Large-scale tethered function assays identify factors that regulate mRNA stability and translation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The functions of most RNA-binding proteins (RBPs) are unknown, hindering the understanding of gene expression regulation.
• Identifying specific roles of RBPs in RNA metabolism is crucial for advancing molecular biology.

Purpose of the Study:

• To develop a comprehensive resource and assay for functionally characterizing human RBPs.
• To identify RBPs that regulate RNA stability and translation.
• To elucidate the specific molecular functions of uncharacterized RBPs, such as UBAP2L.

Main Methods:

• Developed a plasmid resource of 690 human RBPs.
• Utilized luciferase-based 3'-untranslated-region tethered function assays.
• Employed enhanced UV-cross-linking and immunoprecipitation (eCLIP) to identify endogenous mRNA targets.
• Applied polysome profiling and CRISPR-Cas9 fusion assays to study protein function.

Main Results:

• Identified thousands of endogenous mRNA targets regulated by RBPs.
• Characterized UBAP2L as an RNA-binding protein that interacts with mRNA and rRNA via its RGG domain.
• Demonstrated that UBAP2L promotes mRNA translation, particularly for global translation regulators.
• Validated the utility of tethering assays for assigning molecular activities to RBPs.

Conclusions:

• The developed RBP resource and assays enable rapid functional characterization of RNA-binding proteins.
• UBAP2L plays a significant role in promoting mRNA translation.
• This work provides a framework for dissecting the roles of RBPs in gene expression regulation and mRNA metabolism.