Browsing by Author "Zimmerly, Steven"
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- ItemOpen AccessAbiK: A Novel Polymerase That Confers Resistance to Phage Infection(2019-05-15) Soufi, Bahar; Zimmerly, Steven; Hynes, Michael F.; Hansen, David V.The abortive infection K (AbiK) system of Lactococcus lactis is a powerful antiviral defense mechanism that acts in the period after phage DNA enters the cell but before new progeny are released. The N-terminal domain of AbiK encodes a novel DNA polymerase that in vitro uses an internal amino acid to synthesize an un-templated DNA. The C-terminal domain of AbiK, on the other hand, has not been biochemically characterized. In the first of two projects presented in this thesis, strategies were devised to identify the amino acid priming site in AbiK. A close examination of data has narrowed down the priming site to two highly-conserved tyrosines in the N-terminal sequence of AbiK. In the second project, the characterization of the AbiK’s C terminal domain was undertaken to identify its function. This study uncovers a novel RNA modification activity, and provides evidence that AbiK’s C-terminal domain has ribonuclease activity.
- ItemOpen AccessAlternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani(Oxford Journals, 2013-10) McNeil, Bonnie A; Simon, Dawn M; Zimmerly, StevenGroup II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.
- ItemOpen AccessAlternative splicing of an ORF-less group II intron in Clostridium tetani(2013-12-04) McNeil, Bonnie; Zimmerly, StevenGroup II introns are ribozymes that are encoded within all domains of life. They are also capable of mobility through an RNA intermediate. Due to similarities in RNA structure and splicing mechanisms, group II introns are thought to have been the ancestors of nuclear pre-mRNA introns and snRNAs. In this dissertation I report the discovery of a unique ORF-less group II intron, C.te.I1, in the human pathogen Clostridium tetani. The intron is encoded within a surface layer protein region of the C. tetani chromosome and possesses an unusual genomic organization such that a full-length copy of the intron is followed downstream by three copies of the RNA structural domains 5 and 6 (D5/6). This arrangement led to the hypothesis that C.te.I1 is capable of alternative splicing utilizing the downstream copies of D5/6 as alternate 3ʹ splice sites. RNA extractions and RT-PCR support the hypothesis and revealed that the splicing reaction of C.te.I1 links a surface layer protein ORF (CTC00465) in the upstream exon to one of four downstream ORFs that encode transglutaminase-related or protease-related reading frames (CTC00467-CTC00470). Including unspliced transcript, five mRNAs are produced. Sequencing of the exon junctions showed that the 5ʹ splice site utilized by C.te.I1 is shifted 8 nt upstream both in vivo and in vitro. Use of this splice site is critical to alternative splicing as it results in the elimination of the stop codon at the end of CTC00465 and results in the correct ligation of 5ʹ and 3ʹ exon sequences. Site-directed mutagenesis and self-splicing assays for C.te.I1 revealed that the shifted splice site is due to a novel EBS1-IBS1 pairing. Although C.te.I1 is thought to be derived from a mobile Class B (IIB) intron that lost its ORF, the intron was found to have evolved to utilize a IIA-like mechanism of 3ʹ splice site recognition. These changes represent structural adaptations of the intron to its role in alternative splicing. The structural adaptations and splicing of C.te.I1 illustrate the plasticity of group II introns in that they can adapt new RNA structural and catalytic properties which can be utilized to affect gene expression.
- ItemOpen AccessBiochemical characterization of bacterial group II introns: rules for 3' splice site selection and insertion of IIC introns after intrinsic terminator motifs(2007) Robart, Aaron Reigh; Zimmerly, Steven
- ItemOpen AccessBioinformatic and phylogenetic analyses of retroelements in bacteria(2018-11-29) Wu, Li; Zimmerly, Steven; Fraser, Marie Elizabeth; De Koning, A. P. Jason; DeVinney, Rebekah; Russell, Tony S.Retroelements are mobile elements that are capable of transposing into new loci within genomes via an RNA intermediate. Various types of retroelements have been identified from both eukaryotic and prokaryotic organisms. This dissertation includes four individual projects that focus on using bioinformatic tools to analyse retroelements in bacteria, especially group II introns and diversity-generating retroelements (DGRs). The introductory Chapter I gives an overview of several newly-identified retroelements in eukaryotes and prokaryotes. In Chapter II, a general search for bacterial RTs from the GenBank DNA sequenced database was performed using automated methods. It not only enlarged the collection of bacterial reverse transcriptases (RTs), but also revealed several new classes of RTs. In Chapter III, another automated search was performed to identify group II introns. All predicted introns were automatically folded and then manually refined. Other information, such as multiple copies and non-standard intron organisations, were also identified. Next, all introns were subjected in several analyses in order to depict common properties for each class, such as preferences in target sites and DNA strands. Using this enlarged dataset, Chapter IV aimed to resolve the phylogeny of group II introns and investigate whether the intron-encoded protein (IEP) and RNA portions coevolved. Among trees constructed from various datasets, such as using different sequence masks, smaller sampled subsets or morphological features, the hypothesis that the IEP and RNA coevolved was supported by comparisons among most trees, even though it seemed to be rejected by formal topology tests. Finally, Chapter V compiled and systematically classified the most recent set of DGRs, which can be used as a reliable reference to direct future DGR-related studies and experimental designs.
