Tracing the evolution of tissue identity with microRNAs

Comparison of microRNA expression identified tissues present in the last common ancestor of Bilaterians and put evolution of microRNAs in the context of tissue evolution

CONREAL web server: identification and visualization of conserved transcription factor binding sites

The use of orthologous sequences and phylogenetic footprinting approaches have become popular for the recognition of conserved and potentially functional sequences. Several algorithms have been developed for the identification of conserved transcription factor binding sites (TFBSs), which are characterized by their relatively short and degenerative recognition sequences. The CONREAL (conserved regulatory elements anchored alignment) web server provides a versatile interface to CONREAL-, LAGAN-, BLASTZ- and AVID-based predictions of conserved TFBSs in orthologous promoters. Comparative analysis using different algorithms can be started by keyword without any prior sequence retrieval. The interface is available at

CASCAD: a database of annotated candidate single nucleotide polymorphisms associated with expressed sequences

BACKGROUND: With the recent progress made in large-scale genome sequencing projects a vast amount of novel data is becoming available. A comparative sequence analysis, exploiting sequence information from various resources, can be used to uncover hidden information, such as genetic variation. Although there are enormous amounts of SNPs for a wide variety of organisms submitted to NCBI dbSNP and annotated in most genome assembly viewers like Ensembl and the UCSC Genome Browser, these platforms do not easily allow for extensive annotation and incorporation of experimental data supporting the polymorphism. However, such information is very important for selecting the most promising and useful candidate polymorphisms for use in experimental setups. DESCRIPTION: The CASCAD database is designed for presentation and query of candidate SNPs that are retrieved by in silico mining of high-throughput sequencing data. Currently, the database provides collections of laboratory rat (Rattus norvegicus) and zebrafish (Danio rerio) candidate SNPs. The database stores detailed information about raw data supporting the candidate, extensive annotation and links to external databases (e.g. GenBank, Ensembl, UniGene, and LocusLink), verification information, and predictions of a potential effect for non-synonymous polymorphisms in coding regions. The CASCAD website allows search based on an arbitrary combination of 27 different parameters related to characteristics like candidate SNP quality, genomic localization, and sequence data source or strain. In addition, the database can be queried with any custom nucleotide sequences of interest. The interface is crosslinked to other public databases and tightly coupled with primer design and local genome assembly interfaces in order to facilitate experimental verification of candidates. CONCLUSIONS: The CASCAD database discloses detailed information on rat and zebrafish candidate SNPs, including the raw data underlying its discovery. An advanced web-based search interface allows universal access to the database content and allows various queries supporting many types of research utilizing single nucleotide polymorphisms

A search for reverse transcriptase-coding sequences reveals new non-LTR retrotransposons in the genome of Drosophila melanogaster

BACKGROUND: Non-long terminal repeat (non-LTR) retrotransposons are eukaryotic mobile genetic elements that transpose by reverse transcription of an RNA intermediate. We have performed a systematic search for sequences matching the characteristic reverse transcriptase domain of non-LTR retrotransposons in the sequenced regions of the Drosophila melanogaster genome. RESULTS: In addition to previously characterized BS, Doc, F, G, I and Jockey elements, we have identified new non-LTR retrotransposons: Waldo, You and JuanDm. Waldo elements are related to mosquito RTI elements. You to the Drosophila I factor, and JuanDm to mosquito Juan-A and Juan-C. Interestingly, all JuanDm elements are highly homogeneous in sequence, suggesting that they are recent components of the Drosophila genome. CONCLUSIONS: The genome of D. melanogaster contains at least ten families of non-site-specific non-LTR retrotransposons representing three distinct clades. Many of these families contain potentially active members. Fine evolutionary analyses must await the more accurate sequences that are expected in the next future

Expression patterns of intronic microRNAs in Caenorhabditis elegans

BACKGROUND: MicroRNAs (miRNA) are an abundant and ubiquitous class of small RNAs that play prominent roles in gene regulation. A significant fraction of miRNA genes reside in the introns of the host genes in the same orientation and are thought to be co-processed from the host gene mRNAs and thus depend on the host gene promoter for their expression. However, several lines of evidence for independent expression of intronic miRNAs exist in the literature but the extent of this independence remains unclear. RESULTS: We performed a systematic analysis of genomic regions surrounding intronic miRNAs in the nematode Caenorhabditis elegans and found that, in many cases, there are extended intronic sequences immediately upstream of the miRNAs that are well-conserved between the nematodes. We have generated transcriptional green fluorescent protein reporter fusions in transgenic C. elegans lines and demonstrated that, in all seven investigated cases, the conserved sequences show promoter properties and produce specific expression patterns that are different from the host gene expression patterns. The observed expression patterns are corroborated by the published small RNA sequencing data. CONCLUSIONS: Our analysis reveals that the number of intronic miRNAs that do not rely on their host genes for expression is substantially higher than previously appreciated. At least one-third of the same-strand intronic miRNAs in C. elegans posses their own promoters and, thus, could be transcribed independently from their host genes. These findings provide a new insight into the regulation of miRNA genes and will be useful for the analysis of interactions between miRNAs and their host genes.

