<p>Much of the regulation of gene expression occurs at the posttranscriptional level, and much of this regulation is controlled and coordinated by RNA binding proteins (RBPs). Many RBPs have multiple mRNA targets, and the proteins encoded by these targets often share functional relationships, forming posttranscriptional RNA operons. These operons often reflect the function of the RBP, thus determination of the genome-wide targets of RBPs allows insight into their functions.</p> <p>The PUF family of RBPs is characterized by the presence of an extremely well conserved RNA binding domain, typically consisting of 8 repeats of an RNA binding motif, with each repeat binding to one RNA base. PUF proteins are proposed to have an ancestral role in self-renewal of stem cells and have been shown to affect a number of developmental processes. Human and other vertebrate genomes contain two canonical PUF genes, Pum1 and Pum2, and at the outset of this study there was very little known about functions or targets of either protein, especially Pum1.</p> <p>In order to identify the genome-wide targets of human Pum1 we used RNA immunoprecipitation followed by microarray, or RIP-Chip, analysis. RIP-Chip allowed us to identify Pum1 target mRNAs in human HeLa cells. We found that there were numerous functional relationships among the proteins encoded by these mRNAs, forming putative RNA operons. Some of these potential operons are progression of cell cycle, cell differentiation and proliferation, and regulation of transcription. We were also able to find a consensus Pum1 binding motif, UGUAHAUA, in the 3' UTRs of Pum1 target mRNAs. </p> <p>The genome-wide targets of PUF proteins from other species have been previously identified, and by comparing the targets of human Pum1 to targets of Drosophila Pumilio and yeast Puf3, both of which bind to the same RNA sequence as Pum1, we determined that there has been evolutionary rewiring of regulation by Puf proteins. While the PUF RNA binding domain and consensus binding sequence have remained almost identical through evolution, the surrounding protein sequence and the mRNAs bound have changed dramatically, indicating that evolutionary rewiring is occurring in a modular fashion. </p> <p>After identifying Pum1 associated mRNAs, we went on the study the function of Pum1. Through Pum1 knockdown assays we found that Pum1 enhances decay of target mRNAs, and that this effect is likely due to Pum1 enhancing deadenylation of these mRNAs. We also showed by immunofluorescence that Pum1 protein has a cytoplasmic granular subcellular localization and upon oxidative stress relocates to stress granules but not processing bodies. We were, however, unable to detect any difference in Pum1 mRNA targeting after stress. We were also unable to detect any changes in progression through cell cycle after Pum1 knockdown. </p> <p>In this study we identified the genome-wide mRNAs associated with Pum1, determined functional relationships among these targets related to the proposed ancestral role of PUF proteins in self-renewal of stem cells, and identified a sequence motif to which Pum1 binds in these mRNAs. We also demonstrated that Pum1 enhances decay of associated mRNAs, and that this effect is likely due to Pum1 enhancing deadenylation of associated mRNAs. These results provide a description of mRNA targets and mechanisms of action of Pum1 proteins, which will provide a strong foundation for future experiments to further explore the functions of the Pum1, especially as they relate to human stem cells.</p> / Dissertation

Cardiac muscle function necessitates the meticulous assembly and interactions of several cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and transmission forces. Despite extensive studies identifying the protein components responsible for these important aspects of heart development, putative RNA based mechanisms remain poorly understood, even with their demonstrated importance in other tissues. Evidence suggests that post-transcriptional regulation is critical for muscle function, but the molecular players involved (RNA binding proteins and mRNA targets) have remained elusive. We investigated the molecular mechanisms and targets of the muscle-specific Fragile X Related protein-1 (FXR1), an RNA binding protein whose absence leads to perinatal lethality in mice. Loss of FXR1 results in global protein level alterations. Morphological and biochemical analyses of Fxr1^(-/-) mice revealed severe disruption of intercalated disc and costamere architecture and composition. We identified several candidate mRNAs specifically enriched in the FXR1 protein complex. Two targets that likely contribute to the architectural defects are desmoplakin (dsp) and talin2 (tln2). In vitro assays indicate that FXR1 binds to these mRNA targets directly and represses their translation. Additionally, we provide preliminary evidence that the Fxr1^(-/-) mice mimic a hypothyroid state of cardiac gene expression, with alterations in myosin heavy chain and troponin I isoforms. Our findings reveal the first mRNA targets of FXR1 in muscle and support translational repression as a novel mechanism for cardiac muscle development and function.

