No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5'-OH ends phosphorylated by Trl1

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Expression Regulation

Background:

• The No-Go Decay (NGD) pathway is a critical mRNA surveillance mechanism that targets and degrades mRNAs with stalled ribosomes.
• Previous models proposed an endoribonuclease initiating cleavage upstream of the stall site, but direct evidence for fragment production was lacking.

Purpose of the Study:

• To provide direct evidence for endonucleolytic cleavage events in the NGD pathway.
• To characterize the precise location and requirements of these cleavage events.
• To identify the factors involved in the subsequent degradation of NGD-targeted RNAs.

Main Methods:

• Utilized 3'-ribozyme-expressing mRNAs to generate truncated transcripts in vivo, mimicking natural truncated mRNAs that trigger NGD.
• Analyzed endonucleolytic cleavage events at single-nucleotide resolution using Hel2-dependent assays.
• Investigated the role of RNA kinases and exoribonucleases in NGD RNA degradation.

Main Results:

• Demonstrated Hel2-dependent endonucleolytic cleavages occurring precisely within the mRNA exit tunnel, 8 nucleotides upstream of the first P-site residue.
• Showed that these cleavages release 5'-hydroxylated RNA fragments.
• Identified the RNA kinase Trl1 (Rlg1) as essential for the efficient degradation of NGD RNAs, with 5'-phosphorylation required for exoribonuclease digestion by Xrn1 or Dxo1.

Conclusions:

• This study provides the first direct evidence of specific endonucleolytic cleavages initiating the No-Go Decay pathway.
• The findings elucidate the precise mechanism of cleavage and the requirement for 5'-phosphorylation by Trl1 (Rlg1) for subsequent exoribonucleolytic degradation.
• Identified key molecular players, including Hel2, Trl1/Rlg1, Xrn1, and Dxo1, involved in the NGD surveillance pathway.