The Role of Ribosome Biogenesis in Arabidopsis thaliana Root Development and Epidermal Patterning.

The establishment of distinct patterns of gene expression is essential to the differentiation of specialized cell types during the development of multicellular organisms. To investigate the regulatory networks involved in this cell fate specification, one can turn to the Arabidopsis root epidermis, where a position-dependent pattern of hair and non-hair cells serves as a pertinent and accessible model. Recent studies have implicated ribosomal protein and ribosome assembly factors in regulation of gene expression during development, however, the connection between ribosome biogenesis and root cell-type patterning has previously never been investigated. To that end, this thesis research explores the connection and function of a highly conserved ribosome biogenesis factor with respect to the development and epidermal patterning of the Arabidopsis root epidermis. The Arabidopsis DIM1A gene was identified in a genetic screen for genes required for position-dependent expression of non-hair fate regulator GLABRA2. Our analysis has revealed that functional DIM1A is required for generation of distinct gene expression patterns in developing root-hair and non-hair cells. In addition to defects in root epidermal patterning, the dim1A mutant displays reduced root meristem cell division rate and deficient leaf size, shape, vascular patterning and trichome branching. Furthermore, DIM1A promoter activity and gene product are enriched in rapidly dividing plant tissues, supporting a role for DIM1A in Arabidopsis development and cell differentiation. The Dim1/KsgA family of dimethylases are a highly conserved enzymes involved in post-transcriptional modification of ribosomal RNA (rRNA) and pre-rRNA processing. While the two small subunit RNA base modifications they catalyze are present in almost every known organism, the functional significance of these modifications is currently unknown. My experimental evidence indicates that the dim1A mutant lacks the two 18S rRNA modifications without affecting pre-rRNA processing. In conclusion, I propose that DIM1A and the post-transcriptional rRNA modification it catalyzes are important for generating well-defined patterns of gene expression necessary for establishment of the two distinct cell fates in the Arabidopsis root epidermis.Ph.D.Molecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86319/1/ypancier_1.pd

The KsgA methyltransferase: Characterization of a universally conserved protein involved in robosome biogenesis

The KsgA enzymes comprise an ancient family of methyltransferases that are intimately involved in ribosome biogenesis. Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution, and therefore were present in the last common ancestor. The first member of the family to be described, KsgA from Escherichia coli, was initially shown to be the determining factor for resistance/sensitivity to the antibiotic kasugamycin and was subsequently found to dimethylate two adenosines in 16S rRNA during maturation of the 30S subunit. Since then, numerous other members of the family have been characterized in eubacteria, eukaryotes, archaea and in eukaryotic organelles. The eukaryotic ortholog, Dim1, is essential for proper processing of the pre-rRNA, in addition to and separate from its methyltransferase function. The KsgA/Dim1 family bears sequence and structural similarity to a larger group of S-adenosyl-L-methionine dependent methyltransferases, which includes both DNA and RNA methyltransferases. In this document we report that KsgA orthologs from archaea and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life. We have characterized KsgA structurally, and discuss aspects of KsgA\u27s activity in light of the structural data. We also propose a model for KsgA binding to the 30S subunit, based on solution probing data. This model sheds light on KsgA\u27s unusual regulation and on the dual function of the Dim1 enzymes

MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome

Making proteins in the powerhouse

Understanding regulation of mitochondrial DNA (mtDNA) expression is of considerable interest as mitochondrial dysfunction is important in human pathology and ageing. Similar to the situation in bacteria, there is no compartmentalization between transcription and translation in mitochondria; hence, both processes are likely to have a direct molecular crosstalk. Accumulating evidence suggests that there are important mechanisms for regulation of mammalian mtDNA expression at the posttranscriptional level. Regulation of mRNA maturation, mRNA stability, translational coordination, ribosomal biogenesis and translation itself, all form the basis for controlling oxidative phosphorylation capacity. Consequently, a wide variety of inherited human mitochondrial diseases are caused by mutations of nuclear genes regulating various aspects of mitochondrial translation. Furthermore, mutations of mtDNA, associated with human disease and ageing, often affect tRNA genes critical for mitochondrial translation. Recent advances in molecular understanding of mitochondrial translation regulation will likely provide novel avenues for modulating mitochondrial function to treat human disease.VetenskapsrådetAccepte