The RNA-binding protein HuR controls key cellular processes by binding target mRNAs and regulating them at various post-transcriptional levels. HuR can function as an Internal Ribosome Entry Site (IRES) trans-acting factor that regulates the IRES-mediated translation of XIAP. Since XIAP and Bcl-xL expression was reported to be co-regulated, we investigated whether HuR is also a regulat or of Bcl-xL expression. We found that HuR binds the 3’end of the Bcl-xL 5’UTR in-vitro. In U2OS cells, we showed that loss of HuR by siRNA significantly increased Bcl-xL protein expression while Bcl-2 and Mcl-1 levels remained unchanged. We found that the HuR-dependent Bcl-xL increase was through translation, shown by polysome profiling. Possible transcriptional, stability and splicing changes were eliminated. At the physiological level HuR levels did not impact cell survival but altered mitochondrial morphology, partially through Bcl-xL. Thus, HuR may be involved in maintaining proper mitochondrial function by controlling Bcl-xL expression.

Abnormal aggregation of microtubule associated protein tau is the defining pathological hallmark of tauopathies, which include Alzheimer’s disease (AD) and related frontotemporal dementias (FTLD-tau). However, the cellular events precipitating tau pathogenesis in disease are unknown. Here, we demonstrate a novel mechanism regulating tau aggregation in tauopathies. We have previously shown that RNA binding proteins (RBPs) associated with stress granules (SGs) progressively accumulate with tau in multiple mouse models of tauopathy, as well as in human AD and FTLD-tau brain tissue. We now present a novel functional role for tau in regulating the biology of SGs in neurons. Tau facilitates the rapid formation of SGs in the soma and dendrites in response to exogenous stress, which functions to transiently reprogram protein synthesis to promote cell survival (also known as the ‘translational stress response’). However, the chronic interaction of tau with SG proteins in disease, such as with the SG nucleating protein T cell intracellular antigen 1 (TIA1), promotes tau misfolding and neurotoxicity, which can be modulated in primary neurons by pharmacological or genetic manipulations that increase (i.e. puromycin, TIA1 overexpression) or decrease (i.e. cycloheximide, TIA1 knockdown or knockout) SG formation, respectively. In order to test whether SGs also mediate the progression of tauopathy in vivo, we crossed PS19 transgenic (P301S) tau mice with Tia1-/- or C57BL/6J (background strain) mice. PS19 mice with heterozygous reduction in TIA1 (P301S TIA1+/-) developed less SGs compared to P301S TIA1+/+ mice, which was associated with marked neuronal protection, improved cognitive function, and prolonged lifespan. The behavioral neuroprotection in P301S TIA1+/- mice was associated with decreased accumulation of soluble tau oligomers, and occurred despite the increased presence of neurofibrillary tangles. Our findings suggest that TIA1 stabilizes tau in its oligomeric state, preventing its further assembly into insoluble fibrils, which are less toxic. More importantly, the studies described in this dissertation identify modulation of RBP aggregation in SGs as a promising therapeutic strategy for the treatment of AD and FTLD-tau.

