Transcriptome-wide studies of hypoxic cancer cells identify alternative splicing as a mechanism to inhibit translation / Lauren Kathleen Brady.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Brady, Lauren Kathleen, author.

Contributor:

Koumenis, Constantinos, degree supervisor.

Bartolomei, Marisa S., degree committee member.

Bassing, Craig H., degree committee member.

Fuchs, Serge Y., degree committee member.

Minn, Andy J., degree committee member.

University of Pennsylvania. Department of Cell and Molecular Biology, degree granting institution.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

viii, 98 leaves : illustrations ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2017.

Summary:

Cellular adaptation to hypoxia involves downregulation of energy-consuming processes such as macromolecular synthesis, and leads to tumor aggressiveness and resistance to therapies for many solid cancers. To delineate mechanisms underlying this process, I carried out a transcriptome-wide study to measure hypoxia-mediated changes in gene expression and alternative splicing in in vivo and in vitro models of hypoxic head and neck carcinoma (HNC) cells. These data represent the first nucleotide-resolution study of the hypoxic transcriptome of HNC cells in vivo and in vitro. This investigation uncovered a global downregulation of genes known to regulate RNA processing, including a significant number of genes involved in splicing catalysis. Exon-level analyses classified >1,000 mRNAs to be affected by alternative splicing, and led to the discovery of a unique retained intron in the master regulator of translation initiation, EIF2B5. In this dissertation, I will describe a previously uncharacterized mechanism by which a hypoxia-mediated retained intron in EIF2B5 leads to a truncated isoform that opposes full-length eIF2Bϵ to inhibit translation. A functional investigation of this hypoxia-induced isoform confirmed that expression of the truncated 65kDa isoform of eIF2Bϵ confers a survival advantage to HNC cells under conditions of hypoxia. Moreover, expression of this isoform was observed in solid tumors of patients with HNC in a stage-dependent manner. Additional work to uncover -cis and -trans regulators of EIF2B5 splicing identified several factors that influence intron retention in EIF2B5 : a weak splice site with an alternate splice site at the retained intron, hypoxia-induced expression of the splicing factor SRSF3, and increased binding of total and phospho-Ser2 RNA polymerase II (RNAPII) specifically at the intron retained under hypoxia. Altogether, these data reveal differential splicing as a previously uncharacterized mode of translational control under hypoxia and are supported by a model in which hypoxia-induced changes to co-transcriptional processing lead to selective retention of an intron containing a premature-termination codon in EIF2B5.

Notes:

Ph. D. University of Pennsylvania 2017.

Department: Cell and Molecular Biology.

Supervisor: Constantinos Koumenis.

Includes bibliographical references.

OCLC:

1334673365