Ribo-ITP expands the translatome of limited input samples.

Area of Science:

• Molecular Biology
• Genomics
• Proteomics

Background:

• Translated regions (translons) and micropeptides play roles in cellular regulation and development.
• Conventional methods for translon identification require high sample input, limiting studies in small or difficult-to-collect tissues.

Purpose of the Study:

• To develop and validate a low-input method for identifying translons in challenging biological samples.
• To investigate translon expression patterns and functional roles in specific cell and tissue types.

Main Methods:

• Ribo-ITP (Ribosome Profiling with Integrated Translatome Profiling) was employed for low-input sample analysis.
• Comparative analysis of over a thousand ribosome profiling datasets was performed.
• A translon-dependent GFP reporter system and mutagenesis in mouse embryonic stem cells (mESCs) were used to assess translon function.

Main Results:

• Ribo-ITP successfully identified translons in microdissected hippocampal tissues and single pre-implantation embryos.
• Distinct, sample-specific expression patterns of translons were observed across various cell types.
• Translons initiating at near-cognate start codons were detected, and some were found to negatively impact mESC growth upon mutation.

Conclusions:

• Ribo-ITP is a viable proof-of-concept method for identifying non-canonical translation events from low-input samples.
• This technique expands the accessibility of translon research to previously inaccessible cell and tissue types.
• The study highlights the functional significance of certain translons in cellular processes.