Yeast pentatricopeptide protein Dmr1 (Ccm1) binds a repetitive AU-rich motif in the small subunit mitochondrial ribosomal RNA

Abstract

PPR proteins are a diverse family of RNA binding factors found in all Eukaryotic lineages. They perform multiple functions in the expression of organellar genes, mostly on the post-transcriptional level. PPR proteins are also significant determinants of evolutionary nucleo-organellar compatibility. Plant PPR proteins recognize their RNA substrates using a simple modular code. No target sequences recognized by animal or yeast PPR proteins were identified prior to the present study, making it impossible to assess whether this plant PPR code is conserved in other organisms. Dmr1p (Ccm1p, Ygr150cp) is a S. cerevisiae PPR protein essential for mitochondrial gene expression and involved in the stability of 15S ribosomal RNA. We demonstrate that in vitro Dmr1p specifically binds a motif composed of multiple AUA repeats occurring twice in the 15S rRNA sequence as the minimal 14 nt (AUA) 4 AU or longer (AUA) 7 variant. Short RNA fragments containing this motif are protected by Dmr1p from exoribonucleolytic activity in vitro. Presence of the identified motif in mtDNA of different yeast species correlates with the compatibility between their Dmr1p orthologs and S. cerevisiae mtDNA. RNA recognition by Dmr1p is likely based on a rudimentary form of a PPR code specifying U at every third position, and depends on other factors, like RNA structure.

RNA processing and degradation mechanisms shaping the mitochondrial transcriptome of budding yeasts

“…123 Similarly, compatibility between Dmr1p/Ccm1p and its recognized motif in the 15S rRNA drives speciation in the genus Saccharomyces. 80,124 The great evolutionary diversity of the mitochondrial genomes can limit the application of knowledge gained from model systems such as yeasts to other more distant lineages, including humans. Nevertheless, there are multiple examples when yeast genetics not only helped to understand fundamental mechanisms of the functioning of human mitochondrial genetic system, but even provided direct models of human diseases.…”

“…paradoxus 123 . Similarly, compatibility between Dmr1p/Ccm1p and its recognized motif in the 15S rRNA drives speciation in the genus Saccharomyces 80,124 …”

“…The precursor, transcribed together with tRNA Trp (tW[UCA]Q) and long spacer sequences, is bound by yet another PPR protein, encoded by the DMR1/CCM1 gene, at two recognition sites containing a repetitive 14 nt motif (AUA) 4 AU. 80 Binding of Dmr1p/Ccm1p is required for the correct maturation of the precursor and protects the RNA from the mitochondrial exoribonucleases involved in RNA degradation and quality control. 81,82 The tight connection between RNA processing and translation is not only functional, but also reflected in the physical interactions of the relevant proteins within a large ribosome-associated assemblies known as MIOREX.…”

“…An interplay between RNA processing and stability mediated by a specific protein was also described in the synthesis of small ribosomal subunit RNA (15S rRNA, SSU ). The precursor, transcribed together with tRNA Trp (tW[UCA]Q) and long spacer sequences, is bound by yet another PPR protein, encoded by the DMR1/CCM1 gene, at two recognition sites containing a repetitive 14 nt motif (AUA) 4 AU 80 . Binding of Dmr1p/Ccm1p is required for the correct maturation of the precursor and protects the RNA from the mitochondrial exoribonucleases involved in RNA degradation and quality control 81,82 …”