- ItemOpen AccessCharacterization of a Group II Intron and its Intron-Encoded Protein(2016) Semper, Cameron; Zimmerly, Steven; Chua, Gordon; Ng, KenGroup II introns are a class of mobile genetic elements found in the genomes of bacteria, archaea and in the organellar genomes of some eukaryotes. They are comprised of a large structural RNA with ribozyme activity and a multi-functional intron-encoded protein. The IEP encodes four domains: reverse-transcriptase, maturase, DNA-binding and endonuclease domains. The RNA and IEP interact to form a functional ribonucleoprotien that facilitates the two activities of group II introns, splicing and mobility. The interaction between group IIA introns and their IEPs has been well characterized; however, many of the details regarding the IIC intron-IEP interaction are poorly understood. This dissertation examines the interaction between the B.h.I1 group II intron and its intron-encoded protein. The B.h.I1 intron is a class IIC intron encoding an IEP that lacks the endonuclease domain. The intron is found in the organism Bacillus halodurans and exhibits clear evidence of retroelement behaviour, inserting downstream of intrinsic transcriptional terminators. In order to better characterize the intron-IEP interaction, it was determined that significant improvement to the IEP purification was required. Through a combination of optimization of expression and FPLC the yield and purity of the IEP was improved significantly. With purified components in sufficient quantity, the hypothesis that IIA and IIC IEPs recognize different high-affinity binding sites was examined using an in vitro mobility assay. Ultimately it was determined that IIC IEPs recognize a structurally similar but functionally different high-affinity binding site than is recognized by IIA-encoded IEPs. During binding site experiments, it was determined that the B.h.I1 intron uses the same IEP binding-site for splicing and mobility, reconciling a previously observed inconsistency. A kinetic characterization found that the B.h.I1-IEP interaction occurs with lower affinity compared to the IIA intron and that the B.h.I1 intron-IEP interaction relies more heavily on secondary contacts made outside the high-affinity binding site. Finally, data obtained from cross-linking experiments implicate the IEP in recognition of the 3’ exon, a previously undiscovered function of the IEP in intron mobility. Though subtle, the differences observed for the IIC intron-IEP interaction appear to facilitate the specific requirements of its genomic niche.
- ItemOpen AccessCharacterization of the interaction between a group IIC inton and its 5' exon(2011) Clarke, Nicholas A. C.; Zimmerly, Steven
- ItemOpen AccessDiverse Structures and Strategies of 5′ Exon Recognition in Group II Introns(2018-06-05) Jarding, Ashley Marie; Zimmerly, Steven; Muench, Douglas G.; Noskov, Sergei Yu; Van Marle, Guido; Cousineau, BenoitGroup II introns are a family of mobile elements found in bacteria and bacteria-derived organelles, which are thought to have a direct evolutionary link to spliceosomal introns. These introns can self-splice in vitro thanks to their highly organized ribozyme structure. Studying the diverse structural features of group II introns illuminates the breadth of their structural diversity and resultant unique splicing and mobility capabilities. In the first of three projects presented in this dissertation, bioinformatic approaches were used to identify over 800 new introns in a large scale, systematic collection and analysis of group II introns. This allowed inference of class-specific mobility properties and the description of novel intron arrangements in genomes. Additionally, one new class and 15 unique, previously unclassified introns with novel structural features have been identified. The newly collected introns revealed a conserved structural feature for Class A introns: they appear to have two 5′ exon recognition motifs, whereas all other group II introns are reported to have one. The second project investigates the binary splicing hypothesis, which states that either of these two motifs can be used to recognize the exon. This was investigated and confirmed through both experimental and bioinformatic approaches. This strategy appears to expand the set of homing sites available to an intron by permitting splicing from one sequence and reverse-splicing into another. In the third project, the 5′ exon recognition mechanism for a IIC intron was investigated. IIC introns lack the canonical IBS2-EBS2 interactions found for other group II introns, but have a conserved transcriptional terminator in place of EBS2. In B.h.I1 a 5S rRNA upstream of this motif was shown to have an unexpected effect on the splicing reaction in vitro. Mutagenesis of 5S rRNA showed that the A-loop is critical for splicing. RNA protection experiments to characterize the interactions with the 5′ exon support the terminator stem interacting with Domain I while 5S has an auxiliary effect on exon ligation. In summary, this work has increased our understanding of the structural diversity of group II introns and corresponding specialized strategies used to recognize the 5′ exon during splicing.
