Transcriptome-wide profiling of multiple RNA modifications simultaneously at single-base resolution

Abstract

The breadth and importance of RNA modifications are growing rapidly as modified ribonucleotides can impact the sequence, structure, function, stability, and fate of RNAs and their interactions with other molecules. Therefore, knowing cellular RNA modifications at single-base resolution could provide important information regarding cell status and fate. A current major limitation is the lack of methods that allow the reproducible profiling of multiple modifications simultaneously, transcriptome-wide and at single-base resolution. Here we developed RBS-Seq, a modification of RNA bisulfite sequencing that enables the sensitive and simultaneous detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide. With RBS-Seq, m5C and m1A are accurately detected based on known signature base mismatches and are detected here simultaneously along with Ψ sites that show a 1–2 base deletion. Structural analyses revealed the mechanism underlying the deletion signature, which involves Ψ-monobisulfite adduction, heat-induced ribose ring opening, and Mg2+-assisted reorientation, causing base-skipping during cDNA synthesis. Detection of each of these modifications through a unique chemistry allows high-precision mapping of all three modifications within the same RNA molecule, enabling covariation studies. Application of RBS-Seq on HeLa RNA revealed almost all known m5C, m1A, and ψ sites in tRNAs and rRNAs and provided hundreds of new m5C and Ψ sites in noncoding RNAs and mRNAs. However, our results diverge greatly from earlier work, suggesting ∼10-fold fewer m5C sites in noncoding and coding RNAs and the absence of substantial m1A in mRNAs. Taken together, the approaches and refined datasets in this work will greatly enable future epitranscriptome studies.

Chemical probe-based Nanopore Sequencing to Selectively Assess the RNA modifications

“…These adduct induced mutations or truncation profiles are used as a proxy identifier of modifications with single-nucleotide resolution. Major shortcomings of the current methods are 1) multi-step sample preparation results in lesser reproducibility, 2) quantifying stoichiometry of the RNA modifications from these methods are not possible owing to the fragmentation of RNA and 3) simultaneously mapping of co-occurring modifications on the single mRNA molecule is difficult 22 .…”

Loss of pseudouridine synthases in the RluA family causes hypersensitive nociception inDrosophila

“…The precise mechanism explaining the involvement of RluA proteins in nociception can only be elucidated by identifying the RNA targets of these enzymes. Several groups have developed methods using next generation sequencing methods to identify the pseudouridine sites in transcriptomes (30-33, 64). Future investigations applying these methods to wild type and RluA mutants in Drosophila will help us to identify the RluA targets and to further define the underlying mechanisms.…”

“…Pseudouridine (Ψ), the C5-glycoside isomer of uridine, was initially found in many positions in rRNA, tRNA and snRNA in all organisms that have been investigated (29). RNA-seq based global pseudouridine profiling has shown the presence of Ψ in many mRNAs and a large number of those sites were found to be dynamically regulated in yeast and human cells (30-33). Dysfunctional pseudouridylation has been linked to several human diseases (34-36).…”

The epitranscriptome in translation regulation: mRNA and tRNA modifications as the two sides of the same coin?

“…In identifying thousands of m 5 C sites throughout the transcriptome RNA-bisulphite sequencing has become the first example for identifying internal mRNA modifications in high throughput. However, the high number of m 5 C sites has been questioned, showing how much care needs to be taken not to misinterpret modification marks that may be the result of single-nucleotide polymorphisms, inefficient chemical reactions or sequencing errors [20,88].…”