Biochemical Characterization of TbALPH1: The Major mRNA Decapping Enzyme in \(Trypanosoma\) \(brucei\)

Kinetoplastida mRNAs contain a unique and highly methylated cap. The first four transcribed nucleotides (AACU) ribose 2’-O and the first (m62A) and fourth (m3U) nucleotides of the miniexon sequence are methylated. In most eukaryotic cells, removal of the mRNA cap is largely accomplished by the Nudix domain protein Dcp2 in a multiprotein complex consisting of specific regulators commonly known as decapping activators or enhancers. In contrast to most eukaryotic organisms, Kinetoplastida lack Dcp2 and its specific regulators, so they rely on a non-Nudix domain protein of bacterial origin, ApaH-like phosphatase, as the main decapping enzyme. ApaH-like phosphatases are widely distributed throughout the eukaryotic kingdom, but only two have been functionally characterized. In S. cerevisiae, ALPH functions as an endopolyphosphatase that cleaves vacuolar poly(P), while in the procyclic stage of Trypanosome brucei, ALPH functions as the major mRNA decapping enzyme. The substrates of eukaryotic ApaH-like phosphatases are not known. To determine the enzyme substrate requirements and physicochemical in vitro conditions of the trypanosome decapping enzyme, ApaH-like phosphatase, TbALPH1 capped and -uncapped synthetic RNA oligos and cap structures were used as substrates. The in vitro physicochemical conditions of TbALPH1 have shown that TbALPH1ΔN exhibits metal-dependent phosphatase and decapping activities. Mn2+ supports the phosphatase activities, while Ni2+, Ca2+, Mg2+ and Fe2+ support the decapping activities with high efficiency. The effect of pH on TbALPH1ΔN showed that the decapping activity is not dramatically affected between pH 2.0 -9.0, but the maximum decapping activity can be achieved between pH 6-8. The temperature requirements are between the physiological temperatures of vertebrates and mammals (27°C and 37°C) and mutational analyses have shown that catalytic domain is sufficient for in vitro decapping activities We have also shown that the substrate requirements for in vitro decapping activities are not restricted to the type of cap, sequence or length of an RNA oligo. Furthermore, through metabolomic analyses and a competition assay, we have shown that TbALPH1ΔN also exhibits dinucleoside polyphosphate activities in vitro when different methylated and non-methylated RNA cap structures are used as substrate. While all tested cap structures can compete with a capped RNA oligo, we confirmed in the metabolome analysis that TbALPH1ΔN can hydrolyse RNA cap structures yielding different cap moieties. In contrast, using a capped RNA oligo, TbALPH1ΔN consistently yielded an RNA with a 5’-diphosphate end as product, regardless of cap type and sequence length. In vitro 5'-3' exonuclease activities using a purified C-terminally GFP-tagged TbXRNA from the insect stage of T. brucei and the commercial yeast XRN1 showed that the GFP-TbXRNA did not exhibit 5'-3' exonuclease activity towards a non-methylated RNA with a 5’-monophosphate end, but a coordinated 5'-3' exonuclease activity towards a non-methylated RNA with a 5’-diphosphate end was only observed in the presence of TbALPH1ΔN. In contrast to GFP-TbXRNA, XRN1 showed activity towards the non-methylated RNA with a 5’-monophosphate end, but 5'-3' exonuclease activity towards a non-methylated RNA was only observed in the presence of a non-methylated RNA with a 5’-monophosphate end. In addition, coordinated 5'-3' exonuclease activity was observed in the presence of a capped RNA oligo and TbALPH1ΔN. The RNA binding activity of TbALPH1ΔN was analysed in a competition assay as well as in a mobility assay. While a non-capped RNA oligo competes with a capped RNA oligo in a concentration-dependent manner, in the mobility assay we observed a direct RNA binding activity that was solely dependent on an active TbALPH1ΔN and the presence of a capped RNA oligo. Finally, we have shown that although eukaryotic ALPHs retain their evolutionary decapping activity in vitro regardless of their localisation prediction, protein localisation is one of the main factors that precludes the differential activities of eukaryotic ALPHs in vivo In this study we have shown that using copolymerized acryloylaminophenylboronic acid (APB) in standard denaturing polyacrylamide gels, we were able to determine the physicochemical properties, substrate requirements and RNA binding activities of TbALPH1ΔN in vitro and conclude that non-cytoplasmic ALPHs proteins retain their evolutionary decapping activities in vitro.