Targeted Quantitative Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Using Stable Isotope-Labeled Peptides. 2022

Tianyu F Qi, and Xiaochuan Liu, and Feng Tang, and Jiekai Yin, and Kailin Yu, and Yinsheng Wang
Environmental Toxicology Graduate Program, University of California Riverside, Riverside, California 92521-0403, United States.

N6-Methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from m6A, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of previously published m6A mapping results revealed the presence of m6A in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.

UI MeSH Term Description Entries
D007554 Isotopes Atomic species differing in mass number but having the same atomic number. (Grant & Hackh's Chemical Dictionary, 5th ed) Isotope
D008780 Methyltransferases A subclass of enzymes of the transferase class that catalyze the transfer of a methyl group from one compound to another. (Dorland, 28th ed) EC 2.1.1. Methyltransferase
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D011560 Pseudouridine A naturally-occurring isomer of URIDINE found in RNA, in which ribosyl is attached to a carbon instead of a nitrogen atom.
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000071516 Alpha-Ketoglutarate-Dependent Dioxygenase FTO A dioxygenase that repairs alkylated DNA and RNA by oxidative demethylation. It has highest activity towards single-stranded RNA containing 3-methyluracil, followed by SINGLE-STRANDED DNA containing 3-methylthymine and specifically demethylates N(6)-methyladenosine (m6A) RNA, the most common internal modification of MESSENGER RNA (mRNA) in higher EUKARYOTES. It contributes to the regulation of the global metabolic rate, energy HOMEOSTASIS, as well as body fat accumulation by regulating the differentiation of ADIPOCYTES into BROWN FAT CELLS or WHITE FAT CELLS. AlkB Homolog 9 Protein,Alpha-Ketoglutarate-Dependent Dioxygenase,Fat Mass and Obesity Associated (FTO) Protein,Fat Mass and Obesity Associated Protein,Alpha Ketoglutarate Dependent Dioxygenase,Alpha Ketoglutarate Dependent Dioxygenase FTO,Dioxygenase, Alpha-Ketoglutarate-Dependent,FTO, Alpha-Ketoglutarate-Dependent Dioxygenase
D000241 Adenosine A nucleoside that is composed of ADENINE and D-RIBOSE. Adenosine or adenosine derivatives play many important biological roles in addition to being components of DNA and RNA. Adenosine itself is a neurotransmitter. Adenocard,Adenoscan
D012313 RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. (Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed) RNA, Non-Polyadenylated,Ribonucleic Acid,Gene Products, RNA,Non-Polyadenylated RNA,Acid, Ribonucleic,Non Polyadenylated RNA,RNA Gene Products,RNA, Non Polyadenylated
D012333 RNA, Messenger RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm. Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated

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