Human ANGEL2, the first eukaryotic 2´,3´-cyclic phosphatase

RNA molecules are multifunctional key players with various essential biological roles. In particular, RNAs display alternative chemical groups at their termini, which are critical for their fate and function. At the 5 end, RNAs could carry a single or triple phosphate group or, instead of a phosphate, a hydroxy (OH) group. Messenger RNAs contain a cap structure, a modification that is added after transcription and is essential for the translation into proteins and for their stability. At the 3 end, RNAs often display an OH-group; however, through de novo synthesis or by enzymatic cleavage, some RNAs end with a cyclic phosphate at the 2 and 3 positions. De novo synthesis is catalyzed by RTCD1, the RNA terminal 3 phosphate cyclase, using a 3-phosphate as a substrate. Enzymes that generate 2,3-cyclic phosphates after cleavage participate in processes such as pre-tRNA splicing removal of a single intron from precursor tRNAs and in the Unfolded Protein Response, which entails an atypical mRNA splicing reaction in the cytoplasm. Our lab is particularly interested in these pathways. In this proposal we would like to investigate the biology of 2,3-cyclic phosphate ended RNAs because we have just purified and identified a fully novel enzymatic activity in human cells that converts 2,3-cyclic phosphates into terminal 2,3-OH groups. The enzyme is called ANGEL2, and it has initially been predicted to contain a different type of enzymatic activity, i.e. the removal of adenosines from poly(A) tails normally present at mRNA 3 ends. Having done a thorough biochemical characterization of ANGEL2, we aim at studying its reaction mechanism and its tridimensional structure. We also plan to identify RNA pathways regulated by ANGEL2 and to reveal interacting RNA molecules and proteins. We also suggest studying the function of ANGEL2 in the mouse through the generation of animals that lack the Angel2 gene or that produce greater protein amounts. Finally, we will search for patients with mutations in ANGEL2 and try to understand their symptoms at the molecular level. The enzymatic activity of ANGEL2, converting 2,3-cyclic phosphates into 2,3-OH will also be useful in current protocols designed to sequence elusive 2,3-cyclic phosphate ended RNAs, refractory to cloning using standard procedures. Taken together, ANGEL2 is a novel and unique RNA processing enzyme in mammalian cells. It is imperative to understand its function at the molecular level in the context of the cell.

The central dogma of molecular biology states that DNA is transcribed into mRNA molecules, which are in turn translated into proteins, the working horses of a cell. Scientists have been "decorating" this apparently simple chain of events with both, groundbreaking findings and numerous details. For instance, it is known that not all RNAs generated via transcription from the DNA are used to generate proteins; some RNA molecules have a life on their own. It is also well established that RNAs suffer a wide range of chemical modifications before becoming useful for the cell. The project I have dealt with in the last four years focused on a single, chemical modification that some RNA molecules display in the last nucleotide, and in the enzyme that recognizes it, reacts with it and converts it into another chemical group, with strikingly different biochemical and biological features. The enzyme is called ANGEL2, and was discovered in my laboratory. A highlight of our finding is that, ANGEL2 is not doing what other scientists thought it should do based on its resemblance with other, pretty similar enzymes. Within this project, we have dissected the mechanism of action of ANGEL2 at the atomic level. We even managed, together Prof. Martin Jinek from the University of Zurich, to obtain the structure of ANGEL2 out of a crystal. Our biochemical and his structural expertise achieved a great synergism to finally elucidate the molecular mechanisms employed by ANGEL2. We then did an old trick: we managed to create cells devoid of ANGEL2. Those cells are alive but they are unable to do some specific jobs in the cell, as we expected. However, and these experiments are still ongoing, both the mouse and a small and transparent fish, called Danio rerio or "zebrafish", seem to have a normal life without ANGEL2! More recently, and in parallel with another group, we found that ANGEL2 spends most of its time in a very special "room" of the cell, called mitochondria, where the energy for the cell is produced. ANGEL2 is "imported" into the mitochondria through a very precise mechanism, and we have dissected that aspect, too. We went further to identify a protein that is embracing ANGEL2 very strongly - in molecular biology we say "it binds to it" - and we are currently studying the properties and purpose of such interaction. This project entailed many more experiments that cannot be outlined in this summary; in fact, each sentence is the tip of an iceberg. However, I can say that we have discovered and characterized in very much detail a new enzyme in human cells, and this is to celebrate! The finding was published in Science Magazine, a reflection of its significance.