Understanding the mechanisms by which upstream open reading frames (uORFs) in mRNA transcripts control gene expression is currently the major focus of my laboratory. A substantial component of this work is focused on the uORF-encoded fungal arginine attenuator peptide (AAP). The major goal of this work is to understand the mechanism by which a nascent peptide encoded by this uORF controls the movement of ribosomes on mRNA and regulates gene expression. Control mechanisms mediated by uORFs and nascent peptides exist in mammals, fungi, plants, viruses, and bacteria, but relatively little is known of the molecular details of such control. The AAP is encoded by a uORF in the 5′-leader regions of mRNAs specifying the first enzyme in fungal arginine (Arg) biosynthesis. Synthesis of the AAP rapidly reduces gene expression in response to Arg. AAP-mediated regulation is observed in vivo in both Neurospora crassaand Saccharomyces cerevisiae and in vitro, using fungal, plant and animal extracts. The nascent AAP causes the ribosome to stall when the concentration of Arg is high.

We are using biochemical and genetic approaches to examine how the AAP stalls ribosomes. Our working model for regulation is that the nascent AAP adopts a conformation in the ribosomal tunnel that, with Arg (or a closely related molecule), interferes with decoding at the ribosomal A site, or with another step crucial for elongation or termination, thus stalling the ribosome.

A second crucial aspect of regulation via the AAP is that it also controls mRNA stability in vivo. Both S. cerevisiae CPA1 and N. crassaarg‑2 mRNA levels are affected by nonsense-mediated mRNA decay (NMD). AAP-mediated stalling promotes NMD of the CPA1 mRNA. In the absence of AAP-mediated stalling, NMD of the CPA1 mRNA can also be promoted by increased ribosome occupancy of the uORF. These data support a regulatory model in which ribosome stalling at the uORF termination codon in response to Arg destabilizes CPA1mRNA by increasing the extent of nonsense codon recognition by NMD. This link between AAP-mediated stalling and NMD provides unique opportunities for assessing the cis- and trans-acting elements that contribute to NMD.

Related work explores the functions of other uORFs whose existence was deduced by in silico methods. In collaboration with investigators at the Broad Institute and others, we examined the functions of two such uORFs that were observed to be evolutionarily conserved in three Aspergillus species.  We are now evaluating the functions of Cryptococcus neoformans uORFs displaying varying levels of evolutionary conservation. Experimental study and validation of such conserved elements has lagged far behind their identification.

We have longstanding collaborations to perform large scale analyses of the N. crassa genome. Our studies are now centered on systematically understanding the functions of N. crassa genes and the mechanisms that regulate them.  Currently, our lab is primarily responsible for scientific (not automated) curation of N. crassa genome data housed at the Broad Institute, and therefore evaluates the quality and accuracy of the data for gene models and proposed functions.  We are also developing additional resources that will be useful to curators and the community for data mining, including the implementation of full-text literature searching resources for filamentous fungi using the Textpresso application developed at Caltech.

A second aspect of our functional genomics work is that we are synthesizing N. crassa cDNA libraries to obtain full length cDNAs that will be used for sequencing to improve genome annotation.  The capability to obtain and characterize such cDNA clones is important for all functional genomics efforts, particularly in intron-rich organisms, such as N. crassa, since sequencing of full-length cDNAs provides data critical for understanding mRNA structure not readily obtainable by any other methods.

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