Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.

SAGA is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering TBP to nucleate the pre-initiation complex on DNA, a pivotal event in protein gene expression. Here we present the structure of yeast SAGA with bound TBP where the core is resolved at 3.5 Å resolution (0.143 FSC). It elucidates the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer, deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP where steric hindrance represses binding of spurious DNA. With complementary biochemical analysis our structure points to a mechanism for TBP delivery and release from SAGA that requires TFIIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding specific TBP delivery onto gene promoters and the multiple roles played by SAGA in regulating gene expression. Main TextTranscription of protein-coding genes begins with the formation of a pre-initiation complex (PIC) composed of RNA polymerase II and several general transcription factors (TFs) 1,2 . PIC assembly is nucleated by loading the TATA-binding protein (TBP) onto promoter DNA 3,4 , a focal point for regulated gene expression 5 . Two multi-protein co-activator complexes, TFIID and SAGA can deliver TBP to the gene promoter 6,7 and are required for global gene expression in yeast 8,9 .The 1.6 MDa SAGA complex also stimulates transcription via its two chromatin-modifying enzymatic activities. It is composed of 19 subunits organized in four modules with distinct functions 10 : a histone acetyl transferase (HAT) module 11 , a histone deubiquitinase (DUB) module 12 , the 430 kDa Tra1 subunit which serves as a docking platform for transcriptional factors that recruit SAGA to activating DNA sequences upstream of the promoter 13 , and a 10-subunit central module that is physically connected to all other modules and is also responsible for recruiting TBP to SAGA 10,14 (Extended Data Fig. 1).

Mitochondrial DNA (mtDNA) mutations are an important cause of human disease for which there is no efficient treatment. Our aim was to determine whether the A8344G mitochondrial tRNA(Lys) mutation, which can cause the MERRF (myoclonic epilepsy with ragged-red fibers) syndrome, could be complemented by targeting tRNAs into mitochondria from the cytosol. Import of small RNAs into mitochondria has been demonstrated in many organisms, including protozoans, plants, fungi and animals. Although human mitochondria do not import tRNAs in vivo, we previously demonstrated that some yeast tRNA derivatives can be imported into isolated human mitochondria. We show here that yeast tRNALys derivatives expressed in immortalized human cells and in primary human fibroblasts are partially imported into mitochondria. Imported tRNAs are correctly aminoacylated and are able to participate in mitochondrial translation. In transmitochondrial cybrid cells and in patient-derived fibroblasts bearing the MERRF mutation, import of tRNALys is accompanied by a partial rescue of mitochondrial functions affected by the mutation such as mitochondrial translation, activity of respiratory complexes, electrochemical potential across the mitochondrial membrane and respiration rate. Import of a tRNALys with a mutation in the anticodon preventing recognition of the lysine codons does not lead to any rescue, whereas downregulation of the transgenic tRNAs by small interfering RNA (siRNA) transiently abolishes the functional rescue, showing that this rescue is due to the import. These findings prove for the first time the functionality of imported tRNAs in human mitochondria in vivo and highlight the potential for exploiting the RNA import pathway to treat patients with mtDNA diseases.

In vivo, human mitochondria import 5 S rRNA and do not import tRNAs from the cytoplasm. We demonstrated previously that isolated human mitochondria are able to internalize a yeast tRNA Lys in the presence of yeast soluble factors. Here, we describe an assay for specific uptake of 5 S rRNA by isolated human mitochondria and compare its requirements with the artificial tRNA import. The efficiency of 5 S rRNA uptake by isolated mitochondria was comparable with that found in vivo. The import was shown to depend on ATP and the transmembrane electrochemical potential and was directed by soluble proteins. Blocking the pre-protein import channel inhibited internalization of both 5 S rRNA and tRNA, which suggests this apparatus be involved in RNA uptake by the mitochondria. We show that human mitochondria can also selectively internalize several in vitro synthesized versions of yeast tRNA Lys as well as a transcript of the human mitochondrial tRNA Lys . Either yeast or human soluble proteins can direct this import, suggesting that human cells possess all factors needed for such an artificial translocation. On the other hand, the efficiency of import directed by yeast or human protein factors varies significantly, depending on the tRNA version. Similarly to the yeast system, tRNA Lys import into human mitochondria depended on aminoacylation and on the precursor of the mitochondrial lysyl-tRNA synthetase. 5 S rRNA import was also dependent upon soluble protein(s), which were distinct from the factors providing tRNA internalization.Mitochondria, although containing their own genome, import the vast majority of their macromolecular components from the cytoplasm. If the mechanisms of pre-protein import are well understood, the import of nuclear-coded RNAs into mitochondria was investigated to a much lesser extent. Targeting of RNA into mitochondria though not universal is widely spread among organisms (1-5). Mitochondrial import of transfer RNAs was found in plants, protists, some lower animals, and fungi. The number of imported tRNA species varies from one (in yeast) to the totality (in trypanosomatids), and tRNA import mechanisms seem to differ from one organism to another. We have shown previously that, in the yeast Saccharomyces cerevisiae, import of a single tRNA CUU Lys (further referred to as tRK1) 1 occurs via formation of a complex with the precursor form of the mitochondrial lysyl-tRNA synthetase (pre-MSK) and requires the intactness of pre-protein import apparatus (6 -8).In mammalians, no tRNA import has been reported, but several other RNAs are thought to be targeted into mitochondria. One of these is the RNA component of RNase MRP, a site-specific endoribonuclease supposed to be involved in primer RNA cleavage during replication of mitochondrial DNA and to be present in the organelle in a very low amount (9). It was hypothesized that the process of mitochondrial DNA replication requires a very low number of MRP RNA molecules per mitochondrial genome (10 -12). The presence of MRP RNA in the mitochondria was recent...

Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.

In the yeast Saccharomyces cerevisiae, one of the two cytoplasmic lysine tRNAs, tRNACUULys, is partially associated with the mitochondrial matrix. Mitochondrial import of this tRNA requires binding to the precursor of the mitochondrial lysyl-tRNA synthetase, pre-MSK, and aminoacylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNAmnmLys5s2UUU [where 5-[(methylamino)-methyl]-2-thiouridine is mnm5s2U] is exclusively localized in the cytoplasm. To study import determinants within the tRNACUULys molecule, we introduced a panel of replacements in the original sequences of the imported and nonimported lysine tRNAs that correspond to domains or individual residues that differ between these two isoacceptors. The mutant transcripts were tested for import, aminoacylation, and binding to pre-MSK. Import and aminoacylation efficiencies correlate well for the majority of mutant transcripts. However, some poorly aminoacylated transcripts were rather efficiently imported. Surprisingly, these transcripts retained binding capacity to pre-MSK. In fact, all imported transcripts retained pre-MSK binding capacity but nonimported versions did not, suggesting that this binding, rather than aminoacylation, is essential for import. Substitution of the anticodon arm of tRNACUULys with that of tRNAmnmLys5s2UUU abolished import without affecting aminoacylation. A version of tRNAmnmLys5s2UUU with an anticodon CUU was efficiently imported in vitro and was also found to be imported in vivo. This implies that the anticodon arm, especially position 34, is important for recognition by the import machinery. A nicked tRNACUULys transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNACUULys is imported as a folded molecule.

The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species.

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.