Repeats In Non-coding Regions Research Articles

Mitogenomics reveals phylogenetic relationships of Arcoida (Mollusca, Bivalvia) and multiple independent expansions and contractions in mitochondrial genome size

Arcoida, comprising about 570 species of blood cockles, is an ecologically and economically important lineage of bivalve molluscs. Current classification of arcoids is largely based on morphology, which shows widespread homoplasy. Despite two recent studies employing multi-locus analyses with broad sampling of Arcoida, evolutionary relationships among major lineages remain controversial. Interestingly, mitochondrial genomes of several ark shell species are 2–3 times larger than those found in most bilaterians, and are among the largest bilaterian mitochondrial genomes reported to date. These results highlight the need of detailed phylogenetic study to explore evolutionary relationships within Arcoida so that the evolution of mitochondrial genome size can be understood. To this end, we sequenced 17 mitochondrial genomes and compared them with publicly available data, including those from other lineages of Arcoida with emphasis on the subclade Arcoidea species. Our phylogenetic analyses indicate that Noetiidae, Cucullaeidae and Glycymerididae are nested within a polyphyletic Arcidae. Moreover, we find multiple independent expansions and potential contractions of mitochondrial genome size, suggesting that the large mitochondrial genome is not a shared ancestral feature in Arcoida. We also examined tandem repeats and inverted repeats in non-coding regions and investigated the presence of such repeats with relation to genome size variation. Our results suggest that tandem repeats might facilitate intraspecific mitochondrial genome size variation, and that inverted repeats, which could be derived from transposons, might be responsible for mitochondrial genome expansions and contractions. We show that mitochondrial genome size in Arcoida is more dynamic than previously understood and provide insights into evolution of mitochondrial genome size variation in metazoans.

Intrinsic Disorder in Proteins with Pathogenic Repeat Expansions.

Intrinsically disordered proteins and proteins with intrinsically disordered regions have been shown to be highly prevalent in disease. Furthermore, disease-causing expansions of the regions containing tandem amino acid repeats often push repetitive proteins towards formation of irreversible aggregates. In fact, in disease-relevant proteins, the increased repeat length often positively correlates with the increased aggregation efficiency and the increased disease severity and penetrance, being negatively correlated with the age of disease onset. The major categories of repeat extensions involved in disease include poly-glutamine and poly-alanine homorepeats, which are often times located in the intrinsically disordered regions, as well as repeats in non-coding regions of genes typically encoding proteins with ordered structures. Repeats in such non-coding regions of genes can be expressed at the mRNA level. Although they can affect the expression levels of encoded proteins, they are not translated as parts of an affected protein and have no effect on its structure. However, in some cases, the repetitive mRNAs can be translated in a non-canonical manner, generating highly repetitive peptides of different length and amino acid composition. The repeat extension-caused aggregation of a repetitive protein may represent a pivotal step for its transformation into a proteotoxic entity that can lead to pathology. The goals of this article are to systematically analyze molecular mechanisms of the proteinopathies caused by the poly-glutamine and poly-alanine homorepeat expansion, as well as by the polypeptides generated as a result of the microsatellite expansions in non-coding gene regions and to examine the related proteins. We also present results of the analysis of the prevalence and functional roles of intrinsic disorder in proteins associated with pathological repeat expansions.

Therapeutic Approaches for Dominant Muscle Diseases: Highlight on Myotonic Dystrophy.

Myotonic Dystrophy (DM), one of the most common neuromuscular disorders in adults, comprises two genetically distinct forms triggered by unstable expanded repeats in non-coding regions. The most common DM1 is caused by expanded CTG repeats in the 3'UTR of the DMPK gene, whereas DM2 is due to large expanded CCTG repeats in the first intron of the CNBP gene. Both mutations induce a pathogenic RNA gain-of-function mechanism. Mutant RNAs containing CUG or CCUG expanded repeats, which are retained in the nuclei as aggregates alter activities of alternative splicing regulators such as MBNL proteins and CELF1. As a consequence, alternative splicing misregulations of several pre-mRNAs are associated with DM clinical symptoms. Currently, there is no available cure for this dominant neuromuscular disease. Nevertheless, promising therapeutic strategies have been developed in the last decade. Preclinical progress in DM research prompted the first DM1 clinical trial based on antisense oligonucleotides promoting a RNase-H-mediated degradation of the expanded CUG transcripts. The ongoing Phase 1/2a clinical trial will hopefully give further insights into the quest to find a bona fide cure for DM1. In this review, we will provide an overview of the different strategies that were developed to neutralize the RNA toxicity in DM1. Different approaches including antisense oligonucleotide technologies, gene therapies or small molecules have been tested and validated in cellular and animal models. Remaining challenges and additional avenues to explore will be discussed.

Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans.

Myotonic dystrophy disorders are caused by expanded CUG repeats in non-coding regions. To reveal mechanisms of CUG repeat pathogenesis we used C. elegans expressing CUG repeats to identify gene inactivations that modulate CUG repeat toxicity. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and modulate formation of nuclear RNA foci by CUG repeats. These genes regulated CUG repeat-induced toxicity through distinct mechanisms including RNA export and RNA clearance, suggesting that CUG repeat toxicity is mediated by multiple pathways. A subset is shared with other degenerative disorders. The nonsense-mediated mRNA decay (NMD) pathway plays a conserved role regulating CUG repeat RNA transcript levels and toxicity, and NMD recognition of toxic RNAs depends on 3′UTR GC nucleotide content. Our studies suggest a broader surveillance role for NMD where variations in this pathway influence multiple degenerative diseases.

A distant cis acting intronic element induces site-selective RNA editing

Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABAA receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome.

Algorithmic information theory may explain the pathogenic number of DNA repeats in myotonic dystrophy type 1 (and in similar diseases)

Myotonic Dystrophy Type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by an abnormal number of repeats of the trinucleotide sequence CTG outside of the protein-coding part of DNA -- more than fifty repeats can cause disease. Several other diseases are caused by similar repeats in non-coding regions; in most of these diseases, up to approximately fifty repeats is normal, while pathogenic cases usually have > 50 repetitions. The fact that this level of approximately fifty repeats can be seen in many diseases, with different repeating sequences, indicates that there may be a fundamental explanation for this threshold. In this paper, we conjecture that such an explanation may come from Algorithmic Information Theory. Crudely speaking, this threshold can be viewed as a measure of the redundancy in healthy DNA; this indirect estimate of the measure of redundancy is in good accordance with a more direct estimate { based on the fact that approximately 1=50 of DNA is directly functionally useful (e.g., protein-coding).

Stopping the stutter: Improvements in sequence quality from regions with mononucleotide repeats can increase the usefulness of non–coding regions for DNA barcoding

