Interrogation of Eukaryotic Stop Codon Readthrough Signals by in Vitro RNA Selection

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RNA sequences downstream of stop codons can induce translational readthrough.
• This readthrough generates C-terminally extended proteins with distinct functions.
• Understanding these RNA signals is crucial for identifying novel gene reprogramming events.

Purpose of the Study:

• To comprehensively characterize RNA signals that stimulate translational readthrough.
• To discover novel readthrough events and mechanisms in eukaryotic genes.
• To develop predictive tools for identifying readthrough activity.

Main Methods:

• In vitro translation selection to enrich for RNA readthrough signals from a large randomized library.
• High-throughput sequencing to identify sequence and structural features of active RNA signals.
• Cellular reporter assays to validate the activity of identified readthrough signals.
• Machine learning models trained on HTS data to predict readthrough activity in human sequences.

Main Results:

• Identified key primary sequence and secondary structure features of readthrough stimulatory RNAs.
• Discovered three novel RNA sequence motifs that promote translational readthrough.
• Validated readthrough signal activities in cellular assays.
• Predicted and discovered >1.5% readthrough in four human genes: CDKN2B, LEPROTL1, PVRL3, and SFTA2.

Conclusions:

• RNA signals play a significant role in reprogramming translation by inducing stop codon readthrough.
• The study provides tools and insights for discovering new readthrough events in eukaryotic genomes.
• This work opens avenues for exploring the biological implications of stop codon readthrough in human health and disease.