xv, 106 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Myotonic Dystrophy type I (DM1) is caused by muscleblind protein sequestration to aberrantly expanded CUG repeats. When muscleblind is sequestered it can no longer fulfill its role as an alternative splicing regulator, leading to mis-splicing events in both humans and Drosophila . The muscleblind protein family's RNA binding specificity has been minimally characterized. Only one pre-mRNA target in humans, cardiac troponin T (cTNT), has a known MBNL1 binding site. In order to understand muscleblind's RNA binding specificity and identify a consensus binding motif, systematic evolution of ligands by exponential enrichment (SELEX) was performed on both the Drosophila muscleblind protein, Mbl, and the human ortholog, MBNL1. Drosophila has provided a useful model for studying the disease mechanism of DM1. Studies of Mbl's RNA binding specificity to CUG repeats concluded that replacing the U-U mismatches with different pyrimidine-pyrimidine mismatches was tolerated, but no other mutations were. To understand Mbl's RNA binding specificity, SELEX was performed. After 6 rounds, several sequences were identified that bound with high affinity, all containing the 5'-AGUCU-3' consensus motif. One sequence, SELEX RNA 20 was analyzed further. In addition to the guanosine in the consensus motif of SELEX RNA 20, two other guanosines were shown to be protected by Mbl in a footprinting assay, indicating that Mbl has a strong preference for binding guanosine. Also, two "tail" regions of SELEX RNA 20 were shown to be single stranded and required for binding by Mbl. These results indicate that Mbl is a highly specific RNA binding protein with preference for both single and double stranded guanosine-rich regions. A doped SELEX was performed on MBNL1's binding site from the cTNT pre-mRNA to determine which sequences and structural aspects were important for recognition by MBNL1. Pool 5 RNA sequences bound with high affinity, and the motif 5'-YGCUU-3' was selected. This motif was then used to identify new MBNL1 binding sites in pre-mRNAs regulated by MBNL1, SERCA1 and MBNL1. The identification of this motif and two new MBNL1 sites provide insight into MBNL1-mediated alternative splicing. This dissertation includes both my previously published co-authored material and my unpublished co-authored material. / Adviser: J. Andrew Berglund

The function of two subunits of the putative mitochondrial RNA binding complex (MRB1) associated with RNA editing in parasitic protist Trypanosoma brucei was studied using various in vivo and in vitro methods of molecular biology.

The RNA-binding protein HuR controls key cellular processes by binding target mRNAs and regulating them at various post-transcriptional levels. HuR can function as an Internal Ribosome Entry Site (IRES) trans-acting factor that regulates the IRES-mediated translation of XIAP. Since XIAP and Bcl-xL expression was reported to be co-regulated, we investigated whether HuR is also a regulat or of Bcl-xL expression. We found that HuR binds the 3’end of the Bcl-xL 5’UTR in-vitro. In U2OS cells, we showed that loss of HuR by siRNA significantly increased Bcl-xL protein expression while Bcl-2 and Mcl-1 levels remained unchanged. We found that the HuR-dependent Bcl-xL increase was through translation, shown by polysome profiling. Possible transcriptional, stability and splicing changes were eliminated. At the physiological level HuR levels did not impact cell survival but altered mitochondrial morphology, partially through Bcl-xL. Thus, HuR may be involved in maintaining proper mitochondrial function by controlling Bcl-xL expression.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22007号 / 医科博第105号 / 新制||科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 稲垣 暢也, 教授 髙橋 良輔, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Both genetic and environment factors influence susceptibility to substance use disorders. However, the genetic basis of these disorders is largely unknown. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene for reduced methamphetamine (MA) stimulant sensitivity. Mutation (heterozygous deletion of a small region in the first coding exon) in Hnrnph1 also decreased MA reinforcement, reward, and dopamine release. 5’UTR genetic variants in Hnrnph1 support reduced 5’UTR usage and hnRNP H protein expression as a molecular mechanism underlying the reduced MA-induced psychostimulant response. Interestingly, Hnrnph1 mutant mice show a two-fold increase in hnRNP H protein in the striatal synaptosome with no change in whole tissue level. Proteome profiling of the synaptosome identified an increase in mitochondrial complex I and V proteins that rapidly decreased with MA in Hnrnph1 mutants. In contrast, the much lower level of basal mitochondrial proteins in the wild-type mice showed a rapid, MA-induced increase. Altered mitochondrial proteins associated with the Hnrnph1 mutation may contribute to reductions in MA behaviors. hnRNP H1 is an abundant RNA-binding protein in the brain, involved in all aspect of post-transcriptional regulation. We examined both baseline and MA-induced changes in hnRNP H-RNA interactions to identify targets of hnRNP H that could comprise the neurobiological mechanisms of cellular adaptations occurring following MA exposure. hnRNP H post-transcriptionally regulates a set of mRNA transcripts in the striatum involved in psychostimulant-induced synaptic plasticity. MA treatment induced opposite changes in binding of hnRNP H to these mRNA transcripts between Hnrnph1 mutants versus wild-types. RNA-binding, transcriptome, and spliceome analyses triangulated on hnRNP H binding to the 3’UTR of Cacna2d2, an upregulation of Cacna2d2 transcript, and decreased 3’UTR usage of Cacna2d2 in response to MA in the Hnrnph1 mutants. Cacna2d2 codes for a presynaptic, voltage-gated calcium channel subunit that could plausibly regulate MA-induced dopamine release and behavior. The multi-omics datasets point to a dysregulation of mitochondrial function and interrelated calcium signaling as potential mechanisms underlying MA-induced dopamine release and behavior in Hnrnph1 mutants.

