Reconstructing phylogeny from RNA secondary structure via simulated evolution

DNA sequences of genes encoding functional RNA molecules (e.g., ribosomal RNAs) are commonly used in phylogenetics (i.e. to infer evolutionary history). Trees derived from ribosomal RNA (rRNA) sequences, however, are inconsistent with other molecular data in investigations of deep branches in the tree of life. Since much of te functional constraints on the gene products (i.e. RNA molecules) relate to three-dimensional structure, rather than their actual sequences, accumulated mutations in the gene sequences may obscure phylogenetic signal over very large evolutionary time-scales. Variation in structure, however, may be suitable for phylogenetic inference even under extreme sequence divergence. To evaluate qualitatively the manner in which structural evolution relates to sequence change, we simulated the evolution of RNA sequences under various constraints on structural change

Characteristics of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) rRNA genes of Apis mellifera (Insecta: Hymenoptera): structure, organization, and retrotransposable elements

As an accompanying manuscript to the release of the honey bee genome, we report the entire sequence of the nuclear (18S, 5.8S, 28S and 5S) and mitochondrial (12S and 16S) ribosomal RNA (rRNA)-encoding gene sequences (rDNA) and related internally and externally transcribed spacer regions of Apis mellifera (Insecta: Hymenoptera: Apocrita). Additionally, we predict secondary structures for the mature rRNA molecules based on comparative sequence analyses with other arthropod taxa and reference to recently published crystal structures of the ribosome. In general, the structures of honey bee rRNAs are in agreement with previously predicted rRNA models from other arthropods in core regions of the rRNA, with little additional expansion in non-conserved regions. Our multiple sequence alignments are made available on several public databases and provide a preliminary establishment of a global structural model of all rRNAs from the insects. Additionally, we provide conserved stretches of sequences flanking the rDNA cistrons that comprise the externally transcribed spacer regions (ETS) and part of the intergenic spacer region (IGS), including several repetitive motifs. Finally, we report the occurrence of retrotransposition in the nuclear large subunit rDNA, as R2 elements are present in the usual insertion points found in other arthropods. Interestingly, functional R1 elements usually present in the genomes of insects were not detected in the honey bee rRNA genes. The reverse transcriptase products of the R2 elements are deduced from their putative open reading frames and structurally aligned with those from another hymenopteran insect, the jewel wasp Nasonia (Pteromalidae). Stretches of conserved amino acids shared between Apis and Nasonia are illustrated and serve as potential sites for primer design, as target amplicons within these R2 elements may serve as novel phylogenetic markers for Hymenoptera. Given the impending completion of the sequencing of the Nasonia genome, we expect our report eventually to shed light on the evolution of the hymenopteran genome within higher insects, particularly regarding the relative maintenance of conserved rDNA genes, related variable spacer regions and retrotransposable elements

Evolutionary rates vary among rRNA structural elements

Understanding patterns of rRNA evolution is critical for a number of fields, including structure prediction and phylogeny. The standard model of RNA evolution is that compensatory mutations in stems make up the bulk of the changes between homologous sequences, while unpaired regions are relatively homogeneous. We show that considerable heterogeneity exists in the relative rates of evolution of different secondary structure categories (stems, loops, bulges, etc.) within the rRNA, and that in eukaryotes, loops actually evolve much faster than stems. Both rates of evolution and abundance of different structural categories vary with distance from functionally important parts of the ribosome such as the tRNA path and the peptidyl transferase center. For example, fast-evolving residues are mainly found at the surface; stems are enriched at the subunit interface, and junctions near the peptidyl transferase center. However, different secondary structure categories evolve at different rates even when these effects are accounted for. The results demonstrate that relative rates and patterns of evolution are lineage specific, suggesting that phylogenetically and structurally specific models will improve evolutionary and structural predictions

The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. Our results implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated

Louse (Insecta : Phthiraptera) mitochondrial 12S rRNA secondary structure is highly variable

Lice are ectoparasitic insects hosted by birds and mammals. Mitochondrial 12S rRNA sequences obtained from lice show considerable length variation and are very difficult to align. We show that the louse 12S rRNA domain III secondary structure displays considerable variation compared to other insects, in both the shape and number of stems and loops. Phylogenetic trees constructed from tree edit distances between louse 12S rRNA structures do not closely resemble trees constructed from sequence data, suggesting that at least some of this structural variation has arisen independently in different louse lineages. Taken together with previous work on mitochondrial gene order and elevated rates of substitution in louse mitochondrial sequences, the structural variation in louse 12S rRNA confirms the highly distinctive nature of molecular evolution in these insects

