<p>RPL23a is one of the ~80 ribosomal proteins (r-proteins) of the cytoplasmic ribosome in the model plant <i>Arabidopsis thaliana</i>. The objectives of this research were to establish Arabidopsis RPL23a as a functional r-protein, characterize expression patterns for the two genes (RPL23aA and B) encoding RPL23a using reverse transcription PCR (RT-PCR), and identify regulatory elements controlling the expression of RPL23aA and B. Complementation of a yeast l25 mutant with AtRPL23aA demonstrated that AtRPL23aA can fulfill all the essential functions of L25 in vivo. A survey of various Arabidopsis tissue types showed that, while RPL23aA and B expression patterns both showed increased transcript abundance in mitotically active tissues, RPL23aB transcript levels were generally lower than those of RPL23aA and responded differently to abiotic stresses. In order to determine cis regulatory elements controlling RPL23aA and B expression, the 5 regulatory region (RR) of each gene was characterized via plants carrying a series of 5 RR deletion fragments upstream of a reporter. Transcript start sites and 5 untranslated regions (UTRs) for both RPL23aA and B were also characterized using primer extension, and transcripts from 5 deletion transgenics were amplified using RT-PCR. No correlation was observed between putative cis-acting elements and the expression patterns from the RPL23aA and B deletion transgenics, although a 102 bp sequence in the RPL23aB 5 RR was found to contain pollen-specific elements. 5 leader introns were found in each RPL23a gene, and amplification of transgene transcripts from deletion series plants indicated the importance of post-transcriptional and translational regulation in RPL23aA and B expression. This thesis work is the first demonstration of a plant RPL23a protein as a functional member of the L23/L25 (L23p) conserved r-protein family, and is one of the few in-depth studies of the regulation of r-protein genes in plants. While the majority of previous research on plant r-protein gene expression has focused solely on transcript levels, I show herein that post-transcriptional mechanisms have a critical role in regulating these genes, and thus plant r-protein genes more strongly resemble their mammalian counterparts than those of yeast in terms of structure and regulation.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Bowen-Conradi syndrome is a lethal autosomal recessive disorder affecting Hutterite infants, characterized by severe growth and psychomotor retardation, and leading to death at an average age of thirteen months. Linkage analysis and sequencing identified an A>G mutation in EMG1 as the probable cause of the disease. This gene is implicated in ribosome biogenesis, and the mutation results in an unstable EMG1 protein. The reduction in available EMG1 causes a transient delay in processing of the ribosomal small subunit 18S rRNA, leading to cell cycle delay at G2/M and a subsequent reduction in cell proliferation rates in patient lymphoblasts. A mouse model of Bowen-Conradi syndrome also displayed severe developmental delay, with prominent effects in the cranial central nervous system. Embryos died prematurely during development, probably due to decreased proliferation rates accompanied by apoptosis. These results shed light on the etiology of Bowen-Conradi syndrome, and open the door for development of treatments.

The GTPase elongation factor G (EF-G) is an important player in the complex process of protein synthesis by bacterial ribosomes. Although extensively studied much remains to be learned about this fascinating protein. In the elongation phase, after incorporation of each amino acid into the growing peptide chain, EF-G translocates the ribosome along the mRNA template. In the recycling phase, when the synthesis of a protein has been completed, EF-G, together with ribosome recycling factor (RRF), splits the ribosome into its subunits. We developed the first in vitro assay for measuring the average time of a complete translocation step at any position along the mRNA. Inside the open reading frame, at saturating EF-G concentration and low magnesium ion concentration, translocation rates were fast and compatible with elongation rates observed in vivo. We also determined the complete kinetic mechanism for EF-G- and RRF-dependent splitting of the post-termination ribosome. We showed that splitting occurs only when RRF binds before EF-G and that the rate and GTP consumption of the reaction varies greatly with the factor concentrations. The antibiotic fusidic acid (FA) inhibits bacterial protein synthesis by binding to EF-G when the factor is ribosome bound, during translocation and ribosome recycling. We developed experimental methods and a theoretical framework for analyzing the effect of tight-binding inhibitors like FA on protein synthesis. We found that FA targets three different states during each elongation cycle and that it binds to EF-G on the post-termination ribosome both in the presence and absence of RRF. The stalling time of an FA-inhibited ribosome is about hundred-fold longer than the time of an uninhibited elongation cycle and therefore each binding event has a large impact on the protein synthesis rate and may induce queuing of ribosomes on the mRNA. Although ribosomes in the elongation and the recycling phases are targeted with similar efficiency, we showed that the main effect of FA in vivo is on elongation. Our results may serve as a basis for modelling of EF-G function and FA inhibition inside the living cell and for structure determination of mechanistically important intermediate states in translocation and ribosome recycling.

