Modelling co-transcriptional cleavage in the synthesis of yeast pre-rRNA

AbstractIn this paper we present a quantified model of the synthesis of pre-rRNAs in yeast. The chemical kinetics simulation software Dizzy has been used as both the modelling and simulation framework of our study. The simulations have been used to investigate the mechanism of co-transcriptional cleavage which can occur during the synthesis of pre-rRNAs.Throughout the paper we emphasise the strong role of experimental data both in shaping the model and in guiding the analysis which is carried out. Parameter estimation procedures have been used to fit the model to the data and we discuss the validation of the model against the available experimental data. Simulation based on Gillespie’s algorithm is considered to be the reference method for our analysis and a comparison with other simulators is reported. Finally, we define an extended model, that relaxes one of the assumptions of the initial model

Protein factors involved in the biogenesis of the mitochondrial ribosome

The mammalian mitochondria contain their own genome which encodes thirteen polypeptide components of the oxidative phosphorylation (OxPhos) system, and the mitochondrial (mt-) rRNAs and tRNAs required for their translation. The maturation of the mitochondrial ribosome requires both mt-rRNAs to undergo post-transcriptional chemical modifications, folding of the rRNA and assembly of the protein components assisted by numerous biogenesis factors. The post-transcriptional modifications of the mt-rRNAs include base methylations, 2’-O-ribose methylations and pseudouridylation. However, the exact function of these modifications is unknown. Many mitoribosome biogenesis factors still remain to be identified and characterised. This work aims to broaden our understanding of two proteins involved in mitoribosome biogenesis through the study of the function of an rRNA methyltransferase and a novel biogenesis factor. Firstly, we characterised MRM1 (mitochondrial rRNA methyltransferase 1), a highly conserved 2’-O-ribose methyltransferase. We confirmed that MRM1 modifies a guanine in the peptidyl (P) transferase region of the 16S mt-rRNA that specifically interacts with the 3’ end of the tRNA at the ribosomal P-site. In bacteria, the modification is dispensable for ribosomal biogenesis and cell viability under standard conditions. However, in yeast mitochondria, Mrm1p is vital for ribosomal assembly and function. We generated knockout cells lines using programmable nuclease technology, and characterised the possible effects of MRM1 depletion on mitochondrial translation and mitoribosome biogenesis. We demonstrated that neither the enzyme nor the modification is required for human mitoribosomal assembly and translation in our experimental setup. Secondly, we identified a novel mitochondrially-targeted putative RNA endonuclease, YbeY. Using YbeY knockout cell lines, we showed that depletion of YbeY leads to loss of cell viability and OxPhos function as a consequence of a severe decrease in mitochondrial translation. Northern blotting and transcriptomic analysis using next generation RNA-Seq revealed transcript-specific changes to steady state levels. This analysis identified mt-tRNASer as a potential target of YbeY. We investigated the effect of YbeY deficiency on mitoribosomal assembly by quantitative sucrose gradient fractionation and mass spectrometry. This analysis showed that the mt-SSU is depleted in YbeY knockout cells. Further, immunoaffinity purification identified MRPS11 as a key interactor of YbeY. We propose that YbeY is a multifunctional protein that performs endonucleolytic functions in the mitochondria and also acts as a mitochondrial ribosome biogenesis factor, assisting small subunit assembly through its interaction with MRPS11.Medical Research Counci

Getting Ready to Translate: Cytoplasmic Maturation of Eukaryotic Ribosomes

The ribosome is the 'universal ribozyme' that is responsible for the final step of decoding genetic information into proteins. While the function of the ribosome is being elucidated at the atomic level, in comparison, little is known regarding its assembly in vivo and intracellular transport. In contrast to prokaryotic ribosomes, the construction of eukaryotic ribosomes, which begins in the nucleolus, requires >200 evolutionary conserved non-ribosomal trans-acting factors, which transiently associate with pre-ribosomal subunits at distinct assembly stages and perform specific maturation steps. Notably, pre-ribosomal subunits are transported to the cytoplasm in a functionally inactive state where they undergo maturation prior to entering translation. In this review, I will summarize our current knowledge of the eukaryotic ribosome assembly pathway with emphasis on cytoplasmic maturation events that render pre-ribosomal subunits translation competent

DNA sequence encoded repression of rRNA gene transcription in chromatin

Eukaryotic genomes are packaged into nucleosomes that occlude DNA from interacting with most DNA-binding proteins. Nucleosome positioning and chromatin organization is critical for gene regulation. We have investigated the mechanism by which nucleosomes are positioned at the promoters of active and silent rRNA genes (rDNA). The reconstitution of nucleosomes on rDNA results in sequence-dependent nucleosome positioning at the rDNA promoter that mimics the chromatin structure of silent rRNA genes in vivo, suggesting that active mechanisms are required to reorganize chromatin structure upon gene activation. Nucleosomes are excluded from positions observed at active rRNA genes, resulting in transcriptional repression on chromatin. We suggest that the repressed state is the default chromatin organization of the rDNA and gene activation requires ATP-dependent chromatin remodelling activities that move the promoter-bound nucleosome about 22-bp upstream. We suggest that nucleosome remodelling precedes promoter-dependent transcriptional activation as specific inhibition of ATP-dependent chromatin remodelling suppresses the initiation of RNA Polymerase I transcription in vitro. Once initiated, RNA Polymerase I is capable of elongating through reconstituted chromatin without apparent displacement of the nucleosomes. The results reveal the functional cooperation of DNA sequence and chromatin remodelling complexes in nucleosome positioning and in establishing the epigenetic active or silent state of rRNA genes

