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within the 3' untranslated region (3' UTR) of an mRNA. Several types of regulatory proteins have been identified that bind these control elements, yet little is known about how the binding of these factors leads to functional changes in the behavior of the mRNAs. We are studying how members of the Puf family of proteins are able to bind and differentially regulate the decay rate of several target mRNAs. Puf proteins appear to have a conserved role in stem cell maintenance, cell development, and differentiation, and are found across the eukaryotic kingdom. However, information about how any of these Puf proteins influence the mRNA decay machinery is only beginning to be uncovered.

The yeast Saccharomyces cerevisiae is a model organism for understanding how Puf proteins modulate mRNA lifespans. S. cerevisiae contains six Puf proteins, providing a unique system for studying differential Puf protein target specificity and regulatory function. In addition, the general pathways of mRNA decay in yeast are well described, and multiple lines of evidence suggest that these pathways are conserved among eukaryotes. Our analysis of the mechanisms by which the yeast Puf proteins recognize and regulate the stability of their target mRNAs will greatly contribute to our understanding of Puf-mediated control of gene expression in yeast and other eukaryotes, and to the general principles of 3' UTR control of mRNA decay. A fundamental issue in understanding mRNA-specific regulation by Puf proteins is the determination of the 3' UTR elements involved . We had earlier identified Puf3p as the first transcript-specific regulator of mRNA deadenylation and decay in yeast (Olivas and Parker, EMBO J. , 2000). Therefore, we characterized the role of specific nucleotides within the 3' UTR of the COX17 mRNA that are responsible and sufficient for binding the Puf3 protein and promoting mRNA decay (Jackson et al., RNA , 2004). Our analysis identified two distinct Puf3p binding sites containing a conserved sequence element. The two sites show differential binding affinity to Puf3p in vitro, with sequences flanking the conserved binding element influencing affinity. However, each site is equally sufficient for partial stimulation of COX17 mRNA decay in vivo, while full decay regulation requires both sites. This indicates that multiple Puf3p signals can combine to increase decay activity. No other sequences outside the 3' UTR are required for Puf3p-mediated decay. In addition, we found the repeat domain of Puf3p to be sufficient for both RNA binding and mRNA decay stimulation, supporting a conserved role of the Puf repeat domain as an independent regulator of mRNA metabolism.

Another important issue in understanding mRNA-specific regulation by Puf proteins is determining how different Puf proteins that are well conserved in their repeat domains target different mRNAs for decay. Thus, we identified the Puf3p amino acid positions that are involved in target-specific binding to the COX17 mRNA, as well as protein regions responsible for signaling to the mRNA decay machinery (Houshmandi and Olivas, RNA , 2005). Crystal structure analysis of a human Puf bound to RNA had suggested a modular mode of binding, with three amino acid positions within each of the eight repeats of the repeat domain contacting a single nucleotide in the target RNA (Wang et al., Cell, 2002). However, by mutating predicted RNA-binding positions of Puf3p to those found in Puf5p, we found that a simple set of rules cannot reliably link specific amino acid positions with target specificity. Moreover, we found that particular amino acid positions played different roles within Puf3p and Puf5p for binding to their respective target mRNAs. Finally, we identified an outer surface loop that is dispensable for RNA binding, but required to promote mRNA decay.

The identification of additional genes required for Puf3p-mediated stimulation of mRNA decay gets at the heart of the mechanism by which Puf proteins influence the mRNA decay machinery. To this end, we identified proteins that interact with Puf3p to mediate rapid mRNA decay. Specifically, we identified several mRNA decay factors that interact with Puf3p, including proteins involved in both deadenylation and mRNA decapping. All of these interacting proteins were found to be essential for Puf3p-mediated COX17 decay, and all appear to bind Puf3p independent of RNA. We are currently analyzing the mechanistic role of these protein interactions.

Another project in the lab has focused on the regulation of Puf protein activity. In collaboration with Dr. Harmen Bussemaker's computational biology lab at Columbia University , we made the first experimental report of condition-specific regulation of Puf3p activity (Foat et al., PNAS , 2005). Specifically, we found that Puf3p activity is inhibited by growth in ethanol and rapamycin, though to different extents. We are currently studying the mechanism by which Puf3p activity is altered under different carbon source and other environmental conditions. We have also identified several new targets of Puf3 protein regulation. Using this set of Puf3p target mRNAs, we have established several different growth conditions that alter Puf3p activity on these targets. Moreover, modulation of Puf3p activity occurs very rapidly upon changes in growth conditions, providing support for a post-translational control event.

Other studies have identified mRNA targets of the Puf4 protein, and experiments are underway to analyze the condition-specific regulation of Puf4p on these targets. Our results show that Puf4p activity is strongly inhibited in several carbon source and stress conditions that only mildly affect Puf3p activity. Current work aims to determine whether Puf4p and Puf3p activities are altered by similar mechanisms. Unlike Puf3p, which stimulates decay, our experiments indicate that Puf4p may act to stabilize its target mRNAs. These results open a whole new avenue of research to determine the mechanism by which Puf4p inhibits the mRNA decay machinery.

A complementary direction in the lab has concentrated on analyzing the regulation of mRNA targets by the yeast Puf proteins, Puf1p, Puf2p, and Puf5p. Several mRNAs whose levels and decay rates are affected by these Pufs have been identified. In a novel finding, two mRNA targets were found that are regulated in a combinatorial fashion by both Puf1p and Puf5p. Decay regulation involves multiple binding elements in the 3' UTRs of the mRNAs, and regulatory activity is condition-specific. Future work will involve the analysis of the mechanism of mRNA regulation by these Puf proteins.

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Last Update 1 December 2006.

Owner: Wendy M. Olivas