Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity

Area of Science:

• Molecular Biology
• Gene Regulation
• Mycology

Background:

• Amino acid limitation triggers transcriptional responses in fungi via transcription activators like *Neurospora crassa* CPC1 and *Saccharomyces cerevisiae* GCN4.
• Gene expression is intricately controlled at the translational level, particularly in response to environmental cues such as nutrient availability.

Purpose of the Study:

• To investigate the translational control mechanisms of *Neurospora crassa cpc-1* mRNA.
• To compare the functional roles of upstream open reading frames (uORFs) in *N. crassa cpc-1* with those in *S. cerevisiae GCN4*.
• To explore the significance of non-canonical initiation events in the expression of CPC1.

Main Methods:

• *In vitro* translation assays using *N. crassa* cell extracts.
• Bioinformatic analysis of *cpc-1* homologs across fungal species.
• Analysis of mRNA 5' leader sequences and upstream open reading frames (uORFs).

Main Results:

• *N. crassa cpc-1* uORF1 and uORF2 are functionally analogous to *S. cerevisiae GCN4* uORF1 and uORF4 in translational control.
• The 5' region of *cpc-1* mRNA extends over 700 nucleotides with conserved upstream extensions in homologs.
• Multiple conserved non-AUG near-cognate codons (NCCs) within the extended CPC1 reading frame initiated translation *in vitro* and *in vivo*, producing N-terminally extended CPC1 isoforms.

Conclusions:

• Translational regulation of *cpc-1* by uORFs and NCCs is crucial for controlling CPC1 synthesis in response to amino acid limitation.
• The utilization of NCCs for translation initiation represents a significant, conserved mechanism in fungal gene expression.
• This study highlights the emerging importance of noncanonical initiation events in regulating gene expression, particularly for essential transcription factors.