The non-LTR retrotransposons in Ciona intestinalis: new insights into the evolution of chordate genomes

BACKGROUND: Non-long terminal repeat (non-LTR) retrotransposons have contributed to shaping the structure and function of genomes. In silico and experimental approaches have been used to identify the non-LTR elements of the urochordate Ciona intestinalis. Knowledge of the types and abundance of non-LTR elements in urochordates is a key step in understanding their contribution to the structure and function of vertebrate genomes. RESULTS: Consensus elements phylogenetically related to the I, LINE1, LINE2, LOA and R2 elements of the 14 eukaryotic non-LTR clades are described from C. intestinalis. The ascidian elements showed conservation of both the reverse transcriptase coding sequence and the overall structural organization seen in each clade. The apurinic/apyrimidinic endonuclease and nucleic-acid-binding domains encoded upstream of the reverse transcriptase, and the RNase H and the restriction enzyme-like endonuclease motifs encoded downstream of the reverse transcriptase were identified in the corresponding Ciona families. CONCLUSIONS: The genome of C. intestinalis harbors representatives of at least five clades of non-LTR retrotransposons. The copy number per haploid genome of each element is low, less than 100, far below the values reported for vertebrate counterparts but within the range for protostomes. Genomic and sequence analysis shows that the ascidian non-LTR elements are unmethylated and flanked by genomic segments with a gene density lower than average for the genome. The analysis provides valuable data for understanding the evolution of early chordate genomes and enlarges the view on the distribution of the non-LTR retrotransposons in eukaryotes

CERKL, a retinal disease gene,encodes an mRNA-biding protein that localizes in compact and unstranslated mRNPs associated to microtubules.

The function of CERKL (CERamide Kinase Like), a causative gene of retinitis pigmentosa and cone-rod dystrophy, still awaits characterization. To approach its cellular role we have investigated the subcellular localization and interaction partners of the full length CERKL isoform, CERKLa of 532 amino acids, in different cell lines, including a photoreceptor-derived cell line. We demonstrate that CERKLa is a main component of compact and untranslated mRNPs and that associates with other RNP complexes such as stress granules, P-bodies and polysomes. CERKLa is a protein that binds through its N-terminus to mRNAs and interacts with other mRNA-binding proteins like eIF3B, PABP, HSP70 and RPS3. Except for eIF3B, these interactions depend on the integrity of mRNAs but not of ribosomes. Interestingly, the C125W CERKLa pathological mutant does not interact with eIF3B and is absent from these complexes. Compact mRNPs containing CERKLa also associate with microtubules and are found in neurites of neural differentiated cells. These localizations had not been reported previously for any member of the retinal disorders gene family and should be considered when investigating the pathogenic mechanisms and therapeutical approaches in these diseases

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

BACKGROUND: Annotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms. RESULTS: While characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs. CONCLUSIONS: From protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER

Distilling a visual network of Retinitis Pigmentosa gene-protein interactions to uncover new disease candidates

BACKGROUND: Retinitis pigmentosa (RP) is a highly heterogeneous genetic visual disorder with more than 70 known causative genes, some of them shared with other non-syndromic retinal dystrophies (e.g. Leber congenital amaurosis, LCA). The identification of RP genes has increased steadily during the last decade, and the 30% of the cases that still remain unassigned will soon decrease after the advent of exome/genome sequencing. A considerable amount of genetic and functional data on single RD genes and mutations has been gathered, but a comprehensive view of the RP genes and their interacting partners is still very fragmentary. This is the main gap that needs to be filled in order to understand how mutations relate to progressive blinding disorders and devise effective therapies. METHODOLOGY: We have built an RP-specific network (RPGeNet) by merging data from different sources: high-throughput data from BioGRID and STRING databases, manually curated data for interactions retrieved from iHOP, as well as interactions filtered out by syntactical parsing from up-to-date abstracts and full-text papers related to the RP research field. The paths emerging when known RP genes were used as baits over the whole interactome have been analysed, and the minimal number of connections among the RP genes and their close neighbors were distilled in order to simplify the search space. CONCLUSIONS: In contrast to the analysis of single isolated genes, finding the networks linking disease genes renders powerful etiopathological insights. We here provide an interactive interface, RPGeNet, for the molecular biologist to explore the network centered on the non-syndromic and syndromic RP and LCA causative genes. By integrating tissue-specific expression levels and phenotypic data on top of that network, a more comprehensive biological view will highlight key molecular players of retinal degeneration and unveil new RP disease candidates

Scaling new heights in the genetic diagnosis of inherited retinal dystrophies

During the last 20 years, our group has focused on identifying the genes and mutations causative of inherited retinal dystrophies (IRDs). By applying massive sequencing approaches (NGS) in more than 500 familial and sporadic cases, we attained high diagnostic efficiency (85%) with a custom target gene panel and over 75% using whole exome sequencing (WES). Close to 40% of pathogenic alleles are novel mutations, which demand specific in silico tests and in vitro assays. Notably, missense variants are by far the most common type of mutation identified (around 40%), with small in-frame indels being less frequent (2%). To fill the gap of unsolved cases, when no candidate gene or only a single pathogenic allele has been identified, additional scientific and technical issues remain to be addressed. Reliable detection of genomic rearrangements and copy number variants (partial or complete), deep intronic mutations, variants that cause aberrant splicing events in retina-specific transcripts, functional assessment of hypomorphic missense alleles, mutations in regulatory sequences, the contribution of modifier genes to the IRD phenotype, and detection of low heteroplasmy mtDNA mutations are among the new challenges to be met