Discovery of high-confidence human protein-coding genes and exons by whole-genome PhyloCSF helps elucidate 118 GWAS loci

Mudge, Jonathan M.; Jungreis, Irwin; Hunt, Toby; Gonzalez, Jose M.; Wright, James C.; Kay, Mike; Davidson, Claire; Fitzgerald, Stephen; Seal, Ruth L.; Tweedie, Susan; He, Liang; Waterhouse, Robert M.; Li, Yue; Bruford, Elspeth A.; Choudhary, Jyoti S.; Frankish, Adam; Kellis, M

doi:10.1101/gr.246462.118

Cited by 65 publications

(54 citation statements)

References 94 publications

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“…1c, Supplementary Table S2). These tools are widely-accepted standards for protein-coding gene annotation and for distinguishing protein-coding vs. non-coding genes in human and other species [20][21][22][26][27][28] .…”

Section: Strain Selection Alignment Constraintmentioning

confidence: 99%

“…Input alignments were extracted from the whole-genome alignment and columns containing a gap in the reference sequence were removed. Browser tracks were created as described previously 26 . Scores listed in Supplementary Table S2 were calculated on the local alignment for each ORF or mature protein, excluding the final stop codon, using the default PhyloCSF parameters, including --strategy=mle.…”

Section: Phylocsf Fresco and Other Conservation Metricsmentioning

confidence: 99%

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SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

Jungreis

Sealfon

Kellis

2020

Preprint

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Despite its overwhelming clinical importance for understanding and mitigating the COVID-19 pandemic, the protein-coding gene content of the SARS-CoV-2 genome remains unresolved, with the function and even protein-coding status of many hypothetical proteins unknown and often conflicting among different annotations, thus hindering efforts for systematic dissection of its biology and the impact of recent mutations. Comparative genomics is a powerful approach for distinguishing protein-coding versus non-coding functional elements, based on their characteristic patterns of change, which we previously used to annotate protein-coding genes in human, fly, and other species. Here, we use comparative genomics to provide a high-confidence set of SARS-CoV-2 protein-coding genes, to characterize their protein-level and nucleotide-level evolutionary constraint, and to interpret the functional implications for SARS-CoV-2 mutations acquired during the current pandemic. We select 44 complete Sarbecovirus genomes at evolutionary distances well-suited for protein-coding and non-coding element identification, create whole-genome alignments spanning all named and putative genes, and quantify their protein-coding evolutionary signatures using PhyloCSF and their overlapping constraint using FRESCo. We find strong protein-coding signatures for all named genes and for hypothetical ORFs 3a, 6, 7a, 7b, and 8, indicating protein-coding roles, and provide strong evidence of protein-coding status for a recently-proposed alternate-frame novel ORF within 3a. By contrast, ORF10 shows no protein-coding signatures but shows unusually-high nucleotide-level constraint, indicating it has important but non-coding functions, and ORF14 and SARS-CoV-1 ORF3b, which overlap other genes, lack evolutionary signatures expected for dual-coding regions, indicating they do not produce functional proteins. ORF9b has ambiguous protein-coding signatures, preventing us from resolving its protein-coding status. ORF8 shows extremely fast nucleotide-level evolution, lacks a known function, and was deactivated in SARS-CoV-1, but shows clear signatures indicating protein-coding function worthy of further investigation given its rapid evolution and potential role in replication. SARS-CoV-2 mutations are preferentially excluded from evolutionarily-constrained amino acid residues and synonymously-constrained nucleotides, indicating purifying constraint acting at both coding and non-coding levels. In contrast, we find a conserved region in the nucleocapsid that is enriched for recent mutations, which could indicate a selective signal, and find that several spike-protein mutations previously identified as candidates for increased transmission and several mutations in isolates found to generate higher viral load in-vitro disrupt otherwise-perfectly-conserved amino-acids, consistent with adaptations for human-to-human transmission.

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Section: Strain Selection Alignment Constraintmentioning

confidence: 99%

Section: Phylocsf Fresco and Other Conservation Metricsmentioning

confidence: 99%

SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

Jungreis

Sealfon

Kellis

2020

Preprint

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“…We initially found evidence of alternate-frame translation in POLG as part of a project to identify novel coding regions using PhyloCSF [30]. We had previously developed PhyloCSF [31] (Phylogenetic Codon Substitution Frequencies) to determine whether a given nucleotide sequence is likely to represent a functional, conserved protein-coding sequence by determining the likelihood ratio of its multi-species alignment under coding and non-coding models of evolution that use precomputed substitution frequencies for every possible pair of codons, trained on whole-genome data.…”

Section: Resultsmentioning

confidence: 99%

Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon

et al. 2020

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“…Highly efficient stop codon readthrough is common in the CNS. A large number of genes are predicted to undergo stop codon readthrough in Drosophila (26)(27)(28)47), including many that are expressed or known to function in the nervous system. Given the high-level readthrough we observed with kelch in neuronal tissues, we wondered whether neurons in Drosophila might generally support high-level readthrough.…”

Section: Sequences 3' Of the Uga Codon Stimulate Readthroughmentioning

confidence: 99%

“…ORF2 function. Evidence for extensive stop codon readthrough in Drosophila comes from ribosome footprinting experiments (31) and analysis of codon conservation downstream of the first stop codon in genes (26)(27)(28)47). Based on these data, there are now over 400 genes annotated in FlyBase as readthrough genes with highly conserved extensions among Drosophila species.…”

Section: Evasion Of Nmd Does Not Explain Tissue-specific Readthroughmentioning

confidence: 99%

Tissue-specific dynamic codon redefinition in Drosophila

Hudson

Loughran

Szabo

et al. 2020

Preprint

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Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3' of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue-and cell-specific dynamic stop codon redefinition in Drosophila. Stop codon readthrough | recoding | Drosophila | Kelch | central nervous system (CNS) †Correspondence: j.atkins@ucc.ie or lynn.cooley@yale.edu Hudson et al. | bioRχiv | June 22, 2020 | 1-11

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Discovery of high-confidence human protein-coding genes and exons by whole-genome PhyloCSF helps elucidate 118 GWAS loci

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Cited by 65 publications

References 94 publications

SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

SARS-CoV-2 gene content and COVID-19 mutation impact by comparing 44 Sarbecovirus genomes

Evidence for a novel overlapping coding sequence in POLG initiated at a CUG start codon

Tissue-specific dynamic codon redefinition in Drosophila

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