- ItemOpen AccessEvolution of group II introns(BioMed Central, 2015) Zimmerly, Steven; Semper, CameronPresent in the genomes of bacteria and eukaryotic organelles, group II introns are an ancient class of ribozymes and retroelements that are believed to have been the ancestors of nuclear pre-mRNA introns. Despite long-standing speculation, there is limited understanding about the actual pathway by which group II introns evolved into eukaryotic introns. In this review, we focus on the evolution of group II introns themselves. We describe the different forms of group II introns known to exist in nature and then address how these forms may have evolved to give rise to spliceosomal introns and other genetic elements. Finally, we summarize the structural and biochemical parallels between group II introns and the spliceosome, including recent data that strongly support their hypothesized evolutionary relationship.
- ItemOpen AccessGroup II intron structure characterizations and three-dimensional modeling(2009) Chai, Dinggeng; Zimmerly, Steven
- ItemOpen AccessIn vivo coupling between groupIIC intron insertion and transcriptional termination(2014-09-15) Ahmed, Salim; Zimmerly, StevenGroup II introns are a class of retroelements consisting of a catalytic RNA and an intron-encoded protein (IEP), which is a reverse transcriptase (RT). Group II ribozymes are classified into three subgroups, IIA, IIB and IIC. IIC introns have the novel mobility property of inserting directly after transcriptional terminator motifs rather than into homing sites of defined sequence. This dissertation addresses a direct correlation between intron homing and terminator motifs by using a set of in vivo experiments. Target DNAs were constructed such that the native terminator motif was replaced with sequences from the E. coli tryptophan attenuator or the B. subtilis adenine, SAM, FMN or lysine riboswitches. The data support a model for in vivo mobility in which the intron’s RNP (intron lariat bound by IEP) gains access to its unwound DNA target during the pause of transcriptional termination, allowing it to recognize the DNA stem-loop structure.
- ItemOpen AccessInvestigating the role of splicing in disorders of craniofacial development(2017) Lynch, Danielle C; Parboosingh, Jillian S.; Innes, A Micheil; Bernier, Francois P; Childs, Sarah J.; Rancourt, Derrick E; Zimmerly, Steven; Trainor, Paul AFollowing clinical delineation of a rare disease, identification of the causative gene(s) is a crucial first step towards providing enhanced patient care and understanding disease aetiology. Disease gene discovery, especially when considered in the context of related disorders, can also provide new insight regarding normal development and physiology. Herein, I present two novel disease-causing genes: the ribosomal gene RPLP2 in Nager syndrome (NS) and the spliceosomal gene SNRPB in cerebro-costo-mandibular syndrome (CCMS). The mutations identified in SNRPB are the first example of de-regulated alternative splicing-coupled nonsense-mediated decay as a mechanism for human disease. NS and CCMS are both disorders of first and second pharyngeal arch development, with the jaw and ears being affected. Over the past five years, the association between disorders of craniofacial development and mutations in spliceosomal genes has become apparent with the discovery of SF3B4 in NS, EFTUD2 in mandibulofacial dysostosis type Guion-Almeida, and TXNL4A in Burn McKeown syndrome. The specificity of the phenotypes resulting from mutations in such ubiquitous genes has perplexed the scientific community, but it is at least clear that spliceosomal genes play a more prominent yet subtle role in development than previously thought. The association between Treacher Collins syndrome, which overlaps phenotypically with CCMS and NS, and the ribosomal genes TCOF1, POLR1C, and POLR1D has been known for longer. This work establishes NS as having both spliceosomal and ribosomal defects as a cause. In this thesis I discuss potential links between the mechanisms underlying ribosomal and spliceosomal defects in disorders of craniofacial development, particularly increased sensitivity to reactive oxygen species (ROS).
