Wayne A. Decatur

rRNA modifications and ribosome function

The development of three-dimensional maps of the modified nucleotides in the ribosomes of Escherichia coli and yeast has revealed that most (approximately 95% in E. coli and 60% in yeast) occur in functionally important regions. These include the peptidyl transferase centre, the A, P and E sites of tRNA- and mRNA binding, the polypeptide exit tunnel, and sites of subunit-subunit interaction. The correlations suggest that many ribosome functions benefit from nucleotide modification.

Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution.

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.

A Well-Connected and Conserved Nucleoplasmic Helicase Is Required for Production of Box C/D and H/ACA snoRNAs and Localization of snoRNP Proteins

ABSTRACT Biogenesis of small nucleolar RNA-protein complexes (snoRNPs) consists of synthesis of the snoRNA and protein components, snoRNP assembly, and localization to the nucleolus. Recently, two nucleoplasmic proteins from mice were observed to bind to a model box C/D snoRNA in vitro, suggesting that they function at an early stage in snoRNP biogenesis. Both proteins have been described in other contexts. The proteins, called p50 and p55 in the snoRNA binding study, are highly conserved and related to each other. Both have Walker A and B motifs characteristic of ATP- and GTP-binding and nucleoside triphosphate-hydrolyzing domains, and the mammalian orthologs have DNA helicase activity in vitro. Here, we report that theSaccharomyces cerevisiae ortholog of p50 (Rvb2, Tih2p, and other names) is required for production of C/D snoRNAs in vivo and, surprisingly, H/ACA snoRNAs as well. Point mutations in the Walker A and B motifs cause temperature-sensitive or lethal growth phenotypes and severe defects in snoRNA accumulation. Notably, depletion of p50 (called Rvb2 in this study) also impairs localization of C/D and H/ACA core snoRNP proteins Nop1p and Gar1p, suggesting a defect(s) in snoRNP assembly or trafficking to the nucleolus. Findings from other studies link Rvb2 orthologs with chromatin remodeling and transcription. Taken together, the present results indicate that Rvb2 is involved in an early stage of snoRNP biogenesis and may play a role in coupling snoRNA synthesis with snoRNP assembly and localization.

The 3D rRNA modification maps database: with interactive tools for ribosome analysis.

The 3D rRNA modification maps database is the first general resource of information about the locations of modified nucleotides within the 3D structure of the full ribosome, with mRNA and tRNAs in the A-, P- and E-sites. The database supports analyses for several model organisms, including higher eukaryotes, and enables users to construct 3D maps for other organisms. Data are provided for human and plant (Arabidopsis) ribosomes, and for other representative organisms from eubacteria, archaea and eukarya. Additionally, the database integrates information about positions of modifications within rRNA sequences and secondary structures, as well as links to other databases and resources about modifications and their biosynthesis. Displaying positions of modified nucleotides is fully manageable. Views of each modified nucleotide are controlled by individual buttons and buttons also control the visibility of different ribosomal molecular components. A section called ‘Paint Your Own’ enables the user to create a 3D modification map for rRNA from any organism where sites of modification are known. This section also provides capabilities for visualizing nucleotides of interest in rRNA or tRNA, as well as particular amino acids in ribosomal proteins. The database can be accessed at http://people.biochem.umass.edu/fournierlab/3dmodmap/

RNA‐modifying machines in archaea

It has been known for nearly half a century that coding and non‐coding RNAs (mRNA, and tRNAs and rRNAs respectively) play critical roles in the process of information transfer from DNA to protein. What is both surprising and exciting, are the discoveries in the last decade that cells, particularly eukaryotic cells, contain a plethora of non‐coding RNAs and that these RNAs can either possess catalytic activity or can function as integral components of dynamic ribonucleoprotein machines. These machines appear to mediate diverse, complex and essential processes such as intron excision, RNA modification and editing, protein targeting, DNA packaging, etc. Archaea have been shown to possess RNP complexes; some of these are authentic homologues of the eukaryotic complexes that function as machines in the processing, modification and assembly of rRNA into ribosomal subunits. Deciphering how these RNA‐containing machines function will require a dissection and analysis of the component parts, an understanding of how the parts fit together and an ability to reassemble the parts into complexes that can function in vitro. This article summarizes our current knowledge about small‐non‐coding RNAs in Archaea, their roles in ribosome biogenesis and their relationships to the complexes that have been identified in eukaryotic cells.

Two group I ribozymes with different functions in a nuclear rDNA intron.

DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self‐splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS‐1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self‐splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS‐1. Much of the P2 stem and all of the associated loop could be deleted without effect on self‐splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5′ splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1‐mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron‐encoded endonuclease I‐DirI.

