Browsing by Subject "Noncoding RNA"

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Open Access
Genomic and Epigenomic Attributes of Alpha Satellite Underlying Function Within the Human Centromere Region
(2018) McNulty, Shannon Michelle
The centromere serves as the foundation for the kinetochore and attachment point for spindle microtubules during metaphase. The proper function of this locus is required to ensure chromosome segregation and genomic stability. In humans, repetitive alpha satellite DNA underlies the human centromere region and is organized into specific chromatin domains that are maintained by a complex combination of factors. Although the centromere region is generally thought to be specified epigenetically, some evidence suggests that the underlying DNA sequence is also involved in centromere function. To better define links between alpha satellite and function within the human centromere region, we investigated two attributes of alpha satellite DNA: its transcription into noncoding alpha satellite RNAs and genomic variation within the alpha satellite array. Noncoding transcripts produced from alpha satellite DNA are associated with normal centromere and pericentromere function and evidence from other organisms suggests RNAs from this region are pivotal in the centromere and kinetochore assembly cascade and in maintaining the chromatin environments of the centromere region. However, alpha satellite RNAs have not yet been fully characterized and data reflecting the chromosome-specific nature of alpha satellite arrays is lacking. Additionally, genomic variation within alpha satellite arrays has been linked to reduced centromere protein recruitment and chromosome instability, yet the molecular basis for this is unknown. These gaps in knowledge have stymied our understanding of the role of genomic and epigenetic attributes of alpha satellite that affect function within the human centromere region. Thus, this work aims to functionally characterize the role of alpha satellite transcripts and to determine how genomic variation impacts chromosome stability. Utilizing cytological and molecular techniques that allow the differentiation of alpha satellite RNAs from individual chromosomes and arrays, we have demonstrated that each chromosome produces unique noncoding RNAs that localize in cis to their site of production. Both centromeric and pericentromeric alpha satellite arrays produce noncoding RNAs, but these transcripts are spatially and functionally distinct. Alpha satellite RNAs from the centromere bind at least two key centromere proteins: CENP-A and CENP-C, while alpha satellite RNAs from the pericentromere colocalize with SUV39H1. Centromeric alpha satellite RNAs are required for complete loading of new CENP-A-containing nucleosomes, as well as maintenance of CENP-C levels. Genomic variation affects the origin of alpha satellite transcripts, such that highly variant arrays produce a different set of transcripts than wild type arrays. Further, the long-range organization of variation across the alpha satellite array in unstable chromosomes suggests certain spatial organizations of variation are poor platforms for building a stable centromere and kinetochore. Collectively, these findings implicate alpha satellite RNA and genomic variation and/or the interplay of these two elements as essential factors in the function of the human centromere region.
Open Access
Use of Comparative Genomics for Non-coding Rna Prediction and Investigation of Dna Introgression in Yeast
(2008-04-23) Kavanaugh, Laura Anne
The rapid development of large-scale genomic sequencing has dramatically changed the field of genetics, in part through the development of comparative genomics. Fungal comparative genomics is particularly powerful given the large number of genomes currently available, their compact architecture, and their relative ease of genetic manipulation. Fungal comparative genomics was employed in this work to address two related questions. First, it was used along with computational thermodynamic methods to predict non-coding RNA (ncRNA) in Saccharomyces cerevisiae. Sets of positive and negative control genes were evaluated to determine the effect of window sizes and step sizes on the sensitivity of ncRNA identification. The approach was then applied to predict ncRNA genes on chromosome 6 of S. cerevisiae and S. bayanus. Northern blot analysis, rapid amplification of cDNA ends (RACE), and publicly available cDNA library data were used to test the predictions. Strong experimental evidence was accumulated for four new ncRNA genes. Potential structural elements in the 5' and 3' untranslated regions (UTRs) of six annotated protein-coding genes were also identified. This work shows that thermodynamic approaches, coupled with comparative genomics, are powerful tools for predicting structural ncRNA. Second, comparative genomic approaches were employed to identify a non-reciprocal transfer event from Cryptococcus neoformans var. grubii to var. neoformans ~2 million years ago involving a 14 gene (~40 kb) region. The majority of clinical and environmental var. neoformans strains from around the world contain this sequence obtained from var. grubii. The introgression event likely occurred via an incomplete inter-varietal sexual cycle creating a hybrid intermediate where mobile elements common to both lineages mediated the exchange. The subsequent duplication in laboratory strains of a fragment of this same genomic region supports evolutionary theories that instabilities in subtelomeric regions promote adaptive evolution through gene amplification and subsequent adaptation. These data indicate that DNA exchange between closely related sympatric varieties or species may be a recurrent theme in the evolution of fungal species. It further suggests that while evolutionary divergence is the primary force driving speciation, rare introgression events also play a potentially important role.