Structures of MERS1, the 5′ processing enzyme of mitochondrial mRNAs in Trypanosoma brucei

Abstract

Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan, Trypanosoma brucei, are transcribed individually. Also unlike most mitochondrial mRNAs, the 5 ′ ′ ′ ′ ′ end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTPbound and RNA-bound T. brucei MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.

Mitochondrial RNA quality control in trypanosomes

“…A recent study demonstrated that individual mRNAs and rRNAs are synthesized as 3′ extended precursors, while the pyrophosphohydrolase complex, termed the PPsome, converts transcription‐defined 5′ terminus into a monophosphorylated state This complex of MERS1 NUDIX hydrolase and MERS2 PPR binds to specific sequences near mRNA 5′ termini, but not to gRNAs (Figure 3). Individual MERS1 is inactive, likely due to a missing glutamic acid in the active center (Sement et al, 2018), which may be provided in trans by MERS2 (Schumacher, Henderson, & Zeng, 2020). In any event, PPsome inactivation by MERS1 RNAi knockdown (Hashimi et al, 2009; Weng et al, 2008), or in vivo mutations of the two remaining catalytic glutamic acid residues (Sement et al, 2018), selectively destabilize mitochondrial mRNAs, but not gRNAs or rRNAs.…”

“…UV‐crosslinking‐sequencing suggests that MERS2 PPR factor enables the PPsome's in vivo binding specificity for degenerate G‐rich motifs near mRNA 5′ ends and an affinity for RNA substrate. It has been proposed that MERS2 also activates MERS1 hydrolase by inserting a glutamate‐bearing peptide near the missing acidic residue in the RNA binding pocket (Schumacher et al, 2020). Finally, the virtual absence of PPsome binding sites in minicircle‐encoded small RNAs is consistent with the triphosphorylated nature of gRNA 5′ termini (Blum et al, 1990).…”