences in the labs of HHMI investigators and HHMI professors.
In their experiments, Gilbert, Doudna and their colleagues sought to understand the biological function of stretches of messenger RNA called internal ribosome entry sites (IRESs). These sites are in the untranslated regions of messenger RNA (mRNA) molecules that help in hijacking the ribosome to initiate protein synthesis. IRESes effectively
commandeer the protein synthesis machinery, diverting it from making `house-keeping' cellular proteins. Protein synthesis is normally initiated when the ribosome recognizes a characteristic nucleotide cap on the end of the cell's mRNA. But IRESs provide an alternative mechanism of initiation.
Scientists first discovered IRESs in viruses. They found that viruses use IRESs as part of their invasion strategy as a way to circumvent normal controls on infected cells' protein synthesis pathways. While researchers have sought to understand IRES function in the cells of higher organisms, such studies have been largely thwarted by the difficulty of genetically manipulating such cells to study IRESs, said Doudna.
In response to nutrient deprivation, yeast can enter an invasive growth state that is characterized by an elongated shape and ability to invade a solid food source. Gilbert recognized that this invasive growth state in yeast would make a tractable experimental model for the study of IRESs. Scientists can easily manipulate yeast cells and readily observe the production of the invasive growth proteins in starving cells. “Invasive growth is quite easy to measure experimentally, because you can actually see in the petri dish whether the cells are adhering or not,” said Doudna.
To test for possible IRES activity in the mRNAs encoding invasive-growth proteins, Gilbert altered the invasive growth genes to block the normal mechanism by which the protein synthesis machinery can “read” the mRNA. In the normal reading process, the protein machinery attaches to one end of the mRNA and reads it like a ticker tape to translate its genetic code into protein.
With this normal translation mechanism thwarted, the mRNA could only be read if the protein-making machinery localized to an IRES. Indeed, the experiments revealed that several invasive-growth genes switched to using IRESs to produce the invasive growth proteins in starved cells, said Doudna.
“So, Wendy proved that IRESs were used in synthesizing these proteins,” said Doudna. “But the other big problem in this field is that—despite a large number of papers showing IRES activity in various cells—there was no proposed molecular mechanism for how they might work,” she said.
Gilbert performed experiments that revealed that the same protein, called a translation initiation factor, is used in initiating normal protein synthesis and that initiated by IRESs. Gilbert also found that the protein synthesis machinery detects IRESs in the yeast mRNA by recognizing long strings of the nucleotide adenine that are a hallmark signature of many yeast IRESs. In contrast, said Doudna, viral IRESs are recognized in cells by their three-dimensional structure.
To test directly whether IRES sequences were physiologically important, Gilbert engineered yeast cells so that the gene for a protein critical to invasive growth produced mRNA lacking its IRES sequence. She found that these mutant yeast cells with the IRES-deleted mRNA were incapable of invasive growth adaptation. “This was a very important discovery because it showed for the first time that IRESs play a critical physiological function,” said Doudna.
“An important take-home message from this work is that IRESs actually have a physiological function—in this case helping yeast respond to starvation by entering a foraging pathway,” concluded Doudna. “One could certainly imagine that this kind of activity might be going on in other cell types, most intriguingly in human cells,” she added. “Although this is only speculation, IRESs might allow such cells to respond to stress by expressing particular proteins even when general protein synthesis is being downregulated.”