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. 2017 Jun 27;8(3):e00844-17.
doi: 10.1128/mBio.00844-17.

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

Ivaylo P Ivanov  1 , Jiajie Wei  2 , Stephen Z Caster  2 , Kristina M Smith  3 , Audrey M Michel  1 , Ying Zhang  2 , Andrew E Firth  4 , Michael Freitag  3 , Jay C Dunlap  5 , Deborah Bell-Pedersen  2 , John F Atkins  1   6 , Matthew S Sachs  7
Affiliations

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

Ivaylo P Ivanov et al. mBio. .

Abstract

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitroIn vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

Keywords: Neurospora; filamentous fungi; gene regulation; molecular genetics; translational control.

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Figures

FIG 1
FIG 1
N-terminal extensions of Pezizomycotina CPC1. (A) cpc-1–luc mRNA produces a larger product in vitro than luc mRNA. mRNA templates were used to program N. crassa in vitro translation extracts, and [35S]Met-labeled products were analyzed on a 12% NuPAGE gel. Translation of mRNA for N. crassa cpc-1–luc (in which the 5′ leader of cpc-1 plus the first three codons of its mORF are fused in frame to firefly luciferase) and that for luc were compared to a no-mRNA control. The positions of full-length firefly LUC (arrowhead) and the larger CPC1-LUC product (asterisk) are indicated. (B) Schematic diagram of the N. crassa cpc-1 mRNA. Each reading frame is on a separate line. Frame 1 specifies CPC1 (black rectangle). uORF1and uORF2 (blue rectangles) initiate from uAUG1 and uAUG2, respectively, in other reading frames. AUG codons are indicated by green bars, and stop codons are indicated by red bars. NCCs in frame 1 upstream of uORF2 are indicated by magenta bars and are numbered 1 to 8. The approximate position of the 3′-most stop codon upstream of uORF2 and in frame with the main ORF (present in Cordyceps bassiana) is indicated (dashed red bar). The number of sequences used for comparisons is shown in parentheses. Features are drawn to scale. The nucleotide sequence of the N. crassa cpc-1 5′ leader is given in Fig. S6 in the supplemental material. (C to J) Frequency WebLogo of the conservation of the initiation contexts, from −6 to +4, of all N. crassa genes initiated with AUG (C), all A. nidulans genes initiated with AUG (D), Pezizomycotina cpc-1 uAUG1 (E), Pezizomycotina cpc-1 uAUG2 (F), Pezizomycotina cpc-1 mAUG (G), Pezizomycotina NCC6 (CUG) (H), Pezizomycotina NCC7 (AUU) (I), and Pezizomycotina NCC8 (ACG) (J). Letter heights are proportional to the frequency of occurrence of each nucleotide at each position. The crucial positions −3 and +4 are indicated in red underneath the frequency plots. Data used to calculate consensus AUG initiation context for N. crassa and A. nidulans were obtained from the Transterm database (61). (K) The amino acid sequence encoded by the N. crassa mRNA in the CPC1 reading frame, starting from the 5′ end of the mRNA and ending with the first in-frame stop codon. mAUG indicates the annotated cpc-1 initiation codon; upstream NCC6 (uNCC6), uNCC7, and uNCC8 are also indicated. The approximate positions of uORF1 and uORF2 (which are in other reading frames) are indicated by blue lines. The C-terminal bZIP domain of CPC1 is indicated in red font. The regions analyzed in Fig. S3A and B are bracketed by dashed red lines. The highly conserved patch specifically marked in Fig. S3A is bracketed by a dashed orange line. The level of conservation of each residue from the alignment of homologs from 95 species (those used to construct the tree in Fig. S1A) is shown below the amino acid sequence and was generated using ClustalX2. The conservation, expressed as percent amino acid identity, is indicated on the right side of each alignment.
FIG 2
FIG 2
