Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements

Area of Science:

• Molecular Biology
• Genetics

Background:

• Internal ribosome entry site (IRES) elements are crucial for cap-independent translation initiation.
• Identifying novel IRES elements is important for understanding translation regulation and developing new biotechnological tools.

Purpose of the Study:

• To develop and utilize a novel fluorescent protein-based screening method to identify small, synthetic IRES elements in vivo.
• To characterize the activity and properties of the identified synthetic IRES elements.

Main Methods:

• Construction of a bicistronic plasmid library with randomized sequences between enhanced blue and green fluorescent protein (EBFP and EGFP) reporters.
• Delivery of the plasmid library into mammalian cells via protoplast fusion.
• Isolation of cells with functional IRES elements using fluorescence-activated cell sorting (FACS).
• In vitro and in vivo characterization of identified IRES elements, including activity assays and competition studies.

Main Results:

• Identification of several small, synthetic IRES elements using the fluorescent protein-based screen.
• Two novel synthetic IRES elements demonstrated IRES activity comparable to a known viral IRES element (encephalomyocarditis virus IRES variant).
• These synthetic IRES elements functioned independently of context and in multiple cell types.
• The synthetic IRES elements specifically inhibited poliovirus (PV) IRES-mediated translation in vitro, suggesting competition for shared translation factors.

Conclusions:

• The developed fluorescent protein-based screen is effective for identifying novel and functional IRES elements.
• The identified synthetic IRES elements possess significant translational activity and can modulate other IRES activities.
• These findings provide insights into IRES-mediated translation mechanisms and offer potential tools for gene expression control.