RNA helicase regulation of RNA metabolism and gene expression

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Owttrim SUMMARY All organisms must be able to sense and respond to changes in their environment. Two of the most commonly experienced stresses are changes in light and temperature, factors that are crucial for the survival of photosynthetic organisms. In response to these stresses, cells must alter protein abundance in order to survive. Proteins are produced from RNA in a process that requires the RNA to be folded into the correct 3-D structure. RNA binding proteins, termed RNA helicases, facilitate RNA folding in all organisms. The purpose of our research is to determine how changes in the growth environment regulate RNA helicase accumulation and how helicases interact with RNA. To accomplish this, we are using the photosynthetic model system, cyanobacteria, a potentially important source of biofuels and medically active compounds. Cyanobacteria contain a single RNA helicase whose abundance is elevated at 20oC and low at 30oC. We have created mutant cells that lack a functional RNA helicase. These cells do not grow at 20oC and have aberrant expression of proteins that are normally only expressed at 30oC. These results indicate that the helicase is a cold stress gene that unexpectedly functions in cellular response to heat stress. We can rescue the cold stressed cells by returning a functional RNA helicase to the mutant cells. This allows us to test which areas of the RNA helicase are required for function by removing or altering crucial amino acid sequences. Another aspect of the project involves analysis of the mechanism that keeps RNA helicase abundance low at 30oC. Our research indicates the helicase is specifically degraded at 30oC. We are using our mutant helicase proteins to determine which amino acid sequences are required for this degradation and also identify the proteins causing the degradation. Finally, we wish to identify the RNAs and proteins with which the helicase interacts. To do this, we have added a specific amino acid sequence to the helicase, a protein-tag that will allow us to specifically purify the tagged helicase from cyanobacterial cells. We can then identify interacting partners by sequencing the RNAs and proteins that co-purify with the tagged helicase. Together, the data will allow us to formulate an intracellular molecular picture of how the helicase functions and also regulates cyanobacterial response to temperature fluctuation. Understanding these processes may allow us to design temperature-regulated expression platforms, generating cyanobacteria in which protein expression is specifically regulated, diverting material to production of the desired commercial product and not cell growth. Overall, the project will help us to understand how the RNA helicase protein is expressed and functions within cells, allowing insights into how we can manipulate RNA folding to generate more efficient production of beneficial products in cyanobacteria.