Ribosoomide lagundamine bakterites

Väitekirja elektrooniline versioon ei sisalda publikatsiooneRibosoomid on makromolekulaarsed kompleksid, mis koosnevad kahest suurest ja ühest väikesest RNAst ja paljudest erinevatest valkudest. Ribosoomides sünteesitakse kõik valgud, mida organismis leida võib, ning aktiivsete ribsoomide konsentratsioon (ja seega sünteesi kiirus) limiteerib rakkude kasvu kiirust. Ehk teisisõnu, mida kiiremini sünteesitakse uusi ribosoome, seda kiiremini kasvab ja jaguneb ka rakk. Kuna ribosomaalse RNA süntees hõlmab ca 80% raku RNA sünteesi aktiivsusest ja ribosoomi valgud moodustavad kuni veerandi raku valgumassist on selge, et mitte ainult ribosoomide funktsioon valgusünteesil vaid ka nende metabolism on rakulises majapidamises määrava tähtsusega. Tõepoolest, juba mõnda aega on teada, et aeglaselt kasvavates bakterirakkudes tegeleb enamus raku RNA lagundamise võimekusest värskelt sünteesitud ribosomaalse RNA lagundamisega. Sellegipoolest on viimase 50 aasta vältel üldiselt usutud, et kord juba valmis tehtud ja kokku pakitud ribosoomid on äärmiselt stabiilsed ning, et neid lagundatakse vaid tugeva stressi tingimustes. Samuti on meie teadmised ribosoomide lagundamise molekulaarsetest mehhanismidest bakteris üsnagi piiratud. Käesoleva doktoritöö eesmärk on kirjeldada ribosoomide lagundamist kasvavates soolekepikese (Escherichia coli) rakkudes ja heita valgust ribsoomide lagundamise mehhanismidele, molekulaarsetele radadele ning ensüümidele, mis selles protsessis osalevad. Me avastasime üllatusega, et kuigi ribosoome tõepoolest lagundatakse kasvavates bakterirakkudes, toimub see protsess vaid rakukultuuri kasvu aeglustumise perioodil, mis eelneb statsionaarse kasvufaasi saabumisele. Meil ei õnnestunud tuvastada küpsete ribosoomide lagundamist ei ühtlase kiirusega kasvavates ega ka null-kiirusega kasvavates rakkudes. Võimalik, et ribosoomide lagundamine aitab rakke neid ette valmistades eluks statsionaarses faasis, mil ei vajata suurt valgusünteesi võimekust, küll aga vabu komponente, millest elutingimuste paranedes kiiresti uusi makromolekule tootma hakata. Lisaks leidsime, et osad (kuid mitte kõik) ribosoomi RNAd inaktiveerivad mutatsioonid viivad samuti ribsoomide lagundamisele, kuid miskipärast lagundatakse siis nii mutantseid ning inaktiivseid kui metsiktüüpi ning aktiivseid ribosoome. Jällegi viitab see, et ribsoomide lagundamise eesmärk võiks olla üldise ribosoomide konsentratsiooni alandamine rakus. Kui me lisasime ribsoomide lagundamise katsesüsteemi valgusünteesi pärssivat antibiootikumi kloramfenikool, päästsime me sellega ribosoomid lagundamisest. Seda tulemust võib tõlgendada viisil, et de novo valgusüntees on vajalik ribosoomide lagundamisprogrammi käivitamiseks rakus. Testides ribosoomide lagundamise võime osas bakteritüvesid, kus puuduvad erinevad RNAd lagundavad ensüümid, leidsime kaks ensüümi, mille puudumise korral ribosoome ei lagundatud. Neist esimene, RNaas R, lõhub RNAsid alates nende tagumisest ehk 3’ otsast ning tunneb erilist lõbu kõrge sekundaarstruktuuriga RNA-de hävitamisest. RNaas R on ka eelnevalt näidatud osalevat ribosoomide lagundamisel. Teine ensüüm on seevastu suhteliselt vähetuntud endoribonukleeas nimega YbeY, mis lõikab RNAd katki keskelt, mitte ei lagunda seda otstest. See huvitav valk on arvatud osalevat ribsoomide kokkupakkimise kvaliteedikontrollil, kus ta on vajalik kõige viimases etapis, mil tuntakse ära valgusünteesil ebaõnnestuvad ribosoomid ja suunatakse need lagundamisse. Meie katsed viitavad, et seesama valk võib valla päästa ka töökorras olevate ribosoomide lagundamise, tehes ribosoomi RNAsse esimese lõike ning tekitades sellega kaitsetu 3’ otsa, mida tunneb ära RNaas R, mis omakorda suudab ribosoomi RNA täielikult lagundadaRibosomes are macromolecular complexes that consist of two large and one small RNA and of many different small proteins. The ribosome synthesizes all cellular proteins and the concentration of active ribosomes is rate limiting for cell growth. As synthesis or ribosomal RNA encompasses 80% of cellular RNA synthesis activity and the ribosomal proteins can make up half of the cellular protein mass, it is clear that ribosomal metabolism, including ribosomal degradation, makes a worthy object of study. Nevertheless, during the past half century it has been widely believed that mature ribosomes are quite stable in the cells. The major goal of this dissertation is to describe the degradation of mature ribosomes in growing E. coli cells and to shed light on the molecular mechanism of degradation. We discovered that while mature ribosomes are indeed degraded in cells growing in batch cultures, this process is limited to the slowing of growth phase, which precedes entry into the stationary phase. We were unable to detect degradation during constant-rate growth and during early stationary phase. In addition, we found that some, but not all, ribosome-inactivating mutations in 23S rRNA and 16S rRNA led to degradation of both mutant and wild-type ribosomal RNAs. Thus, unlike in yeast, the ribosome degradation in E. coli is a general process that, once initiated, does not discriminate between active and inactive ribosomes. As ribosome degradation is inhibited by the protein synthesis inhibitor chloramphenicol, we further suggest that de novo protein synthesis might be needed for triggering the degradation program. To pinpoint the enzymes responsible for degradation we tested several strains defective for different RNases. We found two RNases, RNaseR and YbeY, whose deletion saved ribosomes from degradation. RNaseR is a well studied 3’ to 5’ exonuclease whose role in degrading heavily structured RNAs, including the rRNAs, is well established. In contrast YbeY is a potential endonuclease recently implicated in a late step ribosomal quality control, which could well be the initiating endonuclease, whose cut(s) in rRNA would present substrates for RNaseR to further scavenge into mononucleotides

