Transcription-wide mapping of dihydrouridine reveals that mRNA dihydrouridylation is required for meiotic chromosome segregation

Abstract

Highlights d Rho-seq detects a range of RNA modifications through differential rhodamine labeling d The dihydrouridine tRNA modification is also present on fission yeast mRNAs d Dihydrouridine on the tubulin mRNA is required for meiotic chromosome segregation d Tubulin mRNA dihydrouridylation is evolutionarily conserved from yeast to human

Epitranscriptomic mapping of RNA modifications at single-nucleotide resolution using rhodamine sequencing (Rho-seq)

“…Until recently, the dihydrouridine (D) RNA modification was exclusively studied within the context of tRNA and rRNA biology. However, preliminary data from our lab and elsewhere led us to consider the possibility that other RNA species could also be dihydrouridilated ( Dudin et al., 2017 ; Fauquenoy et al., 2018 ; Finet et al., 2022 ). To assess whether D extends beyond tRNA is particularly challenging given the close resemblance of D to the canonical nucleotide U.…”

“…We reasoned that regardless of the fluorescent properties of rhodamine, its incorporation should interfere with reverse transcription (RT), leaving a footprint that can be exploited to indirectly observe the presence D. In earlier epitranscriptomic studies, the concept of RT stop footprint was successfully applied to the detection of pseudouridine ( Schwartz et al., 2014 ), inosine ( Suzuki et al., 2015 ) and m 1 A ( Li et al., 2017 ; Safra et al., 2017 ). Similarly, by combining rhodamine labeling of the D modification with high-throughput sequencing, we developed Rho-seq, a method to assess the presence of D at the transcriptome-wide scale ( Finet et al., 2022 ).…”

“…The protocol below describes the specific steps for using Rho-seq to detect dihydrouridine in fission yeast. However, we have also used Rho-seq in prokaryote ( E. coli ) and higher eukaryote (human cell line HCT116) ( Finet et al., 2022 ) and the protocol can be readily applied to any organism provided that the appropriate modification-free controls are available ( Table 1 ).…”

“…Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022) .…”

“…For complete details on the use and execution of this protocol, please refer to Finet et al. (2022) .…”