LncRNA discovery in the Listeria monocytogenes infection model

Magagula, Loretta Q

LncRNA discovery in the Listeria monocytogenes infection model

Master Thesis

2015

Publisher

University of Cape Town

Department

Division of Immunology

Faculty

Faculty of Health Sciences

Abstract

A growing body of evidence indicates that long noncoding RNAs (lncRNAs), the most abundant noncoding RNA (ncRNA) species of the pervasively transcribed mammalain genome, have functional roles in the gene regulation of an array of cellular processes. These observations have since discredited the long standing central dogma formulated by Franscis Crick in 1958, which states that genetic output is entirely conducted by protein. Recent studies collectively indicate that lncRNAs play important functional roles in the transcriptional regulation of a wide array of cellular processes. In the last year alone, a handful of studies have identified lncRNAs linc-Cox2, Lethe, PACER and THRIL as central players in host cell innate immune response against microbial infection. These discoveries and the vast numbers of uncharacterized lncRNAs identified by high-throughput nextgeneration transcriptome sequencing technologies, set a precedence for further investigation and characterization of lncRNAs in infection biology. Importantly, lncRNAs may serve as important diagnostic markers of infection as well as therapeutic targets. These aspect of lncRNAs field although extensively being explored in cancer research, have been neglected in infection biology, particularly in microbial infection. In this study, next-generation technologies were used to identify subtle vairations in transcriptional activity, with particular emphasis to lncRNA differential expression, and uncover their physiological relevance during Listeria monotocytogenes infection. To this end, an RNA-Seq dataset of Listeria-infected HeLa cells was subjected to several variations of data analysis lncRNA discovery pipelines. Potential lncRNA functioning was hypothesized using the Rinn & Chang "guilt by association" approach in which lncRNA functioning was hypothesized based on the known functions of tightly co-expressed protein coding mRNAs. "Guilty" lncRNAs were then knocked down in the HeLa cells using transcription activator-like nucleases (TALENs) to validate their candidacy as infection-regulating lncRNAs. Preliminary investigations conducted in this study have revealed potential Listeria infectionregulating lncRNA candidates. Furthermore, we explored the use of the physiologically relevant cellular model of iPSC-MDMs to validate identified lncRNA candidates. This work provides a framework for lncRNA discovery from RNA-Seq data by iterative and intergrative analysis.

Description

Includes bibliographical references

Keywords

Immunology

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