Alcoholism is a complex neurological disorder characterized by loss of control in limiting intake, compulsion to seek and imbibe ethanol, and chronic craving and relapse. It is suggested that the characteristic behaviors associated with the escalation of drug use are caused by long-term molecular adaptations precipitated by the drug’s continual administration. These lasting activity-dependent changes that underlie addiction-associated behavior are thought, in part, to depend on new protein synthesis and remodeling at the synapses. It is well established that mRNA can be transported to neuronal distal processes, where it can undergo localized translation that is regulated in a spatially restricted manner in response to stimulation. Through two avenues of investigation, the research herein demonstrates that behavioral responses to ethanol result, at least in part, from alterations in the synaptic transcriptome which contribute to synaptic remodeling and plasticity. The synaptoneurosome preparation was utilized to enrich for RNAs trafficked to the synapse. Two complementary methods of genomic profiling, microarrays and RNA-Seq, were used to survey the synaptic transcriptome of DBA/2J mice subjected to ethanol-induced behavioral sensitization. A habituating expression profile, characteristic of glucocorticoid-responsive genes, was observed for a portion of synaptically targeted genes determined to be sensitive to repeated ethanol exposure. Other ethanol-responsive genes significantly enriched for at the synapse were related to biological functions such as protein folding and extra-cellular matrix components, suggesting a role for local regulation of synaptic functioning by ethanol. In a separate series of experiments, it was shown that altered trafficking of Bdnf, an ethanol-responsive gene, resulted in aberrant ethanol behavioral phenotypes. In particular, mice lacking dendritically targeted Bdnf mRNA exhibited enhanced sensitivity to low, activating doses and high, sedating doses of ethanol. Together these experiments suggest that ethanol has local regulatory effects at the synapse and lays the foundation for further investigations into the role of the synaptic transcriptome in ethanol-responsive behaviors. Supported by NIAA grants R01AA014717, U01 AA016667 and P20AA017828 to MFM, F31AA021035 to MAO, and NIDA T32DA007027 to WLD.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of proteins intitially characterised in the late 1980’s by their presence in complexes that form on nascent RNA transcripts. This definition was purely operational, and was based on protein isolation techniques available at that time. Since then, the tendency to refer to and view the hnRNPs as a protein family has become increasingly prevalent, although there has been no systematic sequence- or structure-based study of their evolutionary history. While the hnRNPs share some structural characteristics (modular structure, presence of RNA-binding domains) and functional properties (binding to RNA, involvement in multiple steps of RNA processing), these criteria also apply to other types of RNA binding proteins (RBPs), such as the SR and ELAV families of proteins. Thus, we have adopted a more methodical and rigorous approach to the classification of hnRNPs and other RBPs, through the phylogenetic analysis of their sequences and domains. Besides establishing phylogenetic relationships and simplifying nomenclature, studying the evolutionary divergence of the hnRNPs is important for understanding their functional features. The hnRNP A/B subfamily is comprised of paralogues A1, A2/B1, A3 and A0, which exhibit a high level of similarity at both the sequence and structural level. While they are often treated as functional homologues, they are not functionally identical. Hence, we undertook a detailed comparison of their sequences, and found that the introduction of novel splicing signals or mutation of existing sequence elements has led to changes in alternative splicing patterns between the paralogues, which may affect the regulation of their expression and their RNA binding properties. In addition, we also investigated species-specific alternative splicing of the hnRNPs A/B, which has significant implications for the interpretation of current research, since different research groups tend to use different model organisms in their experiments. Hence, exploration of the sequence divergence of the hnRNPs A/B has provided some clues as to how their functional differences arose, and also highlighted the need to take species-specific splicing into consideration. Alternative splicing can create functional variation not only between paralogues, but also between splice variants. hnRNP A2/B1, which has a well-established role in mRNA trafficking in neuronal cells, has four spliceoforms. In order to study the contribution of each isoform to this process, we investigated isoform-specific variations in intracellular localisation, and expression in different developmental stages and species. We found that in rat, minor isoform A2b was the predominant isoform in the cytoplasm, and may be the key player in mRNA trafficking. These findings demonstrate the importance of considering individual isoforms (including those expressed in low abundance) when studying the function of alternatively spliced proteins, especially when the function is restricted to a particular subcellular compartment. In addition to its cytoplasmic role in mRNA trafficking, hnRNP A2/B1, and the other hnRNPs A/B, have multiple nuclear functions, including packaging of nascent transcripts, nuclear export of mRNA, regulation of alternative splicing and telomere maintenance. These processes take place in discrete regions within the nucleus, and thus we examined the subnuclear distribution of the hnRNPs A/B. We found that hnRNP A1 had a localisation pattern distinct from that of A2/B1 and A3, and that these patterns were spatially and temporally regulated. Hence, the evolutionary divergence of the hnRNPs A/B has affected the localisation, expression and splicing patterns of these proteins, which we have examined at multiple levels, including comparisons across all hnRNPs, within the hnRNP A/B paralogues, and between the hnRNP A2/B1 splice variants. As the hnRNPs A/B are involved in almost every step in RNA processing, this functional diversity has significant implications for transcriptomic complexity. Furthermore, our findings highlight the importance of taking species- and isoform-specific differences into account when investigating protein function. In conclusion, this study of the hnRNPs A/B provides a conceptual framework for exploring the relationships between sequence, structural and functional divergence, which may be applicable to protein families in general.

The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications. / Ph. D.

Cuscuta pentagona is an obligate parasitic plant that hinders production of crops throughout the world. Parasitic plants have unique morphological and physiological features, the most prominent being the haustorium, a specialized organ that functions to connect them with their host's vascular system. The Cuscuta haustorium is remarkable in that it enables mRNA movement to occur between hosts and parasite, but little is known about the mechanisms regulating cross-species mRNA transfer or its biological significance to the parasite. These questions were addressed with genomics approaches that used high throughput sequencing to assess the presence of host mRNAs in the parasite as well as parasite mRNAs in the host. For the main experiment Cuscuta was grown on stems of Arabidopsis thaliana and tomato (Solanum lycopersicon) hosts because the completely sequenced genomes of these plants facilitates identification of host and parasite transcripts in mixed mRNA samples. Tissues sequenced included the Cuscuta stem alone, the region of Cuscuta-host attachment, and the host stem adjacent to the attachment site. The sequences generated from each tissue were mapped to host reference genes to distinguish host sequences, and the remaining sequences were used in a de novo assembly of a Cuscuta transcriptome. This analysis revealed that thousands of different Arabidopsis transcripts, representing nearly half of the expressed transcriptome of Arabidopsis, were represented in the attached Cuscuta. RNA movement was also found to be bidirectional, with a substantial proportion of expressed Cuscuta transcripts found in host tissue. The mechanism underlying the exchange remains unknown, as well as the function of mobile RNAs in either the parasite or host. An approach was developed to assay potential translation of host mRNAs by detecting them in the Cuscuta translatome as revealed by sequencing polysomal RNA and ribosome-protected RNA. This work highlights RNA trafficking as a potentially important new form of interaction between hosts and Cuscuta. / Ph. D.