Sonja J. Prohaska

Evolution of Vault RNAs

Vault RNAs (vtRNAs) are small, about 100 nt long, polymerase III transcripts contained in the vault particles of eukaryotic cells. Presumably due to their enigmatic function, they have received little attention compared with most other noncoding RNA (ncRNA) families. Their poor sequence conservation makes homology search a complex and tedious task even within vertebrates. Here we report on a systematic and comprehensive analysis of this rapidly evolving class of ncRNAs in deuterostomes, providing a comprehensive collection of computationally predicted vtRNA genes. We find that all previously described vtRNAs are located at a conserved genomic locus linked to the protocadherin gene cluster, an association that is conserved throughout gnathostomes. Lineage-specific expansions to small vtRNA gene clusters are frequently observed in this region. A second vtRNA locus is syntenically conserved across eutherian mammals. The vtRNAs at the two eutherian loci exhibit substantial differences in their promoter structures, explaining their differential expression patterns in several human cancer cell lines. In teleosts, expression of several paralogous vtRNA genes, most but not all located at the syntenically conserved protocadherin locus, was verified by reverse transcriptase-polymerase chain reaction.

The amphioxus Hox cluster: characterization, comparative genomics, and evolution

The amphioxus Hox cluster is often viewed as archetypal for the chordate lineage. Here, we present a descriptive account of the 448 kb region spanning the Hox cluster of the amphioxus Branchiostoma floridae from Hox14 to Hox1. We provide complete coding sequences of all 14 previously described amphioxus sequences and give a detailed analysis of the conserved noncoding regulatory sequence elements. We find that the posterior part of the Hox cluster is so highly derived that even the complete genomic sequence is insufficient to decide whether the posterior Hox genes arose by independent duplications or whether they are true orthologs of the corresponding gnathostome paralog groups. In contrast, the anterior region is much better conserved. The amphioxus Hox cluster strongly excludes repetitive elements with the exception of two repeat islands in the posterior region. Repeat exclusion is also observed in gnathostomes, but not protostome Hox clusters. We thus hypothesize that the much shorter vertebrate Hox clusters are the result of extensive resolution of the redundancy of regulatory DNA after the genome duplications rather than the consequence of a selection pressure to remove nonfunctional sequence from the Hox cluster.

Noisy: Identification of problematic columns in multiple sequence alignments

MotivationSequence-based methods for phylogenetic reconstruction from (nucleic acid) sequence data are notoriously plagued by two effects: homoplasies and alignment errors. Large evolutionary distances imply a large number of homoplastic sites. As most protein-coding genes show dramatic variations in substitution rates that are not uncorrelated across the sequence, this often leads to a patchwork pattern of (i) phylogenetically informative and (ii) effectively randomized regions. In highly variable regions, furthermore, alignment errors accumulate resulting in sometimes misleading signals in phylogenetic reconstruction.ResultsWe present here a method that, based on assessing the distribution of character states along a cyclic ordering of the taxa, allows the identification of phylogenetically uninformative homoplastic sites in a multiple sequence alignment. Removal of these sites appears to improve the performance of phylogenetic reconstruction algorithms as measured by various indices of tree quality. In particular, we obtain more stable trees due to the exclusion of phylogenetically incompatible sites that most likely represent strongly randomized characters.SoftwareThe computer program noisy implements this approach. It can be employed to improving phylogenetic reconstruction capability with quite a considerable success rate whenever (1) the average bootstrap support obtained from the original alignment is low, and (2) there are sufficiently many taxa in the data set – at least, say, 12 to 15 taxa. The software can be obtained under the GNU Public License from http://www.bioinf.uni-leipzig.de/Software/noisy/.

Computational RNomics of drosophilids.

BackgroundRecent experimental and computational studies have provided overwhelming evidence for a plethora of diverse transcripts that are unrelated to protein-coding genes. One subclass consists of those RNAs that require distinctive secondary structure motifs to exert their biological function and hence exhibit distinctive patterns of sequence conservation characteristic for positive selection on RNA secondary structure.The deep-sequencing of 12 drosophilid species coordinated by the NHGRI provides an ideal data set of comparative computational approaches to determine those genomic loci that code for evolutionarily conserved RNA motifs. This class of loci includes the majority of the known small ncRNAs as well as structured RNA motifs in mRNAs. We report here on a genome-wide survey using RNAz.ResultsWe obtain 16 000 high quality predictions among which we recover the majority of the known ncRNAs. Taking a pessimistically estimated false discovery rate of 40% into account, this implies that at least some ten thousand loci in the Drosophila genome show the hallmarks of stabilizing selection action of RNA structure, and hence are most likely functional at the RNA level. A subset of RNAz predictions overlapping with TRF1 and BRF binding sites [Isogai et al., EMBO J. 26: 79–89 (2007)], which are plausible candidates of Pol III transcripts, have been studied in more detail. Among these sequences we identify several clusters of ncRNA candidates with striking structural similarities.ConclusionThe statistical evaluation of the RNAz predictions in comparison with a similar analysis of vertebrate genomes [Washietl et al., Nat. Biotech. 23: 1383–1390 (2005)] shows that qualitatively similar fractions of structured RNAs are found in introns, UTRs, and intergenic regions. The intergenic RNA structures, however, are concentrated much more closely around known protein-coding loci, suggesting that flies have significantly smaller complement of independent structured ncRNAs compared to mammals.

