The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3′-UTRs of yeast mitochondrial mRNAs

Abstract

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5′ capping and 3′ polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3′-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3′-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.

Long-read direct RNA sequencing of the mitochondrial transcriptome ofSaccharomyces cerevisiaereveals condition-dependent intron turnover

“…Potential target proteins may be identified by comparing the differential expression of RNA binding proteins observed between glucose-and ethanolgrown cultures (43,106,107). From this comparison, YBR238C, mitochondrial paralog of Rmd9p that stabilizes mitochondrial RNA at their dodecamer sequence (67,68), was 1.8-times more abundant in glucose-than ethanol-grown cultures (Figure S 14). MtRNA binding protein levels between ethanol and glucose were also compared relative to Cox1p expression, as this gives insight in the different levels of protein required between ethanol and glucose to mature a similar level of COX1 mRNA.…”

Principles of mitoribosomal small subunit assembly in eukaryotes

“…23 – 26 ). LC–MS/MS analysis of both yeast complexes further revealed the presence of co-purified mtSSU assembly factors Mtg3 (homologous to human NOA1) and Rmd9, a protein so far only implicated in the protection of 3′ ends of mitochondrial mRNAs 34 , 35 (Supplementary Data 2 and 3 and Supplementary Fig. 20 ).…”

“…The 3′ rRNA extension in yeast contains a sequence (5′-AAUAUUCUU-3′) that partially matches a motif that is sequence specifically recognized by Rmd9 (ref. 35 ). These data suggest that Rmd9 may be involved in yeast 15S rRNA 3′ end maturation in an early and likely conformationally dynamic assembly intermediate.…”

How RNases Shape Mitochondrial Transcriptomes

“…In yeast, most mRNAs terminate with a dodecamer sequence element. The PPR protein Rmd9 was found to bind this element and stabilize mRNAs [ 118 ]. This stabilization occurs by blocking the action of mtEXO at least in vitro.…”