RNA Processing in Eukaryotes

Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS Torsten Neuhof 1 , Ralf Dieckmann 1, *, Irina S. Druzhinina 2 , Christian P. Kubicek 2 , Tiina Nakari-Seta ¨ la ¨ 3 , Merja Penttila ¨ 3 and Hans von Do ¨ hren 1 1 TU Berlin, Institut fu ¨ r Chemie, FG Biochemie und Molekulare Biologie, Berlin, Germany 2 FB Gentechnik und Angewandte Biochemie, Institut fu ¨ r Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, TU Wien, Vienna, Austria 3 VTT Technical Research Centre of Finland, Espoo, Finland Hydrophobins are small proteins thought to be ubi- quitous in filamentous fungi. They are usually present on the outer surfaces of cell walls of hyphae and coni- dia. Here, they mediate interactions between the fun- gus and the environment, such as surface recognition during pathogenic interactions with plants, insects or other fungi, and also in symbiosis. The size of hydro- phobins ranges from approximately 75 to 400 amino acid residues; they contain eight positionally conserved cysteine residues, and can be divided into two classes according to their hydropathy profiles and spacing between the conserved cysteines [1]. The anamorphic fungal genus Trichoderma (Hypocre- ales, Ascomycota) contains cosmopolitan soil-borne fungi with economic importance as biocontrol agents and producers of beneficial metabolites and enzymes. In addition, Trichoderma spp. have recently been reported to occur as endophytes, eliciting positive plant responses against potential pathogens [2]. Hydrophobins are likely to play a role in this process, and a hydrophobin gene has in fact recently been isolated that leads to overpro- duction of hydrophobins during endophytic interactions between Trichoderma asperellum and cucumber roots [3]. However, hydrophobins may also be involved in the Keywords fungal biomarker; hydrophobin; intact-cell MS; MALDI-TOF MS; Trichoderma Correspondence H. von Do ¨ hren, TU Berlin, Institut fu ¨ r Chemie, FG Biochemie und Molekulare Biologie, Franklinstr. 29, 10587 Berlin, Germany Fax: +49 30 314 24783 Tel: +49 30 314 22697 E-mail: Doehren@chem.tu-berlin.de *Present address AnagnosTec, Gesellschaft fu ¨ r Analytische Biochemie und Diagnostik mbH, Potsdam- Golm, Germany (Received 19 September 2006, revised 27 November 2006, accepted 6 December 2006) doi:10.1111/j.1742-4658.2007.05636.x Intact-cell MS (ICMS) was applied for the direct detection of hydropho- bins in various species and strains of Hypocrea ⁄ Trichoderma. In both myce- lia and spores, dominating peaks were identified as hydrophobins by detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis of knockout mutants, and comparison with protein databases. Strain-speci- fic processing was observed in the case of Hypocrea jecorina (anamorph Trichoderma reesei). An analysis of 32 strains comprising 29 different spe- cies of Trichoderma and Hypocrea showed hydrophobin patterns that were specific at both at the species and isolate (subspecies) levels. The method therefore permits rapid and direct detection of hydrophobin class II com- positions and may also provide a means to identify Trichoderma (and other fungal) species and strains from microgram amounts of biomass without prior cultivation. Abbreviations HFB, hydrophobin; ICMS, intact-cell MALDI-TOF MS. FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 841 mechanism of mycoparasitism as well as the coloniza- tion of decaying wood. Our RNA Processing in Eukaryotes RNA Processing in Eukaryotes Bởi: OpenStaxCollege After transcription, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein synthesis machinery mRNA Processing The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA Eukaryotic mRNAs last for several hours, whereas the typical E coli mRNA lasts no more than five seconds Pre-mRNAs are first coated in RNA-stabilizing proteins; these protect the pre-mRNA from degradation while it is processed and exported out of the nucleus The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5' and 3' ends of the molecule, and the removal of intervening sequences that not specify the appropriate amino acids In rare cases, the mRNA transcript can be “edited” after it is transcribed Evolution Connection RNA Editing in TrypanosomesThe trypanosomes are a group of protozoa that include the pathogen Trypanosoma brucei, which causes sleeping sickness in humans ([link]) Trypanosomes, and virtually all other eukaryotes, have organelles called mitochondria that supply the cell