The Development Of Peptide Ligands To Target H69 Rrna

ABSTRACT THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69 by DANIELLE NICOLE DREMANN December 2015 Advisor: Prof. Christine S. Chow Major: Chemistry (Biochemistry) Degree: Doctor of Philosophy In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to increase the efficiency of peptide ligand selection in which novel peptides with high affinity and selectivity could be identified for future projects. This assay was done using flow cytometry, instrumentation capable of identifying beads, bound to the target RNA conjugated to a fluorophore, based on fluorophore emission, and sorting them into 96-well plates for MS analysis. The last part of the research focused on aminoglycoside-H69 RNA interactions. ESI MS was used to obtain binding affinities and stoichiometries of 2AP- and A-containing H69 RNAs. The findings revealed that the binding mode had not changed between these two sets of RNAs, which revealed the value for using ESI MS in combination with other techniques, such as fluorescence, to give a complete picture of the binding mode (stoichiometry, affinity, selectivity) in comparison with conformational changes that may occur upon binding. Further exploration of aminoglycoside-H69 RNA interactions took place with the H69 peptide NQVANHQ-NH2 to determine whether a fragment-based drug design approach could be used to create small compounds for future in vivo applications

Synthesis, Screening and Cocrystallization of Adenosine Based Inhibitors with Methyltransferases, ErmC\u27 and KsgA

Antibiotic resistance threatens to throw mankind back into an era when infectious disease was the predominant cause of death. In an effort to mitigate this danger, we targeted ErmC’ and KsgA. Both methylate N6-adenosine of ribosomal RNA, though each serve different roles in their bacterial host. ErmC’ dimethylates A2058 on 23S rRNA, conferring resistance to MLSB antibiotics (macrolides, lincosamides, streptogramin B). KsgA aids in ribosome assembly, binding inactive 30S until dimethylating A1518/A1519 of 16S rRNA. Like most methyltransferases, ErmC’ and KsgA use cofactor S-adenosylmethionine (SAM) as their methyl source, which binds adjacent to their specific adenosine substrate. ErmC’ inhibitors could restore MLSB antibiotics against infections with this resistance mechanism. KsgA inhibitors could form novel antibiotics that stall 30S assembly. Previous studies reported a potent ErmC’ inhibitor, N6-cyclopentyl adenosine (1), binding to the substrate pocket with cyclopentyl bridging into the SAM pocket. We expanded this study by synthesizing 1 and 22 other N6-substituted analogs to explore more favorable interactions within the SAM pocket. When these compounds (1mM) were screened against ErmC’ and KsgA, some showed greater inhibition than 1. Two of these inhibitors that were crystallized with ErmC’, N6-8-octylamine adenosine (2.60Å) and N6-phenethyl adenosine (2.40Å), unexpectedly docked into the SAM pocket with their 5’-C pointing towards the substrate pocket. New compounds were made to exploit this orientation by adding substituents off the 5’-C to probe the substrate pocket. Through a five step synthesis, the 5’-OH of 1 was substituted with an amine linked to benzyl (30), phenethyl (31), propylphenyl (32) or butylphenyl (33). When 30-33 were screened using 20μM SAM, ErmC’ showed greater inhibition (relative to 1), while KsgA showed virtually none. However, when ErmC’ was tested using 0.5μM SAM, inhibition from 30-33 was nearly unchanged, whereas 1 became significantly more potent than 30-33, suggesting 30-33 were not binding to the SAM pocket. Preliminary data showed that raising 23S concentrations lowered inhibition from 32-33, while inhibition from 1, 30 and 31 was nearly unchanged, suggesting that at least 32-33 bound within the substrate pocket