Axel Mosig

Evolutionary patterns of non-coding RNAs

A plethora of new functions of non-coding RNAs (ncRNAs) have been discovered in past few years. In fact, RNA is emerging as the central player in cellular regulation, taking on active roles in multiple regulatory layers from transcription, RNA maturation, and RNA modification to translational regulation. Nevertheless, very little is known about the evolution of this “Modern RNA World” and its components. In this contribution, we attempt to provide at least a cursory overview of the diversity of ncRNAs and functional RNA motifs in non-translated regions of regular messenger RNAs (mRNAs) with an emphasis on evolutionary questions. This survey is complemented by an in-depth analysis of examples from different classes of RNAs focusing mostly on their evolution in the vertebrate lineage. We present a survey of Y RNA genes in vertebrates and study the molecular evolution of the U7 snRNA, the snoRNAs E1/U17, E2, and E3, the Y RNA family, the let-7 microRNA (miRNA) family, and the mRNA-like evf-1 gene. We furthermore discuss the statistical distribution of miRNAs in metazoans, which suggests an explosive increase in the miRNA repertoire in vertebrates. The analysis of the transcription of ncRNAs suggests that small RNAs in general are genetically mobile in the sense that their association with a hostgene (e.g. when transcribed from introns of a mRNA) can change on evolutionary time scales. The let-7 family demonstrates, that even the mode of transcription (as intron or as exon) can change among paralogous ncRNA.

Structure and Function of the Smallest Vertebrate Telomerase RNA from Teleost Fish

Telomerase extends chromosome ends by copying a short template sequence within its intrinsic RNA component. Telomerase RNA (TR) from different groups of species varies dramatically in sequence and size. We report here the bioinformatic identification, secondary structure comparison, and functional analysis of the smallest known vertebrate TRs from five teleost fishes. The teleost TRs (312-348 nucleotides) are significantly smaller than the cartilaginous fish TRs (478-559 nucleotides) and tetrapod TRs. This remarkable length reduction of teleost fish TRs correlates positively with the genome size, reflecting an unusual structural plasticity of TR during evolution. The teleost TR consists of a compact three-domain structure, lacking most of the sequences in regions that are variable in other vertebrate TR structures. The medaka and fugu TRs, when assembled with their telomerase reverse transcriptase (TERT) protein counterparts, reconstituted active and processive telomerase enzymes. Titration analysis of individual RNA domains suggests that the efficient assembly of the telomerase complex is influenced more by the telomerase reverse transcriptase (TERT) binding of the CR4-CR5 domain than the pseudoknot domain of TR. The remarkably small teleost fish TR further expands our understanding about the evolutionary divergence of vertebrate TR.

Label-free imaging of drug distribution and metabolism in colon cancer cells by Raman microscopy

Targeted cancer therapies block cancer growth and spread using small molecules. Many molecular targets for an epidermal growth factor receptor (EGFR) selectively compete with the adenosine triphosphate-binding site of its tyrosine kinase domain. Detection of molecular targeted agents and their metabolites in cells/tissues by label-free imaging is attractive because dyes or fluorescent labels may be toxic or invasive. Here, label-free Raman microscopy is applied to show the spatial distribution of the molecular targeted drug erlotinib within the cell. The Raman images show that the drug is clustered at the EGFR protein at the membrane and induces receptor internalization. The changes within the Raman spectrum of erlotinib measured in cells as compared to the free-erlotinib spectrum indicate that erlotinib is metabolized within cells to its demethylated derivative. This study provides detailed insights into the drug targeting mechanism at the atomic level in cells. It demonstrates that Raman microscopy will open avenues as a non-invasive and label-free technique to investigate pharmacokinetics at the highest possible resolution in living cells.

Evolution of the vertebrate Y RNA cluster

Relatively little is known about the evolutionary histories of most classes of non-protein coding RNAs. Here we consider Y RNAs, a relatively rarely studied group of related pol-III transcripts. A single cluster of functional genes is preserved throughout tetrapod evolution, which however exhibits clade-specific tandem duplications, gene-losses, and rearrangements.

