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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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Pan-viral ORFs discovery using massively parallel ribosome profiling.

Shira Weingarten-Gabbay1,2,3, Matthew R Bauer4, Alexandra C Stanton1,5,6

  • 1Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Science (New York, N.Y.)
|June 12, 2025
PubMed
Summary
This summary is machine-generated.

Scientists discovered thousands of new viral open reading frames (ORFs) using massively parallel ribosome profiling (MPRP). These findings reveal novel viral peptides and regulatory elements, expanding vaccine targets and understanding viral mechanisms.

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Area of Science:

  • Virology
  • Genomics
  • Immunology

Background:

  • Defining viral proteomes is essential for understanding viral life cycles and immune responses.
  • The full extent of translated regions in viral genomes is largely unknown.

Purpose of the Study:

  • To develop and apply a method for identifying novel viral open reading frames (ORFs).
  • To characterize the landscape of translated regions in human-associated viral genomes.

Main Methods:

  • Massively parallel ribosome profiling (MPRP) was employed to analyze tens of thousands of designed oligonucleotides.
  • MPRP was used to identify and map ORFs across numerous viral genomes.

Main Results:

  • 4208 unannotated ORFs were identified in 679 human-associated viral genomes.
  • Viral peptides from noncanonical ORFs were presented on human leukocyte antigen (HLA) class I molecules.
  • Hundreds of upstream ORFs (uORFs) were found, potentially regulating viral protein translation.

Conclusions:

  • The discovery of numerous viral ORFs expands the known viral proteome and potential vaccine targets.
  • Identified ORFs and uORFs offer insights into viral cis-regulatory sequences and translation modulation.
  • This work enhances our understanding of viral biology and immune recognition across diverse viral families.