- ItemOpen AccessMolecular Genetic Characterization of Pumilio Genes in the Fission Yeast Schizosaccharomyces pombe(2016) Henry, Darren; Chua, Gordon; Muench, Doug; Zimmerly, Steven; Grewal, Savraj; Labbe, SimonThe regulation of gene expression in eukaryotes is a complex process that is controlled at many levels. Posttranscriptional regulation is performed by RNA-binding proteins that can effect the processing, localization and decay rates of mRNAs. One such family of RNA-binding proteins is Pumilio and FBF (Puf), which are conserved across eukaryotes. In this work, we have started to functionally characterize the nine puf+ genes found in the fission yeast Schizosaccharomyces pombe through the use of fluorescence localization studies, transcriptomics, RNA immunoprecipitation microarrays (RIP-chip) and molecular genetic techniques. Deletion strains of the classical puf+ genes were created and observed to have no phenotype, while strains overexpressing these genes did. This characterization also led to the finding that the puf+ genes of S. pombe are intracellularly localized in three distinct patterns of nucleolar, cytoplasmic and granular. The localizations were observed under heat shock conditions and only the cytoplasmic Pufs relocalized to foci in the cell that appeared to correspond to a stress granule marker. It was also observed that the Puf gene pfr1+ is involved in regulating flocculation, adhesion and invasive growth as the deletion strain cannot be induced to flocculate or invasively grow, while overexpression causes constitutive flocculation. Putative mRNA targets of Pfr1 regulation include pvg1+ and SPBPB7E8.01, which were identified by expression microarrays and RIP-chip analysis. These putative target genes were found to be repressors of flocculation, while the posttranscriptional regulation genes ccr4+ and ste13+were shown to be required for flocculation. The analysis of puf2 and puf4 using expression microarrays and RIP-chip to identify putative mRNA targets of these puf+ genes was also performed. This analysis indicated that puf2+ may be involved in regulating hexose import while puf4+ could be involved in the regulation of iron homeostasis. The results of this study have substantially contributed to our understanding of Puf function and posttranscriptional regulation in S. pombe.
- ItemOpen AccessNext-Generation Sequencing of vlsE Recombinational Switching in the Lyme Spirochete(2017) Verhey, Theodore; Chaconas, George; Schriemer, David; Samuels, Scott; Zimmerly, Steven; DeVinney, Rebekah; de Koning, Jason; McGhee, JamesBorrelia burgdorferi and other spirochetes that cause Lyme disease effectively evade the acquired immune response through antigenic variation. The VlsE antigen is expressed on the spirochete surface during mammalian infection. Virtually unlimited numbers of variants are generated through segmental gene conversion events at the vlsE gene from a series of nearby silent cassette sequences that are homologous to the variable region of vlsE. In contrast to other antigenic variation systems, the molecular mechanism for this switching is unknown. Switching at vlsE is dependent on mammalian host factors and the only known requirement is the RuvAB branch migrase; RecA and many of the other homologous recombination proteins in B. burgdorferi are not required. In this study, we developed a new assay for switching at vlsE based on PacBio sequencing and an analytical pipeline that allows the analysis of tens of thousands of full-length variants. We developed the first fully-automated and unbiased method to accurately identify switch events and non-templated mutations from sequence data. This software also contains a large suite of dataset management and analysis tools to quantify many aspects of switch events and the switching process in the dataset. Following a time-course of B. burgdorferi infection in immunocompetent and immunocompromised mice in a variety of tissues, we uncover a series of new insights into recombinational switching at vlsE. We demonstrate that although switching requires mammalian host factors, the rate of vlsE switching is unaffected by the presence or absence of the required immune system. We identify residues that undergo diversifying and stabilizing selection in the VlsE protein in the presence of acquired immunity. We also report accurate rates of recombination and nontemplated mutation, the size and origin of switch events, a role for local sequence homology in promoting switching, and that switch events accumulate in a clustered rather than uniform fashion. We also quantify a secondary mechanism of sequence variation by demonstrating that polymerase slippage generates in-frame, surface-localized insertions and deletions that contribute to VlsE variability.
