A versatile toolbox for determining IRES activity in cells and embryonic tissues

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• Gene expression is regulated at the translational level, with internal ribosomal entry sites (IRESes) playing a key role.
• The precise functions of IRES elements in gene regulation and development have been challenging to characterize rigorously.

Purpose of the Study:

• To develop and present a novel toolbox of technologies for investigating IRES-mediated gene regulation.
• To elucidate the functions of IRES RNA elements, including Hox IRESes, in cellular and embryonic contexts.

Main Methods:

• Development of a technology suite including IRES-mediated translation of circular RNA (circRNA) reporters.
• Application of single-molecule messenger (m)RNA isoform imaging, PacBio long-read sequencing, and isoform-sensitive mRNA quantification.
• Analysis of IRES elements in both cellular and embryonic model systems.

Main Results:

• Demonstration of IRES-dependent translation occurring within circRNAs.
• Characterization of the relative expression, localization, and translation of IRES-containing mRNA isoforms in specific embryonic tissues.
• Successful application of the developed technologies to study a range of cellular IRES RNA elements.

Conclusions:

• The presented technologies offer a powerful new resource for dissecting IRES-mediated gene regulation.
• This work provides critical insights into the roles of IRES elements in controlling protein expression during embryonic development.
• The findings pave the way for deeper understanding of translational control mechanisms.