Browsing by Author "Semper, Cameron"
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- 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 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 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