Contribution of cpc-1 uORF1 and uORF2 to the regulation of translation from the mAUG in N. crassa cell extracts. (A) Effects of eliminating cpc-1 uORF1 and uORF2 on translation from the mAUG. Constructs (numbered 1 to 4) contained the UAA stop codon (red bar) to eliminate translation from upstream in-frame NCCs and the indicated mutations to uORF start codons to eliminate initiation from them (uORF1 AUG to AAA and/or uORF2 AUG to ACA). Capped and polyadenylated mRNA (6 ng) was used to program N. crassa translation reaction mixtures (10 µl). LUC activity produced from mRNAs 2 to 4 obtained after 30 min of incubation at 26°C was calculated relative to the activity produced from mRNA 1. Mean values and standard deviations from three independent experiments, each performed in triplicate, are given as normalized relative light units (RLU). In addition, [35S]Met-labeled translation products from translation reactions programmed with mRNAs 1 to 4 or with no mRNA were analyzed on 12% NuPAGE gels, and a representative gel is shown. The position of radiolabeled LUC produced from the mAUG is indicated. (B) Toeprint analysis indicates reinitiation following translation of cpc-1 uORF1 but not uORF2. cpc-1–luc mRNA (60 ng) was used to program 20-µl N. crassa cell-free translation reaction mixtures. WT mRNA containing the wild-type cpc-1 5′ leader and the mRNAs used in panel A were analyzed in parallel along with controls. Reaction mixtures were incubated at 26°C min with cycloheximide (CYH) added either prior to incubation (T0) or after 10 min of incubation (T10) as indicated. Radiolabeled primer CPC101 was used to examine ribosomes at uORF1 and uORF2; primer ZW4 was used to examine ribosomes at the mORF. The original data from which the toeprint signals were excised are shown in Fig. S7. (C) Discriminating translation from N. crassa cpc-1 NCCs and mAUG in vitro. Capped and polyadenylated mRNA (6 ng) was used to program N. crassa translation reaction mixtures (10 µl) with the indicated constructs. Firefly luciferase activity from each mRNA obtained after 30 min of incubation at 26°C was calculated relative to synthesis from the WT construct. Mean values and standard deviations from three independent experiments, each performed in triplicate, are plotted.
FIG 3
FIG 3
Evidence that N. crassa NCC5 to NCC8 initiate translation in vitro. Synthetic RNAs (60 ng) for the indicated constructs were used to program 10 µl of cell-free translation systems from N. crassa (left) or wheat germ (right). Reaction mixtures were incubated for 30 min at 26°C. Radiolabeled products were analyzed on 12% NuPAGE gels. Open circles, translation products eliminated upon mutation of NCC5 to NCC8; the product predicted to be initiated from NCC8 also increased when NCC8 was changed to AUG (lane 8). Arrowhead, position of mAUG-initiated translation product (mORF). Brackets, translation products larger than the mORF produced in the absence of an in-frame UAA stop codon.
FIG 4
FIG 4
Discriminating translation from N. crassa cpc-1 NCCs and mAUG in vivo. Constructs 1 to 4 were placed at the N. crassa his-3 locus (three independent transformants of each). LUC activities were measured, and values were plotted relative to WT. Black bars, LUC activities normalized to total extracted protein; gray bars, LUC activities normalized first to total protein and then to luc mRNA/cox-5 mRNA levels. Mean values and standard deviations for all measurements are derived from three independent experiments, each using all independent transformants.
FIG 5
FIG 5
Ribosome profiling evidence for translation of the cpc-1 N-terminal extension. The ribosome-protected fragment count (cutoff at 50) for each position 5′ to 3′ along the cpc-1 mRNA is shown on top. Peaks that can be assigned to specific initiation codons, as labeled in Fig. 1B and K, are indicated in red. A schematic map of the ORF organization of cpc-1, drawn to scale, is shown below the ribosome-protected fragment count. The uORFs are represented by blue rectangles. The eight in-frame NCCs upstream of uORF2 are represented by magenta bars. The mORF is represented by a black rectangle. The reading frame of each feature relative to the mORF is indicated. The reading frame information derived from the ribosome-protected fragments for 5 specific regions along the cpc-1 mRNA, as well as the tabulated data for all N. crassa coding sequences (CDSs), is shown at the bottom. The tabulation is done by scoring the first nucleotide of each ribosome-protected fragment relative to the reading frame of the annotated coding sequence (first to last ribosome A-site codons), using a 15-nucleotide 5′ offset—the proportions of total fragments mapped to frame 1 are shown by green bars, those mapped to frame 2 are shown by red bars, and those mapped to frame 3 are shown by yellow bars. The number of reads used to tabulate the data in each histogram is indicated as “N.”
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