with chemical energy Mitochondria are organelles that express their own DNA and are believed to be the remnants of a symbiotic relationship between a eukaryote and an engulfed prokaryote The mitochondrial DNA of trypanosomes exhibit an interesting exception to The Central Dogma: their pre-mRNAs not have the correct information to specify a functional protein Usually, this is because the mRNA is missing several U nucleotides The cell performs an additional RNA processing step called RNA editing to remedy this 1/6 RNA Processing in Eukaryotes Trypanosoma brucei is the causative agent of sleeping sickness in humans The mRNAs of this pathogen must be modified by the addition of nucleotides before protein synthesis can occur (credit: modification of work by Torsten Ochsenreiter) Other genes in the mitochondrial genome encode 40- to 80-nucleotide guide RNAs One or more of these molecules interacts by complementary base pairing with some of the nucleotides in the pre-mRNA transcript However, the guide RNA has more A nucleotides than the pre-mRNA has U nucleotides to bind with In these regions, the guide RNA loops out The 3' ends of guide RNAs have a long poly-U tail, and these U bases are inserted in regions of the pre-mRNA transcript at which the guide RNAs are looped This process is entirely mediated by RNA molecules That is, guide RNAs—rather than proteins—serve as the catalysts in RNA editing RNA editing is not just a phenomenon of trypanosomes In the mitochondria of some plants, almost all pre-mRNAs are edited RNA editing has also been identified in mammals such as rats, rabbits, and even humans What could be the evolutionary reason for this additional step in pre-mRNA processing? One possibility is that the mitochondria, being remnants of ancient prokaryotes, have an equally ancient RNAbased method for regulating gene expression In support of this hypothesis, edits made to pre-mRNAs differ depending on cellular conditions Although speculative, the process of RNA editing may be a holdover from a primordial time when RNA molecules, instead of proteins, were responsible for catalyzing reactions 5' Capping While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to the 5' end of the growing transcript by a phosphate linkage This moiety (functional group) protects the nascent mRNA from degradation In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosomes 2/6 RNA Processing in Eukaryotes 3' Poly-A Tail Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail This modification further protects the pre-mRNA from degradation and signals the export of the cellular factors that the transcript needs to the cytoplasm Pre-mRNA Splicing Eukaryotic genes are composed of exons, which correspond to protein-coding sequences (ex-on signifies that they are expressed), and intervening sequences called introns (int-ron denotes their intervening role), which may be involved in gene regulation but are removed from the pre-mRNA during processing Intron sequences in mRNA not encode functional proteins The discovery of introns came as a surprise to researchers in the 1970s who expected that pre-mRNAs would specify protein sequences without further processing, as they had observed in prokaryotes The genes of higher eukaryotes very ...Query Processing in RDF/S-based P2P Database Systems George Kokkinidis, Lefteris Sidirourgos and Vassilis Christophides Institute of Computer Science - FORTH Vassilika Vouton, PO Box 1385, GR 71110, Heraklion, Greece and Department of Computer Science, University of Crete GR 71409, Heraklion, Greece {kokkinid, lsidir, christop}@ics.forth.gr 1 Introduction Peer-to-p ee r (P2P) computing is currently attracting enormous attention, spurred by the popularity of file sharing systems such as Napster [31], Gnutella [15], Freenet [9], Morpheus [30] and Kazaa [25]. In P2P systems a very large number of autonomous computing nodes (the peers) pool together their resources and rely on each other for data and services. P2P computing introduces an interesting paradigm of decentralization going hand in hand with an increasing self-organization of highly autonomous peers. This new paradigm bears the potential to realize computing systems that scale to very large numbers of participating nodes while ensuring fault-tolerance. However, existing P2P systems off er very limited data management facil- ities. In most of the cases, searching relies on simple selection conditions on attribute-value pairs or IR-style string