DNA barcoding is now well established in animals based on sequences from a 648 base pair (bp) portion of the mito chondrial coding gene cytochrome oxidase 1 (COl) (Hebert & al., 2003). In contrast, it has proved more difficult to identify a suitable DNA barcoding locus (or loci) for land plants (reviewed by Hollingsworth, 2008). The generally low substitution rate of plant mitochondrial DNA (e.g., Fazekas & al., 2008) has led to investigations into the relative performance and information content of different loci from the plastid genome as alternative DNA barcodes for plants (e.g., Chase & al., 2007; Kress & Er ickson, 2007; Fazekas & al., 2008; Lahaye & al., 2008; CBOL Plant Working Group, 2009; Ford & al., 2009; Hollingsworth & al, 2009). At the third International Barcode of Life Conference in Mexico City (November 2009), the Consortium for the Barcode of Life (CBOL) announced that the standard core DNA bar code for land plants would consist of a two locus combination comprising portions of the protein-coding plastid genes rbcL and matK, to be supplemented with additional loci as required. In making this recommendation, two potential problems were recognized. Firstly, successful amplification and sequencing of the matK barcoding region can be difficult in some taxa with existing primers, and further primer and protocol development is required for this locus. Secondly, the rbcL+matK barcode will not lead to 100% species discrimination in many plant groups, and additional loci beyond this core-barcode will be needed to increase levels of species discrimination in these cases. In recognition of these problems, an 18-month review period on the performance of the rbcL + matK barcode has been established (completion due in mid-2011). During this review period, the executive committee of CBOL recommended that the plant barcoding community continue to collect data from other strongly performing candidate barcoding loci such as the non-coding plastid intergenic spacer trnH-psbA and the internal transcribed spacers (ITS) of nuclear ribosomal DNA. One concern that has been raised regarding the use of non coding plastid regions such as trnH-psbA as DNA barcodes is the presence of microsatellite repeats which can make it dif ficult to obtain bi-directional sequences in some samples (Faze kas & al, 2008; CBOL Plant Working Group, 2009; Devey & al, 2009). Verification of the sequenced strand through bidi rectional sequencing is desirable for the maintenance of data quality standards, as recommended by the CBOL Database Working Group for sequences destined to receive annotation with the reserved keyword 'Barcode' by the INSDC (DDBJ, EMBL & GenBank). However, PCR amplicons derived from regions containing microsatellites can produce poor qual ity sequence chromatograms. Slippage of the polymerase at microsatellite regions during PCR results in a 'stutter' effect observed in the chromatogram, with decreasing sequence qual ity associated with increasing repeat length. The reduction in sequence quality from regions with mononucleotide runs with less than ten repeats is generally moderate and ambigu ous base calls by the software are usually few, requiring little editing by hand. However, as the repeat number increases, the number of ambiguous bases increases disproportionately. This results in a longer amount of time required for editing (with a corresponding reduction in confidence of the true sequence) to the point where sequence data cannot be used at all past the repeat. The net effect is often a contig with overlapping data only at the repeat, and a shortened read length due to missing data at the ends (Fig. 1). In more extreme cases, where mul tiple mononucleotide repeats occur within a given amplicon, disruption of forward and reverse sequencing reads at differ ent mononucleotide runs can lead to only partial sequences of the region with no overlap, thus preventing the construction of a sequence contig. The problem caused by mononucleotide repeats in non-coding regions was noted by the CBOL Plant Working Group (2009) when evaluating the attributes of differ ent barcoding loci for plants, and this contributed towards the selection of an entirely coding core plant barcode. Fixing the 'mononucleotide repeat problem' was considered less tractable than the challenge of improving primer universality for matK. Recently, however, an evaluation of PCR methods and DNA polymerases, focusing on the reduction of the stutter ef fects resulting from mononucleotide repeats, has demonstrated that the use of particular polymerases can reduce the effect of slipped strand mispairing in PCR (Fazekas & al., 2010). This recent study tested the performance of different PCR profiles, reaction chemistries and polymerases on sequences from the trnH-psbA spacer from 25 plant samples. These samples were specifically selected because they contain mononucleotide re peats which had previously resulted in ambiguous base calls and low-quality sequence traces with conventional PCR and

Comparative Genomic Analysis of Three Strains of Ehrlichia ruminantium Reveals an Active Process of Genome Size Plasticity

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.

Cloning and characterization of myogenic regulatory genes in three Ictalurid species

We report sequence, tissue expression and map-position data for myogenin, MYOD1, myostatin and follistatin in three Ictalurid catfish species: channel catfish (Ictalurus punctatus), blue catfish (I. furcatus) and white catfish (Ameiurus catus). These genes are involved in muscle growth and development in mammals and may play similar roles in catfish. Amino acid sequences were highly conserved among the three Ictalurid species (>95% identity), moderately conserved among catfish and zebrafish (approximately 80% identity), and less conserved among catfish and humans (approximately 40-60% identity) for all four genes. Gene structure (number of exons and introns and exon-intron boundaries) was conserved between catfish and other species for all genes. Myogenin and MYOD1 expression was limited to skeletal muscle in juvenile channel catfish, similar to expression patterns for these genes in other fish and mammalian species. Myostatin was expressed in a variety of tissues in juvenile channel catfish, a pattern common in other fish species but contrasting with data from mammals where myostatin is primarily expressed in skeletal muscle. Follistatin was expressed in juvenile catfish heart, testes and spleen. All four genes contained polymorphic microsatellite repeats in non-coding regions and linkage analysis based on inheritance of these microsatellite loci was used to place the genes on the channel catfish linkage map. Information provided in this study will be useful in further studies to determine the role these genes play in muscle growth and development in catfish.

Dominantly inherited ataxias

The autosomal dominant ataxias continue to bewilder us as the enormity of their genetic heterogeneity continues to unfold. The Human Genome Organization website now lists 22 such ataxias, not including dentatorubral-pallidoluysian atrophy. The early genetic discoveries in this field included several disorders caused by CAG repeat expansions within coding regions of the respective genes. More recent discoveries have included unstable expansions of nucleotide repeats in noncoding regions of genes as well as point mutations that have formed the basis of progressive dominant ataxias. This article summarizes the clinical and genetic features of the currently identified dominant ataxias.