The role of RNA Polymerase II-dependent transcription elongation in the cross-talk between mRNA synthesis and decay.

The main molecule in gene expression is messenger RNA (mRNA) which transfers the information contained in genes in the nucleus to the cytoplasm where it is translated into proteins that carry out cellular functions. mRNA levels are determined through its synthesis, by the RNA polymerase II, and degradation, which involves the Ccr4-Not complex and Xrn1. It has become increasingly apparent that the mRNA concentration in a cell is maintained at a particular level even through stressful situations. The way the cell is able to do this is by a cross-talk between the machinery responsible for its transcription and that responsible for its degradation. In this work we have attempted to unravel the mechanisms by which this cross-talk occurs. For this complex task, we first studied how transcription and degradation was affected after deleting a single gene known to be involved in either one of these mechanisms. This study confirmed the existence of a strong feedback between mRNA synthesis and decay, and also helped us uncover some of the elements important for this cross-talk. The most interesting finding was the correlation between transcription elongation and mRNA degradation, suggesting that it is directly relevant for cross-talk. Second, we mathematically modelled and computationally simulated this coupling between transcription and mRNA decay. Thanks to in silico experimentation, we found that two proteins involved in degradation (Ccr4-Not and Xrn1) were most likely also involved in transcription, and therefore the feedback mechanism. This result complements that of the first study and places both Ccr4-Not and Xrn1 as important proteins for cross-talk. Finally, we analysed the exonuclease Xrn1 in depth through genome-wide experiments. This study allowed us to conclude that Xrn1 is directly involved in transcription and influence both early and late RNA polymerase II-dependent transcription elongation. The results of this thesis have enabled us to come up with a model for how the cross-talk could work in yeast cells and allowed us to envision new hypotheses to explain the novel results.Premio Extraordinario de Doctorado U

Co-transcriptional splicing in two yeasts

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length

Combined experimental and computational approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinalis

Non-protein-coding RNAs represent a large proportion of transcribed sequences in eukaryotes. These RNAs often function in large RNA–protein complexes, which are catalysts in various RNA-processing pathways. As RNA processing has become an increasingly important area of research, numerous non-messenger RNAs have been uncovered in all the model eukaryotic organisms. However, knowledge on RNA processing in deep-branching eukaryotes is still limited. This study focuses on the identification of non-protein-coding RNAs from the diplomonad parasite Giardia intestinalis, showing that a combined experimental and computational search strategy is a fast method of screening reduced or compact genomes. The analysis of our Giardia cDNA library has uncovered 31 novel candidates, including C/D-box and H/ACA box snoRNAs, as well as an unusual transcript of RNase P, and double-stranded RNAs. Subsequent computational analysis has revealed additional putative C/D-box snoRNAs. Our results will lead towards a future understanding of RNA metabolism in the deep-branching eukaryote Giardia, as more ncRNAs are characterized