Codon usage biases co-evolve with transcription termination machinery to suppress premature cleavage and polyadenylation
- The University of Texas Southwestern Medical Center, United States
- Yunnan University, China
- East China University of Science and Technology, China
Figures
Figure 1 with 1 supplement
Codon deoptimization of the amino-terminal end of the frq ORF abolished the expression of full-length frq mRNA.
(A) A diagram showing the frq locus. (B) Race tube analysis showing the conidiation rhythm of the frqKO, wt-frq, frq-deopt1, and frq-deopt2 strains. The strains were first cultured in constant light …
Figure 1—figure supplement 1
DNA sequences of 5’ end of the frq open reading frame region for the indicated constructs.
Nucleotides mutated only in frq-deopt1 are shown in green, nucleotides mutated only in frq-deopt2 are shown in yellow; nucleotides mutated both in frq-deopt1 and frq-deopt2 are shown in red.
Figure 2 with 1 supplement
Codon deoptimization of frq results in premature transcription termination.
(A) Northern blot showing the presence of truncated frq mRNA species in both de-optimized strains using an RNA probe targeted to 5’ end of frq mRNA (indicated in ; Figure 2—figure supplement 1E). * …
Figure 2—figure supplement 1
Mechanism of the codon usage-mediated gene expression changes at the frq locus
(A) A diagram showing the frq locus. Primer set one is used to detect H3K9me3 levels (B) and WC-2 levels (C); primer set two is used to detect frq pre-mRNA levels (D). (B) ChIP analysis showing …
Figure 3 with 1 supplement
Rare codons promote while optimal codons suppress premature transcription termination of frq.
(A) A diagram showing the constructs created to map codons important for premature transcription termination. The triangle indicates the location of the mapped poly(A) sites. Black bars indicate the …
Figure 3—figure supplement 1
Codon deoptimization of frq abolish circadian clock function and reduction of frq expression.
(A) Race tube analysis showing the conidiation phenotypes of the wt-frq, frq-deopt3, frq-deopt4, frq-deopt5, frq-deopt6, frq-deopt7, and frq-deopt4* strains in constant darkness (DD). The strains …
Figure 4 with 1 supplement
Genome-wide identification of premature transcription termination events in ORF of endogenous Neurospora genes.
(A) 2P-seq results on NCU09435 (top) and NCU00931 (bottom) genes showing the transcription termination events in the 3’ UTR and ORF. (B) Northern blot analyses showing the presence of both …
Figure 4—figure supplement 1
Analyses of the 2P-seq results in Neurospora.
(A) Scatter analysis showing the reproducibility of two biological replicates of our 2P-seq of nuclear poly(A)-containing RNAs of Neurospora. (B) 2P-seq reads mapped in the coding region of NCU09435 …
Figure 5 with 3 supplements
Strong genome-wide correlations between codon usage and premature transcription termination events.
(A) Scatter plot of normalized ORF/3’ UTR termination events (log10) vs. CBI. r = −0.64, p<2.2 × 10−16, n = 2957. (B) Scatter analysis showing the correlation of normalized ORF/3’ UTR termination …
Figure 5—figure supplement 1
Sequence alignment of NCU09435-wt and NCU09435-opt constructs (A) and sequence alignment of NCU02034-wt and NCU02034-deopt constructs (B).
Nucleotides that are mutated in the optimized NCU09435 gene are shown in red. The arrow indicates the poly(A) site mapped by 2P-seq. The black line in (A) indicates a potential PAS motif present …
Figure 5—figure supplement 2
Nucleotide composition (U top panel and A bottom panel) surrounding PAS motifs in 3’ UTR (A) and coding region (B).
True PAS means at least a poly(A) site was identified by 2P-seq within the 5–35 nt downstream of PAS motif, and False PAS has no poly(A) site was identified in this region. 0 indicates the position …
Figure 5—figure supplement 3
Correlations between relative codon usage frequency and relative synonymous codon adaptiveness.
(A) Scatter plot analysis of relative codon usage frequency (RCUF) with relative synonymous codon adaptiveness (RSCA) of all codons with at least two synonymous codons in Neurospora. r = −0.66, …
Figure 6
Transcription termination events in Schizosaccharomyces pombe.
(A) Nucleotide sequence composition surrounding the poly(A) sites located in 3’ UTR (left) and in ORF (right) in S. pombe. (B) Genome-wide PAS scores for 3’UTR-PAS and ORF-PAS. (C) Scatter analysis …
Figure 7 with 2 supplements
Premature transcription termination events in ORFs in mouse C2C12 cells.
(A) Nucleotide sequence composition surrounding poly(A) sites in 3’ UTR (left) and in ORFs (right) in mouse C2C12 cells. (B) PAS scores for 3’UTR-PAS and ORF-PAS. (C) Scatter analysis showing the …
Figure 7—figure supplement 1
Sequence analyses of poly(A) sites in mouse C2C12 cells.