The Small Subunit rRNA Modification Database

The Small Subunit rRNA Modification Database provides a listing of reported post-transcriptionally modified nucleosides and sequence sites in small subunit rRNAs from bacteria, archaea and eukarya. Data are compiled from reports of full or partial rRNA sequences, including RNase T1 oligonucleotide catalogs reported in earlier literature in studies of phylogenetic relatedness. Options for data presentation include full sequence maps, some of which have been assembled by database curators with the aid of contemporary gene sequence data, and tabular forms organized by source organism or chemical identity of the modification. A total of 32 rRNA sequence alignments are provided, annotated with sites of modification and chemical identities of modifications if known, with provision for scrolling full sequences or user-dictated subsequences for comparative viewing for organisms of interest. The database can be accessed through the World Wide Web at http://medlib.med.utah.edu/SSUmods

Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns

<p>Abstract</p> <p>Background</p> <p>Group I introns are one of the four major classes of introns as defined by their distinct splicing mechanisms. Because they catalyze their own removal from precursor transcripts, group I introns are referred to as autocatalytic introns. Group I introns are common in fungal and protist nuclear ribosomal RNA genes and in organellar genomes. In contrast, they are rare in all other organisms and genomes, including bacteria.</p> <p>Results</p> <p>Here we report five group I introns, each containing a LAGLIDADG homing endonuclease gene (HEG), in large subunit (LSU) rRNA genes of cyanobacteria. Three of the introns are located in the LSU gene of <it>Synechococcus </it>sp. C9, and the other two are in the LSU gene of <it>Synechococcus lividus </it>strain C1. Phylogenetic analyses show that these introns and their HEGs are closely related to introns and HEGs located at homologous insertion sites in organellar and bacterial rDNA genes. We also present a compilation of group I introns with homing endonuclease genes in bacteria.</p> <p>Conclusion</p> <p>We have discovered multiple HEG-containing group I introns in a single bacterial gene. To our knowledge, these are the first cases of multiple group I introns in the same <it>bacterial </it>gene (multiple group I introns have been reported in at least one phage gene and one prophage gene). The HEGs each contain one copy of the LAGLIDADG motif and presumably function as homodimers. Phylogenetic analysis, in conjunction with their patchy taxonomic distribution, suggests that these intron-HEG elements have been transferred horizontally among organelles and bacteria. However, the mode of transfer and the nature of the biological connections among the intron-containing organisms are unknown.</p

The role of the RNA-binding protein Hfq in the model pathogen Salmonella Typhimurium

Hfq is a RNA-binding protein which exists in homohexamers in vivo. Based on its folding, containing the highly conserved Sm1 and Sm2 motifs, it belongs to the growing family of Sm and Sm-like (Lsm) proteins. It has been shown, that Hfq is a pleiotropic regulator in bacteria which is involved in a broad variety of functions. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium Even though hfq has turned out to have no severe influence on the growth or the viability of the pathogenic bacterium Salmonella Typhimurium under laboratory conditions, we could show that it is strongly involved in the regulation of pathogenicity. A Δhfq mutant leads to loss of effector protein expression and secretion and thereby to reduced invasion of non-phagocytic cells and to reduced ability of intracellular replication in macrophages. Based on these observations, loss of infectivity in a mouse-model of infection could be proven. Further studies revealed not only lack of secreted proteins in the Δhfq mutant, but showed severe changes in the overall protein pattern when compared to its isogenic wild type strain, with an overrepresentation of membrane and membrane-associated proteins. Concerning the virulence phenotype, we have been able to restore effector protein expression (even if not their secretion) by overexpression of one of the major transcription factors involved in expression of virulence genes encoded in Salmonella pathogenicity island 1 (SPI1), namely HilA. Additionally, we could show that alteration in mRNA stability is causing for example the increase of the major outer membrane protein, OmpD or the decrease in the flagellar protein, FliC. Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq Our analysis represents a demonstration for usage of high throughput pyrosequencing (HTPS) in bacteria to determine the large regulon of the pleiotropic regulator, Hfq. The combination of transcriptomics with co-immunoprecipitation (coIP) of direct binding partners of Hfq and subsequent cDNA library synthesis and its sequencing allowed the dissection of genes directly influenced by Hfq and downstream effects based on deregulation of transcription factors. By analysis of RNA co-immunoprecipitated with Hfq compared to control coIPs in Salmonella Typhimurium lysates we were able to determine specific enrichment factors for a large set of mRNAs as well as sRNAs. Comparison with the transcriptomic data showed that Hfq regulates multiple major transcription factors, like a transcription factor of SPI1, HilD, and the major transcription factor, FlhD2C2, regulating the large class of flagellar genes in Salmonella and other bacterial species. By overexpression of these transcription factors we could restore phenotypes of a Δhfq mutant, e.g. loss of effector protein expression and secretion and reduced expression of the class III flagellar gene, FliC. Concerning sRNA expression in Salmonella, we found 10 new sRNAs in this pathogen and were able to verify the expression of a large set of sRNAs that have been known to be conserved in the model organism, Escherichia coli. Aside noncoding RNAs also two mRNAs encoding for small open reading frames (ORFs) in E. coli could be detected in the coIP RNA sample from Salmonella Typhimurium