Minimization and complete loss of general transcription factor proteins in the intracellular parasite Encephalitozoon cuniculi.

Genome compaction is a common evolutionary feature of parasites. The unicellular, obligate intracellular parasite Encephalitozoon cuniculi has one of smallest known eukaryotic genomes, and is nearly four times smaller than its distant fungi relative, the budding yeast Saccharomyces cerevisiae. Comparison of the proteins encoded by compacted genomes to those encoded by larger genomes can reveal the most highly conserved features of the encoded proteins. In this study, we identified the proteins comprising the RNA polymerases and their corresponding general transcription factors by using several bioinformatic approaches to compare the transcription machinery of E. cuniculi and S. cerevisiae. Surprisingly, our analyses revealed an overall reduction in the size of the proteins comprising transcription machinery of E. cuniculi, which includes the loss of entire regions or functional domains from proteins, as well as the loss of entire proteins and complexes. Unexpectedly, we found that the E. cuniculi ortholog of Rpc37 (a RNA Polymerase III subunit) more closely resembles the H. sapiens ortholog of Rpc37 than the S. cerevisiae ortholog of Rpc37, in both size and structure. Overall, our findings provide new insight into the minimal core eukaryotic transcription machinery and help define the most critical features of Pol components and general transcription factors.

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 ß-subunits (Gpß1, -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 ß-subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpHß were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpHß 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 lGpHß and lGpA2 in in vitro pituitary culture. Recombinant lamprey GpH was constructed by tethering the N terminal of lGpA2 to the C terminal of lGpHß 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.

Identification and expression of GnRH2 and GnRH3 in the black sea bass (Centropristis striata), a hermaphroditic teleost.

We cloned two cDNAs for two gonadotropin-releasing hormones, GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH), respectively, from the black sea bass (Centropristis striata). Black sea bass are protogynous hermaphroditic teleosts that change from females to males between 2 and 5 years of age. Similar to other GnRH precursors, the precursors of black sea bass GnRH2 and GnRH23 consisted of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide. Our analyses failed to identify GnRH1. GnRH3 precursor transcript was more widely distributed in a variety of tissues compared with GnRH2. Further examination of GnRH expression and gonadal histology was done in black sea bass from three different size groups: small (11.4-44.1 g), medium (179.4-352.2 g) and large (393.8-607.3 g). Interestingly, GnRH3 expression occurred only in the pituitaries of males in the small and medium groups compared with expression of GnRH2. Future functional studies of the sea bass GnRHs will be valuable in elucidating the potential underlying neuroendocrine mechanisms of black sea bass reproduction and may ultimately contribute to management advances in this commercially important fish.

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.

Expression of three GnRH receptors in specific tissues in male and female sea lampreys Petromyzon marinus at three distinct life stages.

Two recently cloned gonadotropin-releasing hormone (GnRH) receptors (lamprey GnRH-R-2 and lamprey GnRH-R-3) along with lamprey (l) GnRH-R-1 were shown to share similar structural features and amino acid motifs common to other vertebrate receptors. Here we report on our findings of RNA expression of these three GnRH receptors in the three major life stages (larval, parasitic, and adult phases) of the sea lamprey, Petromyzon marinus, a basal vertebrate. For each stage, we examined the expression of messenger RNA encoding the receptors in the brain, pituitary, gonad, heart, muscle, liver, eye, intestine, kidney, skin, thyroid, gill, and endostyle by RT-PCR. In adult lampreys, the spatial expression of the three receptors in the brain and pituitary was investigated by in situ hybridization. In general, the receptors were more widely expressed in adult tissues as compared to parasitic-phase tissues and least widely expressed in the larval tissues. There were noted differences in male and female lampreys in the adult and parasitic phases for all three receptors. The data showed the presence of all three receptor transcripts in brain tissues for adult and parasitic phases and all three receptor transcripts were expressed in the adult pituitaries, but not in the parasitic pituitaries. However, in the larval phase, only lGnRH-R-1 was expressed in the larval brain and pituitary. In situ hybridization revealed that lGnRH-R-2 and -3 were expressed in the pineal tissue of adult female lampreys while lGnRH-R-1 was expressed in the pineal in adult male lampreys, all restricted to the pineal pellucida. In summary, these data provide an initial comparative analysis of expression of three lamprey GnRH receptors suggesting differential regulation within males and females at three different life/reproductive stages.

