Ribosomal, mitochondrial and bacterial (Wolbachia) reference sequences for Dipetalonema gracile obtained from a wild pied tamarin (Saguinus bicolor) host in Manaus, Brazil / Sequências ribossômicas, mitocondriais e bacterianas (Wolbachia) de referência para Dipetalonema gracile obtidas de um hospedeiro sauim-de-coleira (Saguinus bicolor) em Manaus, Brasil

The primates that inhabit the rainforest surrounding the city of Manaus (Amazonas, Brazil) have long been recognised as potentially important reservoirs of emerging and re-emerging infectious diseases (ERIDs). PCR amplification of filarial sequences from wild-caught Simulium oyapockense has been used to incriminate potentially important Amazon-region ERID bridge vectors by showing they had previously fed on non-human primates. The broader use of filarial parasite sequences for the incrimination of biting insects as potentially important zoonotic disease vectors is limited by a paucity of primate-derived filarial parasite reference sequences which can be matched to the PCR amplified sequences obtained from insect-vector vectors. Here we have used shotgun sequencing to obtain reference data from an adult Dipetalonema gracile parasite which was found infecting a wild pied tamarin (Saguinus bicolor) in a peripheral region of Manaus. We report the parasite´s complete mitochondrial genome (which is 13,647 base pairs in length), 894,846 base pairs of its Wolbachia genome and 6,426 base pairs of its ribosomal DNA locus (spanning from the start of its 18S subunit to the end of its 28S subunit). Despite being critically endangered, S. bicolor is commonly encountered around the periphery of Manaus and in urban forest fragments. The reported sequences may be a useful reference tool for identifying ERID bridge vectors and potentially provide some insights into the amount and the nature of contact between primate pathogen reservoirs and the residents of Manaus.(AU)

Os primatas que habitam a floresta tropical ao redor da cidade de Manaus (Amazonas, Brasil) há muito são reconhecidos como reservatórios potencialmente importantes de doenças infecciosas emergentes e reemergentes. Sequências de DNA de parasitas filariais detectadas por PCR em amostras de Simulium oyapockense foram usadas para demonstrar que eles haviam se alimentado anteriormente de primatas não humanos e, dessa maneira, incriminar vetores-ponte da região amazônica. O uso mais amplo de detecção de parasitas filariais para a incriminação de vetores-ponte é limitado por uma escassez de sequências referência de parasitas filarias obtidas de hospedeiros. Aqui nós usamos o sequenciamento tipo shotgun para obter dados de referência de um parasita adulto Dipetalonema gracile encontrado infectando um sauim-de-coleira, Saguinus bicolor no entorno de Manaus. Relatamos o genoma mitocondrial completo do parasita (que tem 13.647 pares de bases de comprimento), 894.846 pares de bases de seu genoma de Wolbachia e 6.426 pares de bases de seu locus de DNA ribossômico (desde o início de sua subunidade 18S até o final de sua subunidade 28S). Apesar de criticamente ameaçado, S. bicolor é comumente encontrado no entorno de Manaus e em fragmentos florestais urbanos. As sequências relatadas podem ser uma ferramenta de referência útil para identificar vetores ponte e potencialmente fornecer algumas informações sobre o contato entre reservatórios de patógenos de primatas e os moradores de Manaus.(AU)

Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.

In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria-specific protein extensions at the C-terminus (C) or N-terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria-specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria-specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria-specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.

Mitochondrial ribosome bL34 mutants present diminished translation of cytochrome c oxidase subunits.

Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.

MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach.

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.