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The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis-acting elements, trans-acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA) sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

Thesis advisor: Laura Hake / The completion of meiosis in Xenopus oocytes requires the coordinated translation of stored mRNAs. CPEB, the cytoplasmic polyadenylation element binding protein, controls the translation of developmentally important early-class maternal mRNAs. Resumption of meiosis through stimulation with progesterone leads to the phosphorylation and activation of CPEB. This results in the lengthening of the poly(A) tails and translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). XGef, a putative guanine nucleotide exchange factor, binds to and is required for CPEB activation. Translation of c-mos, a MAPK kinase kinase, is controlled by CPEB, and activation of the Mos/MAPK pathway is required for meiotic maturation. In addition, the synthesis of Ringo protein, an atypical cdk binding protein and activator, is required for progesterone-induced maturation, though Ringo is able to stimulate resumption of meiosis independent of progesterone. Although much work has been done to understand the key events leading to activation of maturation promoting factor (MPF) and meiotic maturation, the events immediately following progesterone stimulation remain unclear, particularly regarding the role of XGef. The work that follows describes experiments performed to further understand the role of XGef in meiotic maturation through both Ringo and MAPK activity. It was found that XGef and Ringo interact directly and form a complex throughout early meiosis. XGef is involved in Ringo’s influence during meiosis, specifically through MEK-activation of MAPK. Notably, XGef functions in a common pathway and complex with Ringo most likely to influence CPEB phosphorylation and activation. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Thesis advisor: Laura E. Hake / Cytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo. / Thesis (BS) — Boston College, 2003. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Cytoplasmic polyadenylation-element binding protein (CPEB) is required for the translational regulation in multiple cell types. CPEB is known to play important roles in early germ cell development, in neuronal synaptic plasticity, and in the process of cellular senescence. CPEB is able to control translation by first interacting with a specific sequence of mRNA known as the CPE site. CPEB recognizes a specific sequence of mRNA, called the cytoplasmic polyadenylation element. This is a uracil rich sequence that is located on the 3' UTR of mRNA. Once CPEB is bound to the CPE site, CPEB can interact with other proteins. CPEB is most notably known for interacting with a cleavage and polyadenylation specificity factor (CPSF), with a poly(A)-specific ribonuclease, and with a poly(A) polymerase in the Gld2 family. This complex of proteins controls polyadenylation on the 3' end of mRNA. By controlling the lengthening of the poly(A) tail, translation can be regulated. CPEB is believed to contain two RNA recognition motifs and a zinc binding region on the N-terminus. The zinc binding region contains six cysteine and two histidine residues that bind to two zinc atoms in a tetrahedral geometry. Using NMR spectroscopy, the structure of zinc binding region of CPEB1 was determined. This protein was shown to bind to two zinc ions in a cross-braced topology. The zinc binding region of CPEB was also determined that the correct classification for this zinc finger is a ZZ domain.

For many human protein-coding genes, alternative cleavage and polyadenylation (APA) of pre-mRNA generates distinct 3' untranslated regions (3'UTRs) with differing regulatory potential. Widespread 3'UTR shortening via APA occurs in proliferative cell states, including cancer, where it can lead to oncogene overexpression. There has therefore been significant interest in identifying factors which influence poly(A) site choice in different physiological states. The multi-subunit human cleavage factor I complex (CFIm), a core component of the mammalian pre-mRNA cleavage machinery, has been identified as a potential master regulator of APA, as its depletion leads to widespread 3'UTR shortening. However, mechanistic understanding of how CFIm influences poly(A) site selection, and how its activity is regulated, is lacking. In this work, gene editing was used to generate cell lines with substantial, permanent depletion of the 25 kDa or 68 kDa subunits of CFIm (CFIm25 and CFIm68), which exhibited the expected 3'UTR shortening for representative transcripts. Reversal of this 3'UTR shortening by CFIm25 or CFIm68 re-expression provided the basis for a complementation assay, which allowed various aspects of CFIm25 and CFIm68 function to be investigated in vivo. The capacity of CFIm25 to recognise UGUA RNA sequences was shown to make an important contribution to poly(A) site selection transcriptome-wide, and a novel function for the C-terminal arginine/serine-rich (RS domain) of CFIm68 in poly(A) site selection was identified. The potential contribution of CFIm post-translational modification (PTM) to APA regulation was also explored. Novel acetylation sites on CFIm25 and CFIm68 were identified, as well as extensive serine phosphorylation in the CFIm68 RS domain. Complementation analysis revealed that phosphomimetic mutations in this RS domain inhibited distal poly(A) site selection, suggesting a potential role for CFIm68 phosphorylation in APA regulation. Taken together, the findings presented here provide insights into several important determinants of CFIm function, and the complementation assay developed provides a useful tool for future investigations.