The RNA helicase Prp43p is one of the few members of the DEAH-box helicase family that is known to operate in more than one cellular process in Saccharomyces cerevisiae. With roles in ribosome biogenesis and pre-mRNA splicing, Prp43p may be important in maintaining a communication conduit between these two pathways. Our studies provide insights into how Prp43p function is regulated through the use of three cofactors, Ntr1p, Pfa1p, and Gno1p, all of which interact with Prp43p at different steps of pre-mRNA splicing or ribosome biogenesis. Each cofactor contains a unique G-patch domain and our data show that they associate with Prp43p in a mutually exclusive manner. A strong growth defect and RNA processing phenotypes are seen upon overexpression of Pfa1p due to the dominance of Pfa1p interaction with Prp43p. Moreover, excess Pfa1p precludes Prp43p from interacting with either 35S pre-rRNA or U6 snRNA, indicating this one cofactor can negatively regulate Prp43p recruitment into ribosome biogenesis and pre-mRNA splicing pathways, respectively. We have determined that Ntr1p and Gno1p are able to compete with one another for Prp43p occupancy. Similar to Ntr1p, we show that the G-patch domain of Gno1p contributes to its association with Prp43p. To further understand pathway specificity of Prp43p, we characterized conditional prp43 alleles with mutations C-terminal to the conserved RecA domains of Prp43p. These novel alleles affect pre-mRNA splicing and ribosome biogenesis, though none are mutually exclusive. Multiple prp43 alleles are deficient in tri-snRNP formation, a previously uncharacterized phenotype in prp43 mutants. The majority of our prp43 mutants display varying rRNA defects, with some alleles impacting ribosome biogenesis more severely or moderately than known prp43 ATPase mutants. To correlate the processing defects seen in each allele, we have determined the extent of association of the mutants with each G-patch protein. Altogether, our data support a working model for Prp43p in which its substrate specificity, activation, and cellular distribution is coordinated through the efforts of the three G-patch proteins in yeast and sheds light on potential mechanisms of general DExH/D helicase function and regulation. / text

The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts. The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

Vesicular stomatitis virus (VSV) infection causes inhibition of host protein synthesis, in part by sequestering initiation factors required for mRNA cap recognition. The viral mRNAs share a common mRNA structure to those of the host cell, with a 5' cap and 3' polyadenylate tail, but continue to be efficiently translated despite host translational shutoff. This observation suggests that a non-canonical translation pathway is utilized for viral protein synthesis. To investigate this pathway, we performed an RNA interference screen to identify genes required for VSV replication. In contrast to bulk cellular translation, viral translation is hypersensitive to knockdown of a protein constituent of the 60S ribosomal subunit, rpL40. Depletion of rpL40 diminishes VSV protein synthesis by >90% and is restored through complementation with an siRNA-resistant mutant of rpL40. To delineate the mechanism by which rpL40 is required for viral protein synthesis, we reconstituted translation of VSV mRNA in yeast extracts in vitro. In the absence of rpL40, we show that the two ribosomal subunits fail to associate on VSV mRNA, and the small subunit does not scan to the initiation codon. Regulation by rpL40 occurs in context of the large subunit, providing direct evidence for translational control by the ribosome itself. This rpL40- dependent mechanism of translation initiation is broadly conserved within eukaryotes, governed solely through an RNA determinant, and is utilized by several viruses within the order Mononegavirales. To determine whether a subset of cellular transcripts also require rpL40 for translation, we identified polysome-associated mRNAs in yeast by deep sequencing. We demonstrate that in vitro and in vivo translation of candidate mRNAs, including factors involved in stress responses, are inhibited in the absence of rpL40. This finding suggests that rpL40 plays a critical role in transcript-specific translation during cellular stress. Collectively, our work identifies an alternative translation pathway that is specifically dependent on rpL40, revealing a previously unappreciated mechanism of protein synthesis regulation by the ribosome.

Several mechanisms have been proposed for the pol I transcript termination in Schizosaccharomyces pombe. Two well known models are “Pause and Release” and “Torpedo”. Each mechanism explains the role of some of the cis- and trans-factors in transcript termination and the eventual maturation of the ribosomal RNA, but neither mechanism can explain all the experimental observations. A recent study has suggested that each of the two mechanisms can terminate the pol I transcription independently but with significantly less efficiency than the presence of both mechanisms. To help clarify the reasons for the discrepancies in these data, in this study the suggested mechanisms were examined further in three areas by using alternative techniques. First, the effect of uracil concentration or selection times on the transformation frequency of alternative 3’external transcribed spacer (3’ETS) constructs were assessed. Consistent with the previous results a construct containing the full 3’ETS showed the higher transformation frequencies compared with a construct containing only the hairpin or only the termination sites. However, results showed neither the uracil concentration nor selection times have a significant effect on the transformation frequency. Second, to further confirm the “pause and release” mechanism, the termination sites identified by S1 nuclease studies were analyzed using ligation-mediated RT-PCR. The 3’ terminus of the mature 25S rRNA was demonstrated readily but, unexpectedly, the 3’termini of the 3’ETS precursor molecules were not detected, possibly because of their specific structure. Finally, the 3’ extended rRNA precursors were studied by semi-quantitative RT-PCR. These appeared not to correspond with past nuclease protection analyses nor did they demonstrate downstream exonuclease function, observations which question our current understanding of Pol I transcript termination.

No description available.