- ItemOpen AccessNovel RNA structural features of an alternatively splicing group II intron from Clostridium tetani(RNA Society, 2014-06) McNeil, Bonnie A; Zimmerly, StevenGroup II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5′ splice site located 8 nt upstream of the usual 5′ GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1–EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5′ splice site is shown to be affected by structures in addition to IBS1–EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3′ exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.
- ItemOpen AccessA pipeline of programs for collecting and analyzing group II intron retroelement sequences from GenBank(2013-12) Abebe, Michael; Candales, Manuel A; Duong, Adrian; Hood, Keyar S; Li, Tony; Neufeld, Ryan AE; Shakenov, Abat; Sun, Runda; Wu, Li; Jarding, Ashley M; Semper, Cameron; Zimmerly, StevenBackground Accurate and complete identification of mobile elements is a challenging task in the current era of sequencing, given their large numbers and frequent truncations. Group II intron retroelements, which consist of a ribozyme and an intron-encoded protein (IEP), are usually identified in bacterial genomes through their IEP; however, the RNA component that defines the intron boundaries is often difficult to identify because of a lack of strong sequence conservation corresponding to the RNA structure. Compounding the problem of boundary definition is the fact that a majority of group II intron copies in bacteria are truncated. Results Here we present a pipeline of 11 programs that collect and analyze group II intron sequences from GenBank. The pipeline begins with a BLAST search of GenBank using a set of representative group II IEPs as queries. Subsequent steps download the corresponding genomic sequences and flanks, filter out non-group II introns, assign introns to phylogenetic subclasses, filter out incomplete and/or non-functional introns, and assign IEP sequences and RNA boundaries to the full-length introns. In the final step, the redundancy in the data set is reduced by grouping introns into sets of ≥95% identity, with one example sequence chosen to be the representative. Conclusions These programs should be useful for comprehensive identification of group II introns in sequence databases as data continue to rapidly accumulate.
- ItemOpen AccessA pipeline of programs for collecting and analyzing group II intron retroelement sequences from GenBank(BioMed Central, 2013-12-20) Abebe, Michael; Candales, Michael A; Duong, Adrian; Hood, Keyar S; Li, tony; Neufeld, Ryan A E; Shakenov, Abat; Sun, Runda; Wu, Li; Jarding, Ashley M; Semper, Cameron; Zimmerly, Steven
- ItemOpen AccessStructure-function studies of viral RNA-dependent RNA polymerases(2018-09-18) Samanta, Soumya; Ng, Kenneth Kai Sing; Zimmerly, Steven; Noskov, Sergei Yu; Williams, Gareth J.; Glover, MarkHuman norovirus (NV) and hepatitis C virus (HCV) are two major and related human pathogens belonging to the positive-strand RNA virus superfamily. NV belongs in the Caliciviridae family and is a major cause of gastroenteritis outbreaks throughout the world. Although there is substantial interest in the development of vaccines and antiviral therapeutics for norovirus infections, there are currently no effective antiviral treatments. Using a fluorescence-based primer extension assay, I have identified a series of structurally-related compounds that inhibit norovirus polymerase at micromolar concentrations. Previous work in our group has helped to structurally characterize human norovirus polymerase complexes with RNA, nucleoside triphosphates, and divalent metal ions. These structures have revealed the enzyme trapped indifferent conformations that are likely important for the catalytic cycle. Several enzyme-inhibitor complexes with alendronic acid and medronic acid inhibitors have been crystallized, and the structure determination of NV Pol complexes has been undertaken. This thesis presents new results correlating enzyme inhibition kinetics with crystallographic structure determination for several novel inhibitor complexes. In the second part of this thesis, work is described on characterizing interactions between RNA and HCV non-structural protein NS5B, the RNA-dependent RNA Polymerase (RDRP) central to HCV replication. HCV NS5B is highly-conserved and forms the core of the catalytic site of the replicase complex. I have determined the structure of a novel polymerase-RNA complex that shows the binding of a short RNA oligonucleotide to a region of the enzyme near a commonly-targeted site for the binding of allosteric inhibitors.
- ItemOpen AccessStudies of bacterial retroelements: a group II intron and an abortive infection protein(2009) Wang, Chen; Zimmerly, Steven
- ItemOpen AccessStudies of group II introns in bacteria and in yeast mitochondria(2002) Seo, Woo Seok; Zimmerly, Steven