pattern matching. These limitations are acceptable for file-sharing applications, but in order to support highly dynamic, ever-changing, autonomous social organizations (e.g., scientific or educational communities) we need richer facilities in exchanging, querying and integrating (semi-)structured data hosted by peers. To this end, we es- sentially need to adapt the P2P computing paradigm to a distributed data management setting. More precisely, we would like to support loosely coupled communities of peer bases, where each base can join and leave the network at free will, while groups of peers can collaboratively undertake the responsibility of query pro c es sing. The importance of intensional (i.e., schema) information for integrat- ing and querying peer bases has been highlighted by a number of recent projects [4, 34, 17, 1]. A natural candidate for representing descriptive schemata of information resources (ranging from simple structured vocab- ularies to complex reference models [40]) is the Resource Description Frame- work/Schema Language (RDF/S). In particular, RDF/S (a) enables a mod- 2 George Kokkinidis, Lefteris Sidirourgos and Vassilis Christophides ular design of descriptive schemata based on the mechanism of namespaces; (b) allows easy reuse or refinement of existing schemata through subsumption of both class and property definitions; (c) supports partial descriptions since properties associated with a resource are by default optional and repeated and (d) permits super-imposed descriptions in the sense that a resource may be multiply classified under several classes from one or several schemata. These modelling primitives are crucial for P2P data management systems where monolithic RDF/S schemata and resource descriptions cannot be constructed in advance and peers may have only partial descriptions about the available resources. In this chapter, we present the ongoing SQPeer middleware for routing and planning declarative queries in peer RDF/S bases by exploiting the schema of peers. More precisely, we make the following contributions: • In Section 2.1 we illustrate how peers can formulate complex (conjunctive) queries against an RDF/S schema using RQL query patterns [23]. • In Section 2.2 we detail how peers can advertise their base at a fine-grained level. In particular, we are employing RVL view patterns [29] for declaring the parts of an RDF/S schema which are actually (or can be) populated in a peer base. • In Section 2.3 we introduce a semantic routing algorithm that matches a given RQL query against a set of RVL peer Semantic and affective processing in psychopaths: An event-related potential ~ERP! study KENT A. KIEHL, a ROBERT D. HARE, a JOHN J. MCDONALD, a and JOHANN BRINK b,c a Department of Psychology, University of British Columbia, Vancouver, Canada b Department of Psychiatry, University of British Columbia, Vancouver, Canada c Regional Health Center ~Pacific!, Abbotsford, British Columbia, Canada Abstract We tested the hypothesis that psychopathy is associated with abnormal processing of semantic and affective verbal information. In Task 1, a lexical decision task, and in Task 2, a word identification task, participants responded faster to concrete than to abstract words. In Task 2, psychopaths made more errors identifying abstract words than concrete words. In Task 3, a word identification task, participants responded faster to positive than to negative words. In all three tasks, nonpsychopaths showed the expected event-related potential ~ERP! differentiation between word stimuli, whereas psychopaths did not. In each task, the ERPs of the psychopaths included a large centrofrontal negative-going wave ~N350!; this wave was absent or very small in the nonpsychopaths. The interpretation and significance of these differences are discussed. Descriptors: Psychopathy, Language, Event-related potentials, Affective processes, Semantic processes Psychopathy is a personality disorder defined by a constellation of affective, interpersonal, and behavioral characteristics, including, egocentricity, manipulativeness, deceitfulness, shallow affect, lack of empathy, guilt or remorse, and a propensity to violate social and legal expectations and norms ~Hare, 1991, 1993, 1996a!. The fac- tors related to the development and maintenance of the disorder are not well understood, but recent theory and research suggest that the cognitions, language, and experiences of psychopaths appear to lack depth