(A) Top 15 most frequently used PAS motifs found in the A-rich element of 3’ UTR-PAS. (B) Top 15 most frequently used PAS motifs found in the A-rich element of 3’ UTR-PAS ORF-PAS in mouse C2C12 …
Figure 7—figure supplement 2
Codon usage and sequence analyses of poly(A) sites in mouse C2C12 cells.
(A) Scatter plot showing the correlation between normalized codon usage frequency (NCUF) (log2) and relative synonymous codon adaptiveness (RSCA) of all codons with at least two synonymous codons in …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Neurospora crassa) | frequencey (frq) | NA | NCBI Gene ID:3876095 | |
| gene (Neurospora crassa) | NCU09435 | NA | NCBI Gene ID:3874734 | |
| gene (Neurospora crassa) | NCU00931 | NA | NCBI Gene ID:3880910 | |
| strain (Neurospora crassa) | 4200 | PMID:155773 | Strain maintained in Yi Liu's lab | |
| strain (Neurospora crassa) | 303–3 (bd, frq10, his-3) | PMID:8052643 | ||
| strain (Neurospora crassa) | 301–6 (bd, his-3, A) | PMCID: PMC180927 | ||
| antibody | anti-FRQ | PMID:9150146 | Rabbit polyclonal; 1:50 for western blot | |
| antibody | anti-WC-2 | PMID: 11226160 | Rabbit polyclonal; 1:500 for ChIP | |
| antibody | Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody | abcam | ab5095 | Rabbit polyclonal; 1:500 for ChIP |
| antibody | Anti-H3K9me3 | Active Motif | catalog no:39161 | Rabbit polyclonal; 1:500 for ChIP |
| recombinant DNA reagent | pKAJ120 | PMID:8052643 | deoptimized frq gene; seeFigure 1—figure supplement 1 | |
| recombinant DNA reagent | frq-deopt1 | this paper | deoptimized frq gene; seeFigure 1—figure supplement 1 | |
| recombinant DNA reagent | frq-deopt2 | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | frq-deopt3 | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | frq-deopt4 | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | frq-deopt5 | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | frq-deopt6 | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | frq-deopt7 | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | frq-deopt4* | this paper | deoptimized frq gene; see Materials and methods | |
| recombinant DNA reagent | gfp-NCU09435-wt | this paper | wild-type NCU09435 gene in frame with gfp | |
| recombinant DNA reagent | gfp-NCU09435-opt | this paper | optimized NCU09435 gene; see Figure 5—figure supplement 1 | |
| recombinant DNA reagent | gfp-NCU02034-wt | this paper | wild-type NCU02034 gene in frame with gfp | |
| commercial assay or kit | SuperScript III Reverse Transcriptase | Thermo Fisher (Waltham, MA ) | catalog no:18080093 | For 3' RACE and making 2P-seq library |
| commercial assay or kit | TURBO DNA-free Kit | Thermo Fisher (Waltham, MA ) | catalog no: AM1907 | |
| commercial assay or kit | TOPO TA Cloning Kit, Dual promotor for in vitro Transcription | Thermo Fisher (Waltham, MA ) | catalog no: 452640 | |
| commercial assay or kit | Direct-zol RNA miniprep plus | Zymo research | catalog no: R2072 | |
| commercial assay or kit | CircLigase II ssDNA Ligase | Epicentre | catalog no: CL9021K | |
| software, algorithm | TopHat | http://ccb.jhu.edu/software/tophat/index.shtml | RRID:SCR_013035 | |
| software, algorithm | samtools | http://samtools.sourceforge.net/ | RRID:SCR_002105 | |
| software, algorithm | BEDTools | http://bedtools.readthedocs.io/en/latest/ | RRID:SCR_006646 | |
| software, algorithm | codonW | http://codonw.sourceforge.net/ | ||
| software, algorithm | Source code | this paper | scripts to analyze 2P-seq and 3’READS. Including eight steps: read processing, mapping, filtering and downstream analyses that create plot and figures. | |
| software, algorithm | raw sequencing data | this paper | PRJNA419320 | 2P-seq data, including two repeats from nuclear RNA extracts |
| software, algorithm | raw sequencing data | PMID:27401558 | GSE75753 | mouse poly(A)-seq data |
| software, algorithm | raw sequencing data | PMID:26765774 | GSE72574 | yeast poly(A)-seq data |
Additional files
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Supplementary file 1
Strain list used in this study
- https://doi.org/10.7554/eLife.33569.018
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Supplementary file 2
Primers used in this study
- https://doi.org/10.7554/eLife.33569.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.33569.020
