RNA 3’-Phosphate Cyclase RTCD1 in mammalian RNA metabolism

The DNA-made genome is transcribed by specific enzymes into a different type of nucleic acid known as RNA. In fact, there are many types of RNA molecules with myriads of cellular functions, some of them waiting to be revealed. Far from being ready to act, those RNAs will suffer transformations. For example, they become decorated with a large variety of chemical groups, or simply cut and rejoined leading to the loss of particular portions. The field of RNA biology is particularly interested in identifying and characterizing the series of enzymes that modify cellular RNAs. At the core of this project is the characterization of RTCD1, an enzyme that was identified 30 years ago but its function remains mysterious. Our work relies on two critical findings: at the biochemical level (so called in vitro) we discovered that RTCD1 is bound to another enzyme. We know what the other enzyme does, but not its identity. We will therefore purify RTCD1 and try to identify its partner and later characterize it. A second part of the project will be performed in the mouse (in vivo). In a well established procedure, scientists usually discover the function of a protein by generating a mouse that lacks the gene encoding that protein (knock-out mouse). We generated a mouse whose genome lacks the gene encoding the enzyme RTCD1. Luckily the mouse is alive, meaning that the gene is not essential.. We can then study its physiological and behavioral characteristics. To our surprise, the mouse shows no obvious abnormalities. However, looking back at the presumed function of RTCD1 in bacteria, we reasoned that the effects of not having RTCD1 could be uncovered only in conditions of stress or even in viral infections where RTCD1 might modify viral RNAs. The hypothesis seems to be right: in comparison to the normal animal, the knock-out mouse cannot develop an innate immune response against the virus, which driven by the molecule interferon. In the next three years we will try to understand how RTCD1 helps the host cell in recognizing the virus.

Genetic information resides in the deoxiribonucleic acid (DNA) but speaks by being copied or transcribed into different types of ribonucleic acid (RNA). To become functional in the cell, RNAs undergo processing, such as chemical modifications, cleavages, ligations, extensions and controlled degradation by a group of so-called RNA processing factors. The P28445 Project focused on the in vitro and in vivo characterization of the RNA 3- Terminal Phosphate Cyclase RTCD1 also known as RtcA in bacteria an enzyme that converts RNA 3-terminal phosphates (3-P) into 2,3-cyclic phosphates (2,3-cycP) and whose cellular function remains elusive. Beyond RTCD1, it is worth noting that the biochemistry and dynamics of 3-P and 2,3-cycP ended RNAs (in contrast to 3-OH ended RNAs) is still enigmatic. As usual in science, what we planned three years ago took several turns; some goals were achieved while others did not yet materialize or were abandoned for being based on wrong hypotheses. Strikingly, two stories emerged: First, we dissected the interplay between RTCD1 and the tRNA ligase complex (tRNA-LC) with a manuscript in preparation and second, we preliminarily identified the first 2,3-cyclic phosphatase activity in human cells, whose biochemistry and role in vivo will be fully developed in a forthcoming grant which is currently under review. In addition, we have generated the tools, and are close to identifying RNA targets of RTCD1 by sequencing and comparing the RNA of wild type (WT) vs. RTCD1-depleted cells. Those targets might guide us towards specific phenotypes in RTCD1 knockout (KO) mice.