Recently, in a research report published in the international journal Science, scientists from Vienna Medical University and other institutions have identified a special chemical reaction at the end of RNA molecules in human cells for the first time. Previously, researchers have only observed this reaction in bacteria and viruses, and by tracking its source in thousands of proteins, the researchers found that a special enzyme called angel homolog 2 (ANGEL2) may perform this reaction. ANGEL2 plays a key role in regulating the body's response to cell pressure, and it seems to play a major role in the pathological progress of neurodegenerative and metabolic diseases.
RNA molecule is a kind of biological molecule with multiple functions. RNA molecules are frequently modified with a terminal 2',3'>P (2',3'-cyclic phosphate) group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-Ps are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. Enzymes known to convert 2',3'>P into 2',3'-OH include bacteriophage T4 polynucleotide kinase (PNK) and other RNA repair enzymes from the DxDxT acyl phosphatase superfamily (e.g. baculoviral RNA ligase 1 (Rnl1)). Examples from bacteria include Pnkp from the binuclear metallophosphoesterase superfamily and the histidine-aspartate (HD)-Pnk RNA healing enzymes. Nevertheless, such activity has not been detected in eukaryotic cells.
To search for an RNA 2',3'>Pase activity in human cells, researchers established an assay using a radiolabeled, short double-stranded RNA (dsRNA) substrate. The researcher Paola Hentges Pinto said, “We screened candidate enzymes from a complex set of thousands of proteins, and tracked their ability to remove cyclophosphate groups. ANGEL2 is a very popular enzyme, which belongs to the deadenylases family that can perform a completely different reaction at the end of RNA.” And their studies suggested that knockdown of ANGEL2 led to a large decrease in 2',3'>Pase activity. Wild-type ANGEL2 expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli showed intrinsic 2',3'>Pase activity, indicating that ANGEL2 alone performs the reaction.
Deadenylases can remove a string of adenosine from the end of messenger RNAs (mRNAs), while removing specific types of nucleotides can degrade mRNAs. Through structural analysis, the researchers were able to reveal the reaction mechanism of ANGEL2 enzymes and explain why they can remove cyclic phosphate molecules more than adenosine. Overall, the ANGEL2 structure confirms that 3'-terminal phosphatases of the phosphatase (EEP) family contain residues that occlude the +1 nucleotide-binding position, which distinguishes them from the endo-/exonucleases and facilitates correct substrate positioning with respect to the catalytic residues and the divalent cation.
Figure 1. Summary of the effects of ANGEL2 on pathways requiring ligation or hydrolysis of 2',3'-P. (Pinto P H, et al. 2020)
In order to make the protein function, the amino acid chain needs to be correctly folded. When the misfolded non functional proteins accumulate due to cell interference, unfolded protein response (UPR) will be triggered and correct these defects of protein. In mammalian cells, RNA molecules ending with 2',3'>P are substrates for the tRNA ligase complex during pre-tRNA processing and X-box-binding protein 1 (XBP1) mRNA splicing in the UPR. Their study shows that ANGEL2 modulates levels of mature tRNAs and XBP1 mRNA during pre-tRNA processing in vitro and in the UPR, respectively. The ubiquitously expressed ANGEL2 confers an additional regulatory mechanism to globally modulate UPR. This may be an important finding because UPR interference is involved in neurodegenerative and metabolic diseases. The results may help to develop and treat a variety of human diseases associated with UPR. Besides, these work paves the way for studying the role of ANGEL proteins on various RNA species with 2',3'>P termini, mostly derived from tRNAs, mRNAs, and rRNAs.