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Structural basis of the mitochondrial RNA editing cascade in trypanosomes

ABSTRACT: The discovery of RNA editing in trypanosomes introduced the concept of guide RNA (gRNA). To understand how the RECC1 and RECC2 complexes mediate gRNA-directed uridine deletions and insertions, respectively, in mitochondrial mRNAs, we determined their structures and RNA interactions. The RECC cores, consisting of distinct heterotetramers of active and inactive RNase III endonucleases, recognize gRNA–mRNA duplexes. Auxiliary zinc-finger domains distinguish deletion from insertion editing sites, while asymmetrical catalytic centers cleave the mRNA. Three peripheral heterotetramers composed of oligonucleotide-binding (OB-fold) proteins flexibly tether exonuclease, TUTase, and ligase enzymes to the cores, facilitating trimming or extension of cleaved mRNA and strand repair. An architectural tRNA stabilizes the RNase III–OB-fold interface. These findings illuminate the organization of the enzymatic cascade and suggest a mechanism for editing site recognition.

INSTRUMENT(S):

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Clinton Yu

LAB HEAD: Ruslan Afasizhev

PROVIDER: PXD055091 | Pride | 2026-06-15

REPOSITORIES: Pride

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			Action	DRS
	LUMOS_RA_MS2s.xlsx	Xlsx
	RA_ObitrapLUMOS_20230404_11.raw	Raw
	RA_ObitrapLUMOS_20230404_13.raw	Raw
	RA_ObitrapLUMOS_20230404_15.raw	Raw
	RA_ObitrapLUMOS_20240116_03.raw	Raw

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Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5â² triphosphate characteristic of transcription start site and possess U-tail indicative of 3â² processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3â²-5â² DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3â² processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3â²-5â² degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5â² extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation. 1. we first sequenced guide RNA precursor (Gel-fractioned total cellular RNA 600-1500nt was used) transcripts from three replicates to study the their tail features and also validate observation of sense/antisense accumulation upon perturbation of pre-processing complex based on few cases. 2. we then sequenced mitochondrial small RNA and built a reference for small RNAs using our custom algorithm. We then took the mitochondrial small RNA data and uncovered the sense/antisense pair. 3. We then usedÂ CLIP-Seq data toÂ investigateÂ in vivo binding sitesÂ and, together with RNA IP-Seq data to understand what determine the relative abundance of sense and antisense pair of the duplex. 4. We used sequenced small mitochondrial RNA in different RNAi experiments (For RNAi experiments, 30-70nt RNA fraction was gel-isolated from total cellular RNAs) to understand which protein will affect the Utail length in mature small mitochondrial RNA.

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Structural basis of the mitochondrial RNA editing cascade in trypanosomes

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