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

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.

Transcriptome maps of general eukaryotic RNA degradation factors.

RNA degradation pathways enable RNA processing, the regulation of RNA levels, and the surveillance of aberrant or poorly functional RNAs in cells. Here we provide transcriptome-wide RNA-binding profiles of 30 general RNA degradation factors in the yeast Saccharomyces cerevisiae. The profiles reveal the distribution of degradation factors between different RNA classes. They are consistent with the canonical degradation pathway for closed-loop forming mRNAs after deadenylation. Modeling based on mRNA half-lives suggests that most degradation factors bind intact mRNAs, whereas decapping factors are recruited only for mRNA degradation, consistent with decapping being a rate-limiting step. Decapping factors preferentially bind mRNAs with non-optimal codons, consistent with rapid degradation of inefficiently translated mRNAs. Global analysis suggests that the nuclear surveillance machinery, including the complexes Nrd1/Nab3 and TRAMP4, targets aberrant nuclear RNAs and processes snoRNAs.

Yeast mitochondrial protein Pet111p binds directly to two distinct targets in COX2 mRNA, suggesting a mechanism of translational activation.

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5'-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.

Architecture and RNA binding of the human negative elongation factor.

Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.