and affective meaning ~Christianson et al., 1996; Cleck- ley, 1976; Day &Wong, 1996; Gillstrom, 1994; Hare, 1993; Hayes, 1995; Intrator et al., 1997; Patrick, 1994; Williamson, Harpur, & Hare, 1990, 1991!. The proposition that psychopathy is associated with abnormalities in semantic and affective processing is not new. Indeed, Cleckley ~1976! speculated that psychopaths suffer from a form of “semantic aphasia” in which the semantic and emotional components of cognition are disturbed and poorly integrated. These cognitive impairments may be part of the reason why psychopaths are so resistant to psychological treatment ~Rice, Harris, &Cormier, 1992; see Hare, 1993, for a review!. A large part of modern cog- nitive therapy that has been applied to the treatment of psychopaths involves teaching conceptually abstract information ~e.g., empathy, role-playing, rational thinking!. Our clinical observation of these treatment programs has revealed that psychopaths have difficulty comprehending this information ~see also Gillstrom, 1994; William- son, 1991!. Specifically, psychopaths are more likely than others to attempt to interpret abstract information by presenting it in more concrete terms. Understanding the nature of these impairments may lead to alternative, and hopefully superior, forms of treatment. Early empirical research sought to elucidate these cognitive impairments by examining the relationship between psychopathy and hemispheric lateralization. There is a now a relatively large body of evidence that suggests psychopathy is associated with weak or unusually lateralized cerebral hemispheres, specifically relating to processing language stimuli ~Day & Wong, 1996; Hare, 1979; Hare & Jutai, 1988; Hare & McPherson, Nop53p, an essential nucleolar protein that interacts with Nop17p and Nip7p, is required for pre-rRNA processing in Saccharomyces cerevisiae Daniela C. Granato 1 , Fernando A. Gonzales 1 , Juliana S. Luz 1 , Fla ´ via Cassiola 2 , Glaucia M. Machado-Santelli 2 and Carla C. Oliveira 1 1 Department of Biochemistry, Chemistry Institute, University of Sa˜o Paulo, Brazil 2 Department of Cellular and Development Biology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Brazil The factors involved in rRNA processing in eukaryotes assemble cotranscriptionally onto the nascent pre- rRNAs and include endonucleases, exonucleases, RNA helicases, GTPases, modifying enzymes and snoRNPs (small nucleolar ribonucleoproteins). The precursor of three of the four eukaryotic mature rRNAs contains the rRNA sequences flanked by two internal (ITS1 and ITS2) and two external (5¢-ETS and 3¢-ETS) spacer sequences that are removed during processing [1,2]. The pre-rRNA is first assembled into a 90S parti- cle that contains U3 snoRNP and 40S subunit-process- ing factors [3,4]. The early pre-rRNA endonucleolytic cleavages at sites A 0 ,A 1 and A 2 occur within the 90S particles [3,5]. A 2 cleavage releases the first pre60S particle, which differs in composition from the known 90S particle. Pre60S particles contain 27S rRNA, ribo- somal L proteins and many nonribosomal proteins [6]. As they mature, pre60S particles migrate from the nuc- leolus to the nucleoplasm and their content of non- ribosomal factors changes [7,8]. Nip7p was among the proteins identified in the early pre60S particle [6–8], and has been shown to participate in the processing of 27S pre-rRNA to the formation of 25S [9]. Interest- ingly, Nip7p also binds the exosome subunit Rrp43p [10]. The exosome complex is responsible for the de- gradation of the excised 5¢-ETS and for the 3¢)5¢ exo- nucleolytic processing of 7S pre-rRNA to form the mature 5.8S rRNA. The exosome is also involved in the processing of snoRNAs and in mRNA degradation [11–13]. During processing, pre-rRNA undergoes covalent modifications that include isomerization of some uri- dines into pseudouridines and addition of methyl groups to specific nucleotides, mainly at the 2¢-O posi- Keywords rRNA processing; nucleolus; ribosome synthesis; Saccharomyces cerevisiae; pre60S Correspondence C. C. Oliveira, Departamento de Bioquı ´ mica, Instituto de Quı ´ mica, USP, Ave Prof Lineu Prestes, 748 Sa˜o Paulo, SP 05508-000, Brazil Fax: +55 11 3815 5579 Tel: +55 11 3091 3810 (ext 208) E-mail: ccoliv@iq.usp.br (Received 12 February 2005, revised 1 July 2005, accepted 12 July 2005) doi:10.1111/j.1742-4658.2005.04861.x In eukaryotes, pre-rRNA processing depends on a large number of non- ribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two- hybrid system, but its depletion affects the exosome function. ARSING )IFFICULTIES U )HONOLOGICAL )ROCESSING IN I[TALIAN Rodolfo Delmonte Istituto di Linguistica e Didattica Ca' Garzoni-Moro - S.Mareo 3417 Univeesit~ degli Studi di Venezla(I) ABSTRACT A recognition grammar to supply information to a text-to-speech system for the synthesis of Italian must rely heavily upon lexical information, in order to instantiate the appropriate grammatical relations. Italian is an almost free word order language which nonetheless adopts fairly analysable stra- tegies to move major constituents: some of these can strongly affect the functioning of the pho- notogical component. Two basic claims will be made: i. difficulties in associating grammatical functions to constituent structure can be overcome only if Lexical Theory is adopted as a general theoretical framework, and translated into adequate computational formalisms like ATN or CHART; ii. decisions made at previous point affect focus structure construal rules, which are higher level phonologicaI rules which individuate intonation centre, build up adequate Intonational Groups and assign pauses to adequate sites, all being very sensitive to syntactic and semantic i nf ormat i on. We will concentrate on Subject/Object function association to c-structure in Italian, and its relation to ATN formalism, in particular HOLD mechanism and F~Gging. Then we will show how syn- tactic decisions interact with an intonation grammar. We shall also introduce two functional no- tions: STRUCTURE REVERSIBILITY vs. FUNCTIONAL REVERSIBILITY in Italian. 1. INTRODUCTION In a recent paper we presented (Delmonte, 1983) a phonological processor for Italian which has been implemented at the University of Venice and is used in a text-to-speech system (Delmonte et al., 1984) for the synthesis of Italian at the Centre of Computational Sonology of the University of Padua. Recently the system has been equipped with a lexi- con and a morphological analyser (DeImonte et al., 1985) while the parser is on its way to be built, which, since we adopt Lexical-Functiona! Grammar (LFG) (Bresnan, 1982), as background linguistic theory, should take the form of a chart, much in the vein of Kay's (1977,1979,1980) and Kaplan's (1973) functional and general syntactic parsers. At present we are working at the context-free gram- mar and the semantic information to be associated with each lexical entry. As it appears, Italian is a much more comptex language to be analysed when compared with English, German and French. As we shall discuss in the paper, difficulties arise basically because Italian has a relatively much higher freedom in the order of constituents than the above mentioned languages. Also, the phenomenon of the unexpressed Subject or Null Subject, makes the working of a parser a much harder task. In this sense, a chart being unbiased as to what procedure to adopt in the course of the analysis, will allow 136 the parser to benefit both from tcp-down and bottom- up procedures in an efficient way (plus the obvious back-up and parallel processing operations usually required). Besides, both semantic and grammatical features need to be present throughout the parsing process, and they will be used to guide the overall parsing strategy. 2. P~IVE STRUCTURES AND REVERSIBILITY Assuming that the ultimate goal of a parser ts that of accomplishing the analysis of the input text in terms cf underlying grammatical relations, we are usually fronted with the task of assigning thematic roles to functional representations mapped onto con- stituent structures, as well as defining other non trivial semantic relations including ellipsis, pre- dication, coordination, quantifier/negation and mo- dality scope, head to modifier/complement/adjunct relations. All these aspects are relevant to a con- ... pre-mRNA processing step is important for initiating translation? poly-A tail RNA editing splicing 7-methylguanosine cap D What processing step enhances the stability of pre-tRNAs and pre-rRNAs?... amino acid phenylalanine to a growing polypeptide chain The anticodon AAG binds the Codon UUC on the mRNA The amino acid phenylalanine is attached to the other end of the tRNA 5/6 RNA Processing. .. capping and the addition of a poly-A tail—just to generate a single, translatable mRNA molecule Link to Learning See how introns are removed during RNA splicing at this website Processing of tRNAs