Random Integration Transgenesis in a Free-Living Regenerative Flatworm Macrostomum lignano

Regeneration-capable flatworms are highly informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. Transgenesis is a powerful research tool for investigating gene function, but until recently, a transgenesis method was missing in flatworms, hampering their wider adoption in biomedical research. Here we describe a detailed protocol to create stable transgenic lines of the flatworm M. lignano using random integration of DNA constructs through microinjection into single-cell stage embryos

The regenerative flatworm Macrostomum lignano, a model organism with high experimental potential

Understanding the process of regeneration has been one of the longstanding scientific aims, from a fundamental biological perspective, as well as within the applied context of regenerative medicine. Because regeneration competence varies greatly between organisms, it is essential to investigate different experimental animals. The free-living marine flatworm Macrostomum lignano is a rising model organism for this type of research, and its power stems from a unique set of biological properties combined with amenability to experimental manipulation. The biological properties of interest include production of single-cell fertilized eggs, a transparent body, small size, short generation time, ease of culture, the presence of a pluripotent stem cell population, and a large regeneration competence. These features sparked the development of molecular tools and resources for this animal, including high-quality genome and transcriptome assemblies, gene knockdown, in situ hybridization, and transgenesis. Importantly, M. lignano is currently the only flatworm species for which transgenesis methods are established. This review summarizes biological features of M. lignano and recent technological advances towards experimentation with this animal. In addition, we discuss the experimental potential of this model organism for different research questions related to regeneration and stem cell biology

Evidence for post-transcriptional regulation of clustered microRNAs in Drosophila

BACKGROUND: MicroRNAs (miRNA) are short 21-23nt RNAs capable of inhibiting translation of complementary target messenger RNAs. Almost half of D. melanogaster miRNA genes are grouped in genomic clusters. RESULTS: The peculiarities of the expression of clustered miRNAs were studied using publicly available libraries of sequenced small RNAs from different Drosophila tissues. We have shown that although miRNAs from almost all clusters have similar tissue expression profiles (coordinated clusters), some clusters contain miRNAs with uncoordinated expression profiles. The predicted transcription start sites (TSSs) of such clusters are located upstream of the first miRNA, but no TSSs are found within the clusters. The expression profiles of miR and miR* sequences in uncoordinated clustered miRNAs do not correlate while their profiles from the coordinated clustered miRNAs are similar. CONCLUSIONS: The presence of exclusively upstream promoters in miRNA clusters containing uncoordinated miRNAs means that the clusters are transcribed as single transcription units. The difference of tissue expression profiles of uncoordinated miRNAs and the corresponding miRs* suggests a post-transcriptional regulation of their processing or stability. [KEYWORDS: Animals, Drosophila/ genetics, Gene Expression Profiling, MicroRNAs/ genetics/ metabolism, Multigene Family, Promoter Regions, Genetic, RNA Processing, Post-Transcriptional, Transcription Initiation Site]

Resilience to aging in the regeneration-capable flatworm Macrostomum lignano

Animals show a large variability of lifespan, ranging from short-lived as Caenorhabditis elegans to immortal as Hydra. A fascinating case is flatworms, in which reversal of aging by regeneration is proposed, yet conclusive evidence for this rejuvenation-by-regeneration hypothesis is lacking. We tested this hypothesis by inducing regeneration in the sexual free-living flatworm Macrostomum lignano. We studied survival, fertility, morphology, and gene expression as a function of age. Here, we report that after regeneration, genes expressed in the germline are upregulated at all ages, but no signs of rejuvenation are observed. Instead, the animal appears to be substantially longer lived than previously appreciated, and genes expressed in stem cells are upregulated with age, while germline genes are downregulated. Remarkably, several genes with known beneficial effects on lifespan when overexpressed in mice and C. elegans are naturally upregulated with age in M. lignano, suggesting that molecular mechanism for offsetting negative consequences of aging has evolved in this animal. We therefore propose that M. lignano represents a novel powerful model for molecular studies of aging attenuation, and the identified aging gene expression patterns provide a valuable resource for further exploration of anti-aging strategies

TIM29 is required for enhanced stem cell activity during regeneration in the flatworm Macrostomum lignano

TIM29 is a mitochondrial inner membrane protein that interacts with the protein import complex TIM22. TIM29 was shown to stabilize the TIM22 complex but its biological function remains largely unknown. Until recently, it was classified as one of the Domain of Unknown Function (DUF) genes, with a conserved protein domain DUF2366 of unclear function. Since characterizing DUF genes can provide novel biological insight, we used previously established transcriptional profiles of the germline and stem cells of the flatworm Macrostomum lignano to probe conserved DUFs for their potential role in germline biology, stem cell function, regeneration, and development. Here, we demonstrate that DUF2366/TIM29 knockdown in M. lignano has very limited effect during the normal homeostatic condition but prevents worms from adapting to a highly proliferative state required for regeneration