The RNA binding protein heterogeneous nuclear ribonucleoprotein L-Like (hnRNP LL) is known to regulate the alternative splicing of various physiologically important precursor messenger RNAs (pre-mRNAs). It undergoes a wide range of post-translational modifications (PTMs), including phosphorylation, ubiquitination and acetylation. However, the target amino acids and effects of these PTMs on the functions of hnRNP LL have not been characterized so far. In this study, we show for the first time that the endogenous hnRNP LL is phosphorylated upon depolarization. Using phosphopeptide mapping followed by the generation of a custom-made phospho-site specific antibody, we further show that phosphorylation at Ser308 of hnRNP LL is induced by depolarization though it is probably not the major phospho-amino acid target of depolarization/CaMKIV. The residue is critical for the nuclear localization and its phosphorylation essential for the CaMKIV-caused perinucleolar localization of the hnRNP LL protein in HEK293T cells. The residue is likely also critical in the regulation of nuclear functions like pre-mRNA splicing. / February 2016

abstract: The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers. Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed. Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before. Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages. DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014