No description available.

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.

Messenger RNAs (mRNAs) have to undergo a series of post-transcriptional processing steps before translation. One of the post-transcriptional steps - 3' end processing, which consists of cleavage and polyadenylation, is critical for delimiting the 3' end of mRNA and determining regulatory elements for downstream post-transcriptional/translational regulation. Like another well-characterized mRNA processing step - splicing, 3' end processing is very flexible due to the diversity of trans-acting factors and cis-acting elements in the 3' end of mRNA. In recent years, the differential usage of alternative polyA sites (APA) of the same gene, which leads to mRNA isoforms of different 3' UTR, has been increasingly revealed by both experimental and computational studies. More significantly, the global changes of 3' UTR length have been observed in multiple clinical settings, particularly in the cancer cells. However, the depiction of APA phenomenon does not synchronize the efforts to study the mechanism underlying APA biogenesis. In this thesis, we first describe general principle and pipeline to identify APA in different biological or clinical conditions using various high throughput sequencing techniques. After that, we present the work about the global impacts of two RNA binding proteins (ESRP/aCP) and one core 3' end processing factor (CstF64 and its paralog CstF64τ) on the regulation of APA. The APA identification analyses and motif analyses suggest a wide range of APA associated with the expression change of those proteins in different cell lines. In addition, for each protein, we have collect substantial evidence about the mechanism underlying the APA induction. Our findings could provide significant insights into the APA regulation mechanisms. In addition, we also conducted a research on the induction of APA in JEG-3 cells as a response to the change of oxygen supply (Hypoxia and Normoxia). Using a robustness protocol for specifically sequencing 3' end of mRNA, we identified more than 500 APA events and revealed a global shortening pattern of 3' UTR length as a result of hypoxia. The work on APA in this thesis largely increases the understanding of APA regulation by various proteins and provided new evidence for the APA in clinical condition.

No description available.

No description available.

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No description available.

The microtubule-associated protein tau forms pathological neuronal filaments in Alzheimer's disease (AD) and other neurodegenerative disorders, known collectively as tauopathies. Previous studies in transgenic mouse models of AD suggest that reducing tau expression may be safe and beneficial for the prevention or treatment of AD and possibly other tauopathies. As a first step toward identifying novel therapeutic strategies to reduce tau levels, the studies presented in this dissertation aim to investigate the role of the human tau 3'-untranslated region (3'-UTR) in regulating tau expression. Tau expresses two 3'-UTR isoforms, long and short, as a result of alternative polyadenylation. The exact sequence of these two 3'-UTR isoforms was determined by rapid amplification of cDNA 3'-ends (3'-RACE), and the two 3'-UTR isoforms were cloned into a luciferase reporter vector. Using these reporter constructs, the expression of these isoforms was found to be differentially controlled in human neuroblastoma cell lines M17D and SH-SY5Y by luciferase assays and quantitative PCR (qPCR). Through an unbiased screen of tau 3'-UTR deletions and fragments using luciferase reporter constructs, several regions in the long tau 3'-UTR isoform that contain regulatory cis-elements were identified. Additionally, several microRNAs were computationally identified as candidates that might bind the long tau 3'-UTR and thereby differentially control the expression of long versus short tau 3'-UTR isoforms. Screening these candidate microRNAs via luciferase reporter assay identified miR-34a, which was subsequently shown to repress the expression of endogenous tau protein and mRNA in M17D cells using Western blot and qPCR, respectively. Conversely, inhibition of endogenously expressed miR-34 family members leads to increased endogenous tau expression. Taken together, these studies suggest that the expression of the two tau 3'-UTR isoforms is differentially regulated and that this differential regulation is due to the presence of regulatory cis-elements found only in the long tau 3'-UTR isoform, including a binding site for miR-34 family members. Improved understanding of the regulation of tau expression by its 3'-UTR may ultimately lead to the development of novel therapeutic strategies for the treatment of Alzheimer's disease and other tauopathies.