Innovation in gene regulation the case of chromatin computation

Chromatin regulation is understood to be one of the fundamental modes of gene regulation in eukaryotic cells. We argue that the basic proteins that determine the chromatin architecture constitute an evolutionary ancient layer of transcriptional regulation common to all three domains of life. We explore phylogenetically, sources of innovation in chromatin regulation, focusing on protein domains related to chromatin structure and function, demonstrating a step-wise increase of complexity in chromatin regulation. Building upon the highly conserved use of variants of chromosomal architectural proteins to distinguish chromosomal states, Eukarya secondarily acquired mechanisms for writing chemical modifications onto chromatin that constitute persistent signals. The acquisition of reader domains enabled decoding of these complex, signal combinations and a decoupling of the signal from immediate biochemical effects. We show how the coupling of reading and writing, which is most prevalent in crown-group Eukarya, could have converted chromatin into a powerful computational device capable of storing and processing more information than pure cis-regulatory networks.

Ecological plant epigenetics: Evidence from model and non-model species, and the way forward

Growing evidence shows that epigenetic mechanisms contribute to complex traits, with implications across many fields of biology. In plant ecology, recent studies have attempted to merge ecological experiments with epigenetic analyses to elucidate the contribution of epigenetics to plant phenotypes, stress responses, adaptation to habitat, and range distributions. While there has been some progress in revealing the role of epigenetics in ecological processes, studies with non-model species have so far been limited to describing broad patterns based on anonymous markers of DNA methylation. In contrast, studies with model species have benefited from powerful genomic resources, which contribute to a more mechanistic understanding but have limited ecological realism. Understanding the significance of epigenetics for plant ecology requires increased transfer of knowledge and methods from model species research to genomes of evolutionarily divergent species, and examination of responses to complex natural environments at a more mechanistic level. This requires transforming genomics tools specifically for studying non-model species, which is challenging given the large and often polyploid genomes of plants. Collaboration among molecular geneticists, ecologists and bioinformaticians promises to enhance our understanding of the mutual links between genome function and ecological processes.

SynBlast: Assisting the analysis of conserved synteny information

MotivationIn the last years more than 20 vertebrate genomes have been sequenced, and the rate at which genomic DNA information becomes available is rapidly accelerating. Gene duplication and gene loss events inherently limit the accuracy of orthology detection based on sequence similarity alone. Fully automated methods for orthology annotation do exist but often fail to identify individual members in cases of large gene families, or to distinguish missing data from traceable gene losses. This situation can be improved in many cases by including conserved synteny information.ResultsHere we present the SynBlast pipeline that is designed to construct and evaluate local synteny information. SynBlast uses the genomic region around a focal reference gene to retrieve candidates for homologous regions from a collection of target genomes and ranks them in accord with the available evidence for homology. The pipeline is intended as a tool to aid high quality manual annotation in particular in those cases where automatic procedures fail. We demonstrate how SynBlast is applied to retrieving orthologous and paralogous clusters using the vertebrate Hox and ParaHox clusters as examples.SoftwareThe SynBlast package written in Perl is available under the GNU General Public License at http://www.bioinf.uni-leipzig.de/Software/SynBlast/.

Process flow for classification and clustering of fruit fly gene expression patterns

The rapidly growing collection of fruit fly embryo images makes automated Image Segmentation and classification an indispensable requirement for a large-scale analysis of in situ hybridization (ISH) - gene expression patterns (GEP). We present here such an automated process flow for segmenting, classification, and clustering large-scale sets of Drosophila melanogaster GEP that is capable of dealing with most of the complications implicated in the images.

Transcriptional regulation of the human CD97 promoter by Sp1/Sp3 in smooth muscle cells.

The EGF-TM7 receptor CD97 shows different features of expression and function in muscle cells compared to hematopoetic and tumor cells. Since the molecular function and regulation of CD97 are poorly understood, this study aimed at defining its basal transcriptional regulation in smooth muscle cells (SMCs). The computational analysis of the CD97 5-flanking region revealed that the TATA box-lacking promoter possesses several GC-rich regions as putative Sp1/Sp3 binding sites. Transfection studies with serially deleted promoter constructs demonstrated that the minimal promoter fragment resided in the -218/+45 region containing one out of five identified GC-boxes in the leiomyosarcoma cell line SK-LMS-1 and human bronchial smooth muscle cells (HbSMCs). Mutation of the most proximal GC-site in CD97 reporter gene constructs caused a significant decrease in promoter activity. Gel shift assays and chromatin immunoprecipitation revealed that Sp1 and Sp3 bound specifically to the most proximal GC-site. Furthermore, we showed that Sp1 and Sp3 over-expression activates CD97 promoter activity in HEK293 cells. Our data characterize for the first time the activity of the human CD97 promoter which is controlled by Sp1/Sp3 transcription factors in SMCs.

A STORY OF GROWING CONFUSION: GENES AND THEIR REGULATION

High-throughput experiments have produced convicing evidence for an extensive contribution of diverse classes of RNAs in the expression of genetic information. Instead of a simple arrangement of mostly protein-coding genes, the human transcriptome features a complex arrangement of overlapping transcripts, many of which do not code for proteins at all, while others “sample” exons from several different “genes”. The complexity of the transcriptome and the prevalence of noncoding transcripts forces us to reconsider both the concept of the “gene” itself and our understanding of the mechanisms that regulate “gene expression”.