Biology subjects

Dzadz, D. A.

Publications and source records attributed to Dzadz, D. A..

2 recordsLinked to original sources

bioRxiv2026

The trypanosome mRNA decapping enzyme ALPH1 prefers caps without m7G methylation and produces diphosphate-RNA

5'ends of eukaryotic mRNAs are protected by the m7G cap, connected to the mRNA via a three-phosphate-bridge. In mRNA decay, the pyrophosphate bond between the and {beta} phosphate is cleaved by the nudix hydrolase DCP2. Uniquely among eukaryotes, Kinetoplastida lack DCP2 and instead employ the ApaH-like phosphatase ALPH1 for mRNA decapping. ALPH1 consists of an unstructured N-terminus, a catalytic domain and a structured C-terminus that mediates ALPH1 dimerisation. Here, we have analysed Trypanosoma brucei ALPH1 in greater detail. We find that the enzyme has broad substrate specificity and accepts different cap types and even cap analogues. Strikingly, cap-analogues and RNAs without the m7G-methyl group are turned over significantly faster than m7G methylated substrates. Moreover, all methylated and non-methylated cap analogues tested, with at least one additional nucleotide 3' to the NpppN moiety are cleaved at the {beta}-{gamma} pyrophosphate bond, producing the equivalent to a 5 diphosphate-RNA. While the presence of the ALPH1 C-terminal domain is essential for cell viability and increases enzyme activity in vitro, substrate preferences are determined solely by the catalytic domain. Altogether, these ALPH1 enzymatic properties exhibit intriguing differences to the canonical eukaryotic decapping enzyme DCP2, which we critically discuss and which potentially have biotechnological applications.

molecular biology↗

bioRxiv2024

Human FASTK preferentially binds single-stranded and G-rich RNA

Fas-activated serine/threonine kinase (FASTK) is the founding member of the FASTKD protein family, which was shown to regulate the fate of mRNA molecules on multiple levels. The mitochondrial variant of FASTK co-localizes with mitochondrial RNA granules and regulates degradation of mitochondrial mRNAs, whereas the cytoplasmic and nuclear forms of FAST are involved in regulation of alternative splicing, cytoplasmic RNA granule formation and mRNA translation. Despite these multiple roles of FASTK in mRNA biology, the exact rules of RNA recognition by this protein remained undetermined. Here, we demonstrate direct RNA binding by purified human FASTK and show its preference for single-stranded G-rich sites and RNA G-quadruplexes. Addition of FASTK alone was sufficient to achieve protection of mitochondrial mRNAs from degradation by the degradosome. Structural characterization by SAXS showed that FASTK in solution is a monomer with an extended conformation. Point mutagenesis studies supported the structural predictions of an exposed RNA-binding interface in the central helical region, preceded by a smaller, flexibly attached, helical N-terminal domain. We provide the first such extensive in vitro characterization of the RNA binding properties for a representative of the FASTKD protein family, and suggest how these intrinsic properties may underly FASTK function in mRNA metabolism.

molecular biology↗