Drought stress is considered one of the most devastating abiotic stress factors that limit crop productivity for modern agriculture worldwide. There is a large range of physiological and biochemical responses induced by drought stress. The responses range from physiological and biochemical to regulation at transcription and posttranscriptional levels. Post-transcription, the products encoded by eukaryotic genes must undergo a series of modifications to become a mature mRNA. Polyadenylation is an important one in terms of regulation. Polyadenylation impacts gene expression through determining the coding and regulation potential of the mRNA, especially when different mRNAs from the same gene may be polyadenylated at more than one position. This alternative polyadenylation (APA) has numerous potential effects on gene regulation and function. I have studied the impact of drought stress on APA, testing the hypothesis that drought stress may give rise to changes in the usage of poly(A) sites generating different mRNA isoforms. The results showed that usage of poly(A) sites that lie within 5’-UTRs and coding sequence (CDS) changes more than usage of sites in other regions due to drought stress. Alternative polyadenylation is meditated by the polyadenylation complex of proteins that are conserved in eukaryotic cells. The Arabidopsis CPSF30 protein (AtCPSF30), which is an RNA-binding endonuclease subunit of the polyadenylation complex, plays an important role in controlling APA. Previous study showed that poly(A) site choice changes on a large scale in oxidative stress tolerant 6 (oxt6), a mutant lacking AtCPSF30. Within the mutant/WT genotypes, there are three classes of poly(A) site, wild type specific, oxt6 specific, and common (both in wild type and mutant). The wild type specific and oxt6 specific mRNAs make up around 70% of the total of all mRNA species. I hypothesize that the stability of these various mRNA isoforms should be different, and that this is a possible way that AtCPSF30 regulates gene expression. I tested this by assessing the influence poly(A) sites can have on the mRNA isoform’s stability in the wild type and oxt6 mutant. My results show that most mRNA isoforms show similar stability profiles in the wild-type and mutant plants. However, the mRNA isoforms derived from polyadenylation within CDS are much more stable in the mutant than the wild-type. These results implicate AtCPSF30 in the process of non-stop mRNA decay. Messenger RNA polyadenylation occurs in the nucleus, and the subunits of the polyadenylation complex that meditate this process are expected to reside within the nucleus. However, AtCPSF30 by itself localizes not only to the nucleus, but also to the cytoplasm. AtCPSF30 protein contains three predicted CCCH-type zinc finger motifs. The first CCCH motif is the primary motif that is responsible for the bulk of its RNA-binding activity. It can bind with calmodulin, but the RNA-binding activity of AtCPSF30 is inhibited by calmodulin in a calcium-dependent manner. The third CCCH motif is associated with endonuclease activity. Previous studies demonstrated that the endonuclease activity of AtCPSF30 can be inhibited by disulfide reducing agents. These published results suggest that there are proteins that interact with AtCPSF30 and act through calmodulin binding or disulfide remodeling. To test this hypothesis, I screened for proteins that interact with AtCPSF30. For this, different approaches were performed. These screens led me to two proteins-one protein that is tyrosine-phosphorylated and whose phosphorylation state is modulated in response to ABA, which well-known ABA regulates guard cell turgor via a calcium-dependent pathway, and the other is ribosome protein L35(RPL35), which plays an important role in nuclear entry, translation activity, and endoplasmic reticulum(ER) docking. These results suggest that multiple calcium-dependent signaling mechanisms may converge on AtCPSF30, and AtCPSF30 might be directly interact with ribosome protein.

Cancer is the collective term used to describe a diverse group of diseases that share certain hallmarks, which in turn enables the affected cells to sustain an uncontrolled cell growth. Despite the increasing efforts and advances in cancer therapies, cancers are still responsible for approximately 10% of all the deaths worldwide. Furthermore, the increase in the average human lifespan will further contribute to the cancer incidences. This brings the necessity to focus our efforts on early detection and effective diagnosis methods. With the advances in high-throughput genomics technologies, gene expression signatures have gained attention as a novel method in cancer diagnostics. These signatures are identified by simply comparing the expression levels of genes in tumor and control samples. Here, we propose an alternative method based on the probe expression level measurement of 3&rsquo / UTR of candidate genes. We chose breast cancer as a model and performed an in silico analysis on publicly available gene expression datasets of Affymetrix chips to analyse 3&rsquo / UTR shortening during breast cancer situation. Overall, our analysis suggests that shortening of 3&rsquo / UTR is a significant mechanism observed in breast cancer .