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Quantitative analysis of mammalian translation initiation sites by FACS‐seq

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Noderer, William L, Flockhart, Ross J, Bhaduri, Aparna, Diaz de Arce, Alexander J, Zhang, Jiajing, Khavari, Paul A, Wang, Clifford L·2014·Molecular Systems Biology
DOI 10.15252/msb.20145136PMC PMC4299517
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Abstract

An approach combining fluorescence‐activated cell sorting and high‐throughput DNA sequencing (FACS‐seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS‐seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome‐wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.

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  • Earlier MPRAs designed to learn aspects of 5′ UTR cis-regulation relied on FACS or growth selection to stratify libraries by activity.

    “Earlier MPRAs designed to learn aspects of 5′ UTR cis-regulation relied on FACS10,11 or growth selection12 to stratify libraries by activity.”

    From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [10]

  • The analysis recapitulated the importance of a purine (A or G) at position −3 relative to AUG and a G at +4.

    “This analysis recapitulated the importance of a purine (A or G) at position −3 relative to AUG and a G at +4 (Fig. 1e)10,23,24.”

    From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [10]

  • FACS- and growth-selection-based MPRAs require expression of a single library variant per cell that must be transcribed within the cell from a DNA template, making it difficult to distinguish between the effects of transcriptional and translational control.

    “These techniques require the expression of a single library variant per cell that must be transcribed within the cell from a DNA template, making it difficult to distinguish between the effects of transcriptional and translational control.”

    From Human 5′ UTR design and variant effect prediction from a massively parallel translation assay (2019) · cites this paper as [10]

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Authors
Noderer, W. L., Flockhart, R. J., Bhaduri, A., Diaz de Arce, A. J., Zhang, J., Khavari, P. A., & Wang, C. L.
Affiliations
  • Stanford University (US)
  • Stanford Medicine (US)
  • VA Palo Alto Health Care System (US)
Corresponding author
Wang, Clifford L
ORCID
Venue
Molecular Systems Biology
Publisher
Springer Science and Business Media LLC
Published
2014-08-01
Volume
10
Issue
8
Pages
748-748
ISSN
1744-4292 (ISSN-L)
DOI
10.15252/msb.20145136
PMC
PMC4299517
OpenAlex
W2127986230
Cited by
178
References
3
Citations by year
2026: 16 · 2025: 12 · 2024: 19 · 2023: 28 · 2022: 18 · 2021: 13 · 2020: 13 · 2019: 16 · 2018: 8 · 2017: 13 · 2016: 13 · 2015: 9
FWCI
4.40
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Systems Biology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • NIH (5R21AG040360‐02)
  • Department of Energy Office of Science Graduate Fellowship Program
  • Stanford Bio‐X Graduate Student Fellowship
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
translation initiation sites, start codon recognition, translation efficiency, carcinogenesis, driver mutations, truncated protein isoform
MeSH terms
Animals, Flow Cytometry, Humans, Models, Theoretical, Mutation, Peptide Chain Initiation, Translational, RNA, Messenger, Codon, Initiator, Amino Acid Motifs, NIH 3T3 Cells, Cell Line, Tumor, Mice, Genetic Association Studies, High-Throughput Nucleotide Sequencing, Carcinogenesis

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