Arthropod 7SK RNA

The 7SK small nuclear RNA (snRNA) is a key player in the regulation of polymerase (pol) II transcription. The 7SK RNA was long believed to be specific to vertebrates where it is highly conserved. Homologs in basal deuterostomes and a few lophotrochozoan species were only recently reported. On longer timescales, 7SK evolves rapidly with only few conserved sequence and structure motifs. Previous attempts to identify the Drosophila homolog thus have remained unsuccessful despite considerable efforts. Here we report on the discovery of arthropod 7SK RNAs using a novel search strategy based on pol III promoters, as well as the subsequent verification of its expression. Our results demonstrate that a 7SK snRNA featuring 2 highly structured conserved domains was present already in the bilaterian ancestor.

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing–based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function.

Invertebrate 7SK snRNAs.

Abstract7SK RNA is a highly abundant noncoding RNA in mammalian cells whose function in transcriptional regulation has only recently been elucidated. Despite its highly conserved sequence throughout vertebrates, all attempts to discover 7SK RNA homologues in invertebrate species have failed so far. Here we report on a combined experimental and computational survey that succeeded in discovering 7SK RNAs in most of the major deuterostome clades and in two protostome phyla: mollusks and annelids. Despite major efforts, no candidates were found in any of the many available ecdysozoan genomes, however. The additional sequence data confirm the evolutionary conservation and hence functional importance of the previously described 3′ and 5′ stem-loop motifs, and provide evidence for a third, structurally well-conserved domain.

Approximately matching polygonal curves with respect to the Fréchet distance

In this paper we present approximate algorithms for matching two polygonal curves with respect to the Frechet distance. We define a discrete version of the Frechet distance as a distance measure between polygonal curves and show that this discrete version is bounded by the continuous version of the Frechet distance.For the task of matching with respect to the discrete Frechet distance, we develop an algorithm that is based on intersecting certain subsets of the transformation group under consideration. Our algorithm for matching two point sequences of lengths m and n under the group of rigid motions has a time complexity of O(m2n2) for matching under the discrete Frechet distance and can be modified for matching subcurves, closed curves and finding longest common subcurves, Group theoretical considerations allow us to eliminate translation components of affine transformations and to consider matching under arbitrary linear algebraic groups.

Circulation times of prostate cancer and hepatocellular carcinoma cells by in vivo flow cytometry

In metastasis, the cancer cells that travel through the body are capable of establishing new tumors in locations remote from the site of the original disease. To metastasize, a cancer cell must break away from its tumor and invade either the circulatory or lymphatic system, which will carry it to a new location, and establish itself in the new site. Once in the blood stream, the cancer cells now have access to every portion of the body. Here, we have used the “in vivo flow cytometer” to study if there is any relationship between metastatic potential and depletion kinetics of circulating tumor cells. The in vivo flow cytometer has the capability to detect and quantify continuously the number and flow characteristics of fluorescently labelled cells in vivo. We have improved the counting algorithm and measured the depletion kinetics of cancer cells with different metastatic potential. Interestingly, more invasive PC‐3 prostate cancer cells are depleted faster from the circulation than LNCaP cells. In addition, we have measured the depletion kinetics of two related human hepatocellular carcinoma (liver cancer) cell lines, high‐metastatic HCCLM3 cells, and low‐metastatic HepG2 cells. More than 60% HCCLM3 cells are depleted within the first hour. Interestingly, the low‐metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, <40% HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes.

Homology search with fragmented nucleic acid sequence patterns

The comprehensive annotation of non-coding RNAs in newly sequenced genomes is still a largely unsolved problem because many functional RNAs exhibit not only poorly conserved sequences but also large variability in structure. In many cases, such as Y RNAs, vault RNAs, or telomerase RNAs, sequences differ by large insertions or deletions and have only a few small sequence patterns in common. Here we present fragrep2, a purely sequence-based approach to detect such patterns in complete genomes. A fragrep2 pattern consists of an ordered list of position-specific weight matrices (PWMs) describing short, approximately conserved sequence elements, that are separated by intervals of non-conserved regions of bounded length. The program uses a fractional programming approach to align the PWMs to genomic DNA in order to allow for a bounded number of insertions and deletions in the patterns. These patterns are then combined to significant combinations of PWMs. At this step, a subset of PWMs may be deleted, i.e., have no match in the current region of the genome. The program furthermore estimates p- and E-values for the matches. We apply fragrep2 to homology searches for RNase MRP, unveiling two previously unidentified matches as well as reproducing the results of two previous surveys. Furthermore, we complement the picture of vertebrate vault RNAs, a class of ncRNAs that has not received much attention so far.