High-throughput 5′P sequencing enables the study of degradation-associated ribosome stalls
Yujie Zhang, Vicent Pelechano
Abstract
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 in vivo 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.