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.

Evolution of vertebrate GnRH receptors from the perspective of a Basal vertebrate.

This minireview provides the current status on gonadotropin-releasing hormone receptors (GnRH-R) in vertebrates, from the perspective of a basal vertebrate, the sea lamprey, and provides an evolutionary scheme based on the recent advance of whole genome sequencing. In addition, we provide a perspective on the functional divergence and evolution of the receptors. In this review we use the phylogenetic classification of vertebrate GnRH receptors that groups them into three clusters: type I (mammalian and non-mammalian), type II, and type III GnRH receptors. New findings show that the sea lamprey has two type III-like GnRH receptors and an ancestral type GnRH receptor that is more closely related to the type II-like receptors. These two novel GnRH receptors along with lGnRH-R-1 share similar structural features and amino acid motifs common to other known gnathostome type II/III receptors. Recent data analyses of the lamprey genome provide strong evidence that two whole rounds of genome duplication (2R) occurred prior to the gnathostome-agnathan split. Based on our current knowledge, it is proposed that lGnRH-R-1 evolved from an ancestor of the type II receptor following a vertebrate-shared genome duplication and that the two type III receptors resulted from a duplication within lamprey of a gene derived from a lineage shared by many vertebrates.

Molecular cloning and pharmacological characterization of two novel GnRH receptors in the lamprey (Petromyzon marinus).

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (lGnRH-R-2 and lGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands lGnRH-II and -III induced an inositol phosphate (IP) response at lGnRH-R-2 and lGnRH-R-3, whereas the ligand lGnRH-I did not stimulate an IP response. lGnRH-II was a more potent activator of lGnRH-R-3 than lGnRH-III. Stimulation of lGnRH-R-2 and lGnRH-R-3 testing all three lGnRH ligands did not elicit a cAMP response. lGnRH-R-2 has a higher binding affinity in response to lGnRH-III than lGnRH-II, whereas lGnRH-R-3 has a higher binding affinity in response to lGnRH-II than IGnRH-III. lGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that lGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and lGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate.

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 LßT2 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.

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 Proteopedia's use for 3D visualization in structure-related pedagogy.

RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes.

BACKGROUND: Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes. RESULTS: By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions. CONCLUSION: The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.

Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs.

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.

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/

Identifying effects of snoRNA-guided modifications on the synthesis and function of the yeast ribosome.

The small nucleolar RNAs (snoRNAs) are associated with proteins in ribonucleoprotein complexes called snoRNPs ("snorps"). These complexes create modified nucleotides in preribosomal RNA and other RNAs and participate in nucleolytic cleavages of pre-rRNA. The various reactions occur in site-specific fashion, and the mature rRNAs are ultimately incorporated into cytoplasmic ribosomes. Most snoRNAs exist in two structural classes, and most members in each class are involved in nucleotide modification reactions. Guide snoRNAs in the "box C/D" class target methylation of the 2'-hydroxyl moiety, to form 2'-O-methylated nucleotides (Nm), whereas guide snoRNAs in the "box H/ACA" class target specific uridines for conversion to pseudouridine (Psi). The rRNA nucleotides modified in this manner are numerous, totaling approximately 100 in yeast and twice that number in humans. Although the chemistry of the modifications and the factors involved in their formation are largely explained, very little is known about the influence of the copious snoRNA-guided nucleotide modifications on rRNA activity and ribosome function. Among eukaryotic organisms the sites of rRNA modification and the corresponding guide snoRNAs have been best characterized in S. cerevisiae, making this a model organism for analyzing the consequences of modification. This chapter presents approaches to characterizing rRNA modification effects in yeast and includes strategies for evaluating a variety of specific rRNA functions. To aid in planning, a package of bioinformatics tools is described that enables investigators to correlate guide function with targeted ribosomal sites in several contexts. Genetic procedures are presented for depleting modifications at one or more rRNA sites, including ablation of all Nm or Psi modifications made by snoRNPs, and for introducing modifications at novel sites. Methods are also included for characterizing modification effects on cell growth, antibiotic sensitivity, rRNA processing, formation of various rRNP complexes, translation activity, and rRNA structure within the ribosome.