Insight from the lamprey genome: Glimpsing early vertebrate development via neuroendocrine-associated genes and shared synteny of gonadotropin-releasing hormone (GnRH)

Study of the ancient lineage of jawless vertebrates is key to understanding the origins of vertebrate biology. The establishment of the neuroendocrine system with the hypothalamic-pituitary axis at its crux is of particular interest. Key neuroendocrine hormones in this system include the pivotal gonadotropin-releasing hormones (GnRHs) responsible for controlling reproduction via the pituitary. Previous data incorporating several lines of evidence showed all known vertebrate GnRHs were grouped into four paralogous lineages: GnRH1, 2, 3 and 4; with proposed evolutionary paths. Using the currently available lamprey genome assembly, we searched genes of the neuroendocrine system and summarize here the details representing the state of the current lamprey genome assembly. Additionally, we have analyzed in greater detail the evolutionary history of the GnRHs based on the information of the genomic neighborhood of the paralogs in lamprey as compared to other gnathostomes. Significantly, the current evidence suggests that two genome duplication events (both 1R and 2R) that generated the different fish and tetrapod paralogs took place before the divergence of the ancestral agnathans and gnathostome lineages. Syntenic analysis supports this evidence in that the previously-classified type IV GnRHs in lamprey (lGnRH-I and -III) share a common ancestry with GnRH2 and 3, and thus are no longer considered type IV GnRHs. Given the single amino acid difference between lGnRH-II and GnRH2 we propose that a GnRH2-like gene existed before the lamprey/gnathostome split giving rise to lGnRH-II and GnRH2. Furthermore, paralogous type 3 genes (lGnRH-I/III and GnRH3) evolved divergent structure/function in lamprey and gnathostome lineages.

Proteopedia: A status report on the collaborative, 3D web-encyclopedia of proteins and other biomolecules

Proteopedia is a collaborative, 3D web-encyclopedia of protein, nucleic acid and other biomolecule structures. Created as a means for communicating biomolecule structures to a diverse scientific audience, Proteopedia (http://www.proteopedia.org) presents structural annotation in an intuitive, interactive format and allows members of the scientific community to easily contribute their own annotations. Here, we provide a status report on Proteopedia by describing advances in the web resource since its inception three and a half years ago, focusing on features of potential direct use to the scientific community. We discuss its progress as a collaborative 3D-encyclopedia of structures as well as its use as a complement to scientific publications and PowerPoint presentations. We also describe Proteopedias use for 3D visualization in structure-related pedagogy.

Is secretoneurin a new hormone

Numerous small potentially bioactive peptides are derived from the selective processing of the ~600 amino acid secretogranin II (SgII) precursor, but only the 31-42 amino acid segment termed secretoneurin (SN) is well-conserved from sharks to mammals. Both SNa and SNb paralogs have been identified in some teleosts, likely arising as a result of the specific genome duplication event in this lineage. Only one copy of the putative lamprey SgII (188 amino acids) could be identified which gives rise to a divergent agnathan SN that contains the signature YTPQ-X-LA-X(7)-EL sequence typical of the central core of all known SN peptides. In rodent models, SN has regulatory effects on neuroinflammation and neurotransmitter release, and possesses therapeutic potential for the induction of angiogenesis. The wide distribution of SN in neuroendocrine neurons and pituitary cells suggests important endocrine roles. The clearest example of the endocrine action of SN is the stimulatory effects on pituitary luteinizing hormone release from goldfish pituitary and mouse LβT2 gonadotroph cells, indicative of an important role in reproduction. Several lines of evidence suggest that the SN receptor is most likely a G-protein coupled protein. Microarray analysis of SN effects on dispersed goldfish pituitary cells in vitro reveals novel SN actions that include effects on genes involved in notch signaling and the guanylate cyclase pathway. Intracerebroventricular injection of SN increases feeding and locomotory behaviors in goldfish. Given that SgII appeared early in vertebrate evolution, SN is an old peptide with emerging implications as a new multifunctional hormone.

Emergence of an Ancestral Glycoprotein Hormone in the Pituitary of the Sea Lamprey, a Basal Vertebrate

The gnathostome (jawed vertebrates) classical pituitary glycoprotein hormones, FSH, LH, and TSH, consist of a common α-subunit (GpA1) and unique β-subunits (Gpβ1, -2, and -3), whereas a recently identified pituitary glycoprotein hormone, thyrostimulin, consists of GpA2 and GpB5. This paper reports the identification, expression, and function of an ancestral, nonclassical, pituitary heterodimeric glycoprotein hormone (GpH) consisting of the thyrostimulin A2 subunit with the classical β-subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpHβ were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpHβ after the overexpression in transiently transfected COS7 cells using a bipromoter vector. Dual-label fluorescent in situ hybridization and immunohistochemistry showed the coexpression of individual subunits in the proximal pars distalis of the pituitary. GnRH-III (1μΜ) significantly increased the expression of lGpHβ and lGpA2 in in vitro pituitary culture. Recombinant lamprey GpH was constructed by tethering the N terminal of lGpA2 to the C terminal of lGpHβ with a linker region composed of six histidine residues followed by three glycine-serine repeats. This recombinant lamprey GpH activated the lamprey glycoprotein hormone receptor I as measured by increased cAMP/luciferase activity. These data are the first to demonstrate a functional, unique glycoprotein heterodimer that is not found in any other vertebrate. These data suggest an intermediate stage of the structure-function of the gonadotropin/thyroid-stimulating hormone in a basal vertebrate, leading to the emergence of the highly specialized gonadotropin hormones and thyroid stimulating hormones in gnathostomes.