Student Theses and Dissertations

Date of Award

2013

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell Robert Laboratory

Abstract

Eukaryotic cells employ multiple posttranscriptional mechanisms to fine tune gene expression programs in response to external signals. Regulation of messenger RNA and microRNA by RNA-binding proteins is critical to the control of this process. Hence, disruptions of RNA regulatory processes result in neurologic diseases, cancer, and immunologic disorders among other complications. Posttranscriptional control of RNA is particularly important for precise cytokine expression in T cells during adaptive immune responses. We have studied human lymphocyte activation as a model for correlating changes in RNA regulation with dynamic cellular state changes and stress responses. We hypothesize that RNA-binding proteins such as Argonaute (Ago) and HuR play critical roles in shaping the dynamic immune response in T cells. We have previously demonstrated the ability to map Ago-microRNA binding sites using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) {Chi:2009ht}. Here we used HITS-CLIP to define genome-wide maps of both Ago and HuR RNA binding sites in freshly isolated primary human T cells in the resting state and after one hour of stimulation with anti-CD3 and anti-CD28 antibodies. We also applied RNA sequencing and ribosomal profiling to these lymphocytes to establish their transcriptional and translational status. We observed different patterns of both Ago and HuR binding from HITS-CLIP in resting and activated T cells. Comparing the Ago and HuR HITS-CLIP genome-wide maps suggests agonistic and antagonistic actions of these two proteins to confer microRNAmediated regulation of mRNA translation. Furthermore, we overlaid these dynamic RNA binding-protein maps with ribosomal profiles of the target transcripts to investigate translational output as it relates to Ago and HuR regulation. By comparing the changes in both RNA-binding maps with ribosomal profiling results from lymphocytes before and after one hour of T cell activation, we developed a combinatorial dynamic map for these two RNA-binding proteins. Additionally, examining the function of Ago in T cells deficient in HuR helped us understand the relationship between these regulatory proteins in determining the resting and activated lymphocyte states. Our results suggest that differences in protein-RNA interactions for Ago and HuR occur quickly after T cell activation and provide important insight into the specificity of how microRNA translational regulation mediates the immune response.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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