Motivation behind the research
Imagine that you, a detective, are invited to decipher a mysterious message. The only letters in this message are A, U, C and G. While the majority of the letters in the message are printed, a few “U”s are hand-written. You are puzzled by the phenomenon and are trying to figure out how many special “U”s there are.
In life science, one of these mysterious messages is RNA. The information in RNA is composed of an alternating sequence of four bases: adenine (A), uridine (U), cytidine (C) and guanosine (G). A small proportion of these bases are further modified to chemically similar structures. The most abundant and well-known modification is pseudouridylation. Pseudouridine (Ψ) constitutes over 1% of total nucleosides and over 7% of all uridines in ribosomal RNA (rRNA) fractions in almost all RNA-containing species. Most importantly, recent studies revealed that Ψ levels are dynamically changed as responses to different types of cellular stress, including, but not limited to, changes in temperature, oxygen supply, or nutrients. We set out to develop a fast and economic tool for the determination of Ψ levels.
The words (permutations of different types of ribonucleosides) involved in the mysterious message (RNA) were chopped up into individual letters (single ribonucleoside) with an enzyme called RNase T2. They were pressed through a sieve (digested with the enzyme shrimp alkaline phosphatase) to remove the nonspecific chemical groups in the structure. The remaining core structures of “letters” (bases) were further sorted by a tool called high-performance liquid chromatography (HPLC) and detected with an ultraviolet detector (UV). Compared to other reports, our method was time-efficient at minimal cost.
After method development and validation, we applied it for the measurement of Ψ levels in RNA. We found that Ψ levels varied from 0.98% to 1.94% of all ribonucleosides in different types of RNA. Our results are comparable to known facts of Ψ levels in vivo. A consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed after we inhibited the pseudouridylation process in cells.
This is not the only method to determine total Ψ levels. There are a lot of studies in the field of RNA modifications. They could detect not only the modified ribonucleosides of lower abundance, but also the changes of Ψ levels at the specific sites. That being said, every method has its limitations. It is the ultimate purpose of research and the availability of time and research funding that determines the choice of a specific quantification method.
With only the digestion steps prior to analysis, our method provides accurate quantification of Ψ levels without any additional sample derivatization or manipulation. With our assay, the global Ψ levels in a specific RNA pool can be quantified to serve as a quick readout of changes in Ψ levels (either in total RNA or in an isolated RNA pool) in response to different types of cellular stress.
Research Article: Quantification of Pseudouridine Levels in Cellular RNA Pools with a Modified HPLC-UV Assay. Genes (Basel). 2017.