Fine-tuning of translation termination efficiency in Saccharomyces cerevisiae involves two factors in close proximity to the exit tunnel of the ribosome.

In eukaryotes, release factors 1 and 3 (eRF1 and eRF3) are recruited to promote translation termination when a stop codon on the mRNA enters at the ribosomal A-site. However, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. To determine such unknown components, we performed a genetic screen in Saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. For this purpose, we constructed a dedicated reporter strain in which a leaky stop codon is inserted into the chromosomal copy of the ade2 gene. Twenty-five antisuppressor candidates were identified and characterized for their impact on readthrough. Among them, SSB1 and snR18, two factors close to the exit tunnel of the ribosome, directed the strongest antisuppression effects when overexpressed, showing that they may be involved in fine-tuning of the translation termination level.

New bioinformatic tools for analysis of nucleotide modifications in eukaryotic rRNA.

This report presents a valuable new bioinformatics package for research on rRNA nucleotide modifications in the ribosome, especially those created by small nucleolar RNA:protein complexes (snoRNPs). The interactive service, which is not available elsewhere, enables a user to visualize the positions of pseudouridines, 2'-O-methylations, and base methylations in three-dimensional space in the ribosome and also in linear and secondary structure formats of ribosomal RNA. Our tools provide additional perspective on where the modifications occur relative to functional regions within the rRNA and relative to other nearby modifications. This package of new tools is presented as a major enhancement of an existing but significantly upgraded yeast snoRNA database available publicly at http://people.biochem.umass.edu/sfournier/fournierlab/snornadb/. The other key features of the enhanced database include details of the base pairing of snoRNAs with target RNAs, genomic organization of the yeast snoRNA genes, and information on corresponding snoRNAs and modifications in other model organisms.

A systematic, ligation-based approach to study RNA modifications.

Over 100 different chemical types of modifications have been identified in thousands of sites in tRNAs, rRNAs, mRNAs, small nuclear RNAs, and other RNAs. Some modifications are highly conserved, while others are more specialized. They include methylation of bases and the ribose backbone, rotation, and reduction of uridine, base deamination, elaborate addition of ring structures, carbohydrate moieties, and more. We have developed a systematic approach to detect and quantify the extent of known RNA modifications. The method is based on the enzymatic ligation of oligonucleotides using the modified or unmodified RNA as the template. The efficiency of ligation is very sensitive to the presence and the type of modifications. First, two oligo pairs for each type of modification are identified. One pair greatly prefers ligation using the unmodified RNA template over the modified RNA template or vice versa. The other pair has equal reactivity with unmodified and modified RNA. Second, separate ligations with each of the two oligo pairs and the total RNA mixture are performed to detect the presence or absence of modifications. Multiple modification sites can be examined in the same ligation reaction. The feasibility of this method is demonstrated for three 2'O-methyl modification sites in yeast rRNA.

Genome-wide searching for pseudouridylation guide snoRNAs: analysis of the Saccharomyces cerevisiae genome.

One of the largest families of small RNAs in eukaryotes is the H/ACA small nucleolar RNAs (snoRNAs), most of which guide RNA pseudouridine formation. So far, an effective computational method specifically for identifying H/ACA snoRNA gene sequences has not been established. We have developed snoGPS, a program for computationally screening genomic sequences for H/ACA guide snoRNAs. The program implements a deterministic screening algorithm combined with a probabilistic model to score gene candidates. We report here the results of testing snoGPS on the budding yeast Saccharomyces cerevisiae. Six candidate snoRNAs were verified as novel RNA transcripts, and five of these were verified as guides for pseudouridine formation at specific sites in ribosomal RNA. We also predicted 14 new base-pairings between snoRNAs and known pseudouridine sites in S.cerevisiae rRNA, 12 of which were verified by gene disruption and loss of the cognate pseudouridine site. Our findings include the first prediction and verification of snoRNAs that guide pseudouridine modification at more than two sites. With this work, 41 of the 44 known pseudouridine modifications in S.cerevisiae rRNA have been linked with a verified snoRNA, providing the most complete accounting of the H/ACA snoRNAs that guide pseudouridylation in any species.