Post-transcriptional regulations (PTRs) have always been considered features of organisms with higher complexity. However recently, the interest toward the post- transcriptional mechanisms in prokaryotes increased. The bacterial proteome is much more complex compared to the genome size, suggesting a tight and articulate regulation of proteins production, extremely important for the bacterial adaptation to an always changing environment. Bacterial PTRs are responsible of modulation of mRNA stability and decay, translation initiation and elongation, modulation of the access of ribosome to the ribosome binding site and control of termination of the transcript. The main actors in the PTRs are small non-coding RNA (responsible of the inhibition of the transcription) and RNA binding proteins (RBPs), which modulate the translation and half-life of the mRNA. RBPs, are particularly of my interest since I wanted to find a possible orthologous of the eukaryotic Elav-like (Elavl) family of proteins in Acinetobacter baumannii. Elav-like proteins are present in all metazoans and are characterized by two highly conserved sequences: RNP-1 (a quite well conserved hexamer) and RNP-2 (a really well conserved octamer) that are responsible of binding to the mRNA. Each species has a different number of Elavl paralogous that is totally independent from the complexity of the organisms, suggesting a more ancient origin. In particular, I focused on the human paralog HuR (human antigen R). HuR is characterized by three RNA Recognition motif (RRM) -domains, is ubiquitously expressed and is mainly localized into the nucleus (where it is responsible of maturation of the mRNA), but under stress stimuli, can shuttle into the cytoplasm where protect the target mRNA from degradation, by binding AU/U rich sequences (ARE sequences). Its high concentration into the cytoplasm can lead to the overexpression of oncogenes and pro-tumorigenic factors. The choice of Acinetobacter baumannii comes from the increasing worldwide concern toward this pathogen that is becoming multidrug resistant. Indeed, in Italy, more the 50% of nosocomial infections are caused by A. baumannii. I found a putative protein (AB-Elavl), composed by a single RRM domain endowed with similar features of the eukaryotic RRM domain as the presence of a quite well conserved RNP-2 and a less conserved RNP-1. I expressed this protein with recombinant tools and confirmed the production of the protein in the host by western blot and mass spectrometry. I evaluated the binding activity of AB-Elavl testing the EC50 and the Kd with different biochemical assays (EMSA, AlphaScreen and HTRF- FRET) toward three different RNA sequences, in order to test the specificity. By X- RAY and NMR, I confirmed the folded structure that can be overlapped to the HuR’s one and the interaction with the probes tested, highlighting the presence of binding, but with different specificity. I also tested some small molecules developed for interfering in the binding of HuR with the target sequence and found a possible compound able to interact with AB-Elavl, by disrupting the binding with the target probe. All these results suggest an ancient origin of the metazoans’ Elavl family of proteins that probably share a common ancestor with AB-Elavl. More studies should be performed to better understand the role of AB-Elavl in A. baumannii as well as in other bacteria. In fact, I found the presence of other ARE sequence-binding proteins also in Pseudomonas aeruginosa. Interesting would be to check the presence of this protein in all the multidrug resistant ESKAPE bacteria.