Molecular interactions between Hel2 and RNA supporting ribosome-associated quality control

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Ribosome-associated quality control (RQC) pathways are vital for cellular health by monitoring and resolving ribosome stalling during protein synthesis.
• Hel2 (also known as Rqt1) is a key E3 ubiquitin ligase involved in RQC, but its precise recruitment and function remain incompletely understood.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying Hel2 recruitment and function in RQC and related pathways.
• To identify the specific interactions and RNA-binding capabilities of Hel2 within the translating ribosome.

Main Methods:

• In vivo UV-crosslinking coupled with mass spectrometry (UX-MS) to identify Hel2-interacting molecules.
• Crosslinking, ligation, and sequencing of RNAs isolated by immunoprecipitation (CLIP-seq) to map Hel2's RNA binding sites.
• Genetic manipulation (gene deletion and truncation mutants) to assess the functional impact of identified domains and interactions.

Main Results:

• A C-terminal region of Hel2 was identified as an RNA-binding domain, with Hel2 binding to 18S rRNA and translated mRNAs, particularly near the stop codon.
• Truncation of the Hel2 C-terminus disrupted 18S rRNA binding, polysome association, and impaired Hel2 function in RQC and no-go decay (NGD).
• Asc1 was found to act upstream of Hel2, facilitating Hel2's binding to 18S rRNA and mRNA.

Conclusions:

• Hel2 is recruited to translating 40S ribosomal subunits through interactions with 18S rRNA and Asc1.
• The identified 18S rRNA interaction is critical for Hel2's essential roles in RQC and NGD.
• Hel2 likely interacts with mRNA during the translation termination process to facilitate ribosome quality control.