Related Experiment Video
Updated: Jun 4, 2025

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale
Published on: May 17, 2014
Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and
Cole J T Lewis1, Li H Xie2, Shivani Milind Bhandarkar1
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06510, USA.
None:
mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions (UTRs) including alternative isoforms and identify sequences that mediate 200-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs.
Related Concept Videos
Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
Regulation of Expression at Multiple Steps
Regulation of Expression Occurs at Multiple Steps
Transcription results in the generation of precursor (pre-mRNA) that consists of both exons and introns, which needs further processing before being translated to a...
Leaky Scanning
Initiation of Translation
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
What is Gene Expression?

