Transcription initiation defines kinetoplast RNA boundaries

Abstract

Mitochondrial genomes are often transcribed into polycistronic RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that in Trypanosoma brucei the monophosphorylated 5′ ends of functional molecules typify precursor partitioning by an unknown endonuclease. On the contrary, we demonstrate that individual mRNAs and rRNAs are independently synthesized as 3′-extended precursors. The transcription-defined 5′ terminus is converted into a monophosphorylated state by the pyrophosphohydrolase complex, termed the “PPsome.” Composed of the MERS1 NUDIX enzyme, the MERS2 pentatricopeptide repeat RNA-binding subunit, and MERS3 polypeptide, the PPsome binds to specific sequences near mRNA 5′ termini. Most guide RNAs lack PPsome-recognition sites and remain triphosphorylated. The RNA-editing substrate-binding complex stimulates MERS1 pyrophosphohydrolase activity and enables an interaction between the PPsome and the polyadenylation machinery. We provide evidence that both 5′ pyrophosphate removal and 3′ adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3′–5′ exonucleolytic trimming defines the mRNA 3′ end before adenylation. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.

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

“…and Serganov 2015). This would be consistent with analyses showing that mutation of MERS1 residues Glu257 and Glu258 to alanines impairs RNA processing in vivo (Sement et al 2018). However, studies have shown that it is challenging to definitively ascribe a residue as the catalytic base in Nudix hydrolases as substitution of many active site residues can either impair or abrogate binding (Piton et al 2013;Vasilyev and Serganov 2015).…”

“…Data indicate that MERS1 forms a complex with the MERS2 and MER3 proteins and that these interactions activate MERS1 activity (Sement et al 2018). The 946 residue MERS2 protein contains several pentatricopeptide (PPR) motifs, which are interaction motifs that may be involved in binding MERS1 and/or RNA.…”

“…Although kinetoplastid mitochondrial mRNAs are not flanked by tRNAs, it was thought that these transcripts were produced by a similar route as animal and fungal mitochondrial mRNAs. However, recent work in the Aphasizhev laboratory revealed the finding that these transcripts in T. brucei are, in fact, individually synthesized and subsequently processed at their 5 ′ and 3 ′ ends to form mature mRNAs (Sement et al 2018;Mesitov et al 2019). The 5 ′ ends are processed by a pyrophosphohydrolase complex termed the PPsome, which is composed of the so-called mitochondrial edited mRNA stability factors 1-3 (MERS1-MERS3).…”

“…MERS1 is a 395 residue protein, MERS2, a 946 residue peptatricopeptide repeat polypeptide and MERS3, a 189 amino acid protein with no known motifs (Sement et al 2018). These three proteins also interface with kRNA editing and RNA processing complexes including the RNA-editing substrate-binding complex (RESC) and the kinteoplastid polyadenylation complex (KPAC) (Aphasizhev and Aphasizheva 2011;Ammerman et al 2013;Aphasizheva et al 2014;Suematsu et al 2016;Sement et al 2018;Mesitov et al 2019). MERS1 was shown to be the factor responsible for catalyzing the 5 ′ processing event while MERS2 and MERS3 activates its catalysis (Sement et al 2018).…”

“…Moreover, outside its putative Nudix motif, the protein shows no sequence homology with any structurally characterized protein. Studies support that MERS1 binds G-rich RNA sites; crosslinking and immunoprecipitation (CLIP) and crosslinking and affinity purification (CLAP) experiments uncovered a consensus motif of 5 ′ -GAGAGGGG GGUUU-3 ′ that was associated with MERS1 and MERS2 (Sement et al 2018). However, direct RNA binding by MERS1 has not been assessed.…”