The heat-shock response is one of the many complex physiological systems that organisms have developed in order to protect their cells against stress. This response is initiated by the binding of heat shock factor 1 (HSF1) to the promoters of genes containing heat-shock elements (HSEs,) which results in the expression of several proteins, among them the proteo-protective inducible heat-shock protein (hsp70i). Due to HSF1s critical role in this process, an active area of research is trying to understand of how HSF1 executes its function. Considering the rapidity with which the field of cell biology is expanding, in particular the sub-field of nuclear compartmentalization, this study seeks to understand how nuclear structure affects the function of HSF1. Specifically, this study investigates the potential role for the interaction between HSF1 and the translocated promoter region protein (Tpr,) a structural component of the nuclear pore, an interaction initially identified by yeast two-hybrid analysis, in the transcription of hsp70i. Due to Tprs location and its putative function in nucleo-cytoplasmic trafficking, this works seeks to answer to the question, Does Tpr play a role in the export of HSF1-driven mRNAs? In a similar vein, heat-shock transcription factor 2 (HSF2,) a less well-understood member of the heat-shock transcription factor family, also interacts with Tpr in the yeast two-hybrid assay. HSF2 has recently been shown to have an active role during mitosis, when the hsp70i gene is being bookmarked for potential expression that might be needed in early G1, when most genes are unable to be expressed. This body of work also seeks to answer the question of, Does the Tpr/HSF2 interaction have a role in positioning the gene in relation to the nuclear pore after mitosis? This study was performed using both novel and standard in vivo and in vitro molecular biology techniques. It ultimately aims to clarify the less understood, although much broader, subject of how does transcription occur in the three-dimensional space of the nucleus.

The spliceosome is a complex, dynamic ribonucleoprotein (RNP) complex that undergoes numerous conformational changes during its assembly, activation, catalysis and disassembly. Defects in spliceosome assembly are thought to trigger active dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. This study found that weak mutant alleles of SPP382 suppress defects in several proteins required for spliceosome