Antisense Transcripts Delimit Exonucleolytic Activity of the Mitochondrial 3′ Processome to Generate Guide RNAs

Abstract

SUMMARY Small non-coding RNA biogenesis typically involves cleavage of structured precursor by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5′ triphosphate characteristic of the transcription initiation and possess a U-tail indicative of 3′ processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase, DSS1 3′-5′ exonuclease and three additional subunits. This complex, termed mitochondrial 3′ processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature size of 40–60 nucleotides, and secondary U-tail addition. Both strands of the gRNA gene are transcribed into sense and antisense precursors of similar lengths. Head-to-head hybridization of these transcripts blocks symmetrical 3′-5′ degradation at a fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5′ extremity of a primary molecule by uridylation-induced, antisense transcription-controlled 3′-5′ exonucleolytic degradation.

RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting

“…Although the terminal deletions clearly reduced RET1's processivity, the mutations in the putative dsRNA binding groove exerted no effect on UMP additions to a single-stranded synthetic 30-mer. Indeed, current model of gRNA processing predicts that double-stranded RNA recognition is responsible for mitochondrial processome pausing at a fixed distance from the duplex formed by overlapping transcripts (16). This positions zinc finger as plausible sensor of impending dsRNA region rather than a significant ssRNA binding determinant.…”

“…This positions zinc finger as plausible sensor of impending dsRNA region rather than a significant ssRNA binding determinant. To test RET1 activity on a dsRNA that resembles a putative gRNA processing intermediate (double-stranded region with 10–13 nucleotide 3′ overhang (16)), we assembled respective substrates and analyzed reaction products in a high-resolution assay. RET1 enzymatic activity was virtually indistinguishable on ss- or dsRNA with an overhang, while fully dsRNA was unable to support UMP incorporation (Figure 3E).…”

“…Finally, sequencing of mRNA 3′ termini revealed that most mRNAs in the actively respiring insect (procyclic) developmental stage of the parasite are modified by addition of 200–300 nt-long A/U-heteropolymers (14), which indicated RET1's involvement in mRNA 3′ processing and translational activation (15). Subsequent work demonstrated that RET1 is not only required for 3′ uridylation on mature gRNAs, ribosomal RNAs and some mRNAs, but also participates in nucleolytic processing of precursors transcribed from maxicircle and minicircle genomes (16,17). In-depth investigation of mitochondrial high molecular mass TUTase-containing complexes revealed that RET1 is mostly sequestered into a stable particle along with DSS1 3′-5′ exonuclease and three proteins lacking annotated motifs (16).…”

“…Subsequent work demonstrated that RET1 is not only required for 3′ uridylation on mature gRNAs, ribosomal RNAs and some mRNAs, but also participates in nucleolytic processing of precursors transcribed from maxicircle and minicircle genomes (16,17). In-depth investigation of mitochondrial high molecular mass TUTase-containing complexes revealed that RET1 is mostly sequestered into a stable particle along with DSS1 3′-5′ exonuclease and three proteins lacking annotated motifs (16). This ∼900 kDa complex, termed the mitochondrial 3′ processome (MPsome), is responsible for recognition, uridylation and exonucleolytic processing of 800–1200 nt-long gRNA precursors, and U-tailing of mature gRNAs.…”

Investigating RNA editing factors from trypanosome mitochondria

“…Second, filtering out reads that align to nuclear genome is needed to remove contamination by nuclear-encoded transcripts. In addition, the heterogeneity of 5′ start sites, and 3′ ends generated by 3′–5′ exonucleolytic processing and subsequent uridylation needs to be accounted for [21]. For these reasons, the common K-mer based methods are not suited for the assembly of a small RNA reference.…”