GLOBAL INVESTIGATION OF POLY(A) TAIL LENGTH DYNAMICS DURING MACROPHAGE ACTIVATION

230 pagesPost-transcriptional regulation plays important roles in spatial-temporal dynamics of gene expression by controlling mRNA stability, translation efficiency, and mRNA localization. In metazoans, poly(A) tail length control plays crucial roles in almost every aspect of post-transcriptional mRNA regulation, and underlies development, normal homeostasis, and diseases. Most systematic, genome-wide investigations of poly(A) tail length control have been limited to specific biological contexts, such as oocyte fertilization. The absence of zygotic transcription makes oocytes a tractable system to examine changes in poly(A) tail lengths without the confounding influence of new transcripts. However, somatic systems are more challenging to monitor post-transcriptional poly(A) tail length regulation, since new transcripts with longer tails continuously enter the mRNA pool. Therefore, most examples of poly(A) tail length regulation in non-developmental systems have only been shown with a handful of genes in some specific biological contexts. The complexity, relevance and widespread nature of poly(A) tail dynamics are largely unknown for post-embryonic cellular processes.In this thesis, I integrated multiple transcriptomic approaches for exploring post-transcriptional poly(A) tail dynamics in post-embryonic systems. By examining mRNA abundance, nascent transcription, and poly(A) tail length across a time course of macrophage activation, a period of widespread and dynamic changes in the gene expression program, I found that a large fraction of the transcriptome underwent changes in poly(A) tail length, including transient increases for pro-inflammatory genes, with distinct patterns of changes in other sets of genes. Increases in tail length correlated with an increase in mRNA levels regardless of transcriptional activity, and many mRNAs that underwent tail extension encode proteins necessary for immune function and post-transcriptional regulation. Our analyses indicate that many mRNAs undergoing tail lengthening are, in turn, degraded by elevated levels of ZFP36, constituting a post-transcriptional feedback loop that ensures transient regulation of transcripts integral to macrophage activation. Collectively, my thesis work introduces an analytic framework to study post-transcriptional control of poly(A) tail length in transcriptionally active, cellular processes and provides evidence that readenylation can be widely used, exerting a profound effect on gene expression in a non-developmental context.2022-12-0

TED-Seq Identifies the Dynamics of Poly(A) Length during ER Stress

Summary: Post-transcriptional RNA processing is a core mechanism of gene expression control in cell stress response. The poly(A) tail influences mRNA translation and stability, but it is unclear whether there are global roles of poly(A)-tail lengths in cell stress. To address this, we developed tail-end displacement sequencing (TED-seq) for an efficient transcriptome-wide profiling of poly(A) lengths and applied it to endoplasmic reticulum (ER) stress in human cells. ER stress induced increases in the poly(A) lengths of certain mRNAs, including known ER stress regulators, XBP1, DDIT3, and HSPA5. Importantly, the mRNAs with increased poly(A) lengths are both translationally de-repressed and stabilized. Furthermore, mRNAs in stress-induced RNA granules have shorter poly(A) tails than in the cytoplasm, supporting the view that RNA processing is compartmentalized. In conclusion, TED-seq reveals that poly(A) length is dynamically regulated upon ER stress, with potential consequences for both translation and mRNA turnover. : Woo et al. develop TED-seq, a method providing robust, sensitive, and cost-efficient transcriptome-wide measurements of poly(A) length in human cells. Using TED-seq, they show that mRNA poly(A) lengths are dynamic and associated with the control of both protein synthesis and mRNA stability under cell stress. Keywords: ER stress, RNA granules, TED-seq, poly(A) tail, poly(A) length, polyadenylation, translation, stabilit

A Pyrazolo[3,4‑<i>d</i>]pyrimidin-4-amine Derivative Containing an Isoxazole Moiety Is a Selective and Potent Inhibitor of RET Gatekeeper Mutants

Aberrant RET kinase signaling plays critical roles in several human cancers such as thyroid carcinoma. The gatekeeper mutants (V804L or V804M) of RET are resistant to currently approved RET inhibitors such as cabozantinib and vandetanib. We, for the first time, report a highly selective and extremely potent RET inhibitor, <b>6i</b> rationally designed. Compound <b>6i</b> inhibits strongly RET gatekeeper mutants and other clinically relevant RET mutants as well as wt-RET. This substance also significantly suppresses growth of thyroid cancer-derived TT cell lines and Ba/F3 cells transformed with various RET mutants. Docking studies reveal that the isoxazole moiety in <b>6i</b> is responsible for binding affinity improvement by providing additional site for H-bonding with Lys758. Also, <b>6i</b> not only substantially blocks cellular RET autophosphorylation and its downstream pathway, it markedly induces apoptosis and anchorage-independent growth inhibition in TT cell lines while having no effect on normal thyroid Nthy ori-3-1 cells