High-throughput 5'P sequencing enables the study of degradation-associated ribosome stalls.

RNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is connected to the translation process up to the degree that 5'-3' mRNA degradation follows the last translating ribosome. Here, we present an improved high-throughput 5'P degradome RNA-sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable, and uses affordable duplex-specific nuclease-based rRNA depletion. We investigate ribosome stalls focusing on translation termination. By comparing ribosome stalls identified by ribosome profiling, disome-seq and HT-5Pseq, we find that degradation-associated ribosome stalls are often enriched in Arg preceding the stop codon. On the contrary, mRNAs depleted for those stalls use more frequently a TAA stop codon preceded by hydrophobic amino acids. Finally, we show that termination stalls found by HT-5Pseq, and not by other approaches, are associated with decreased mRNA stability. Our work suggests that ribosome stalls associated with mRNA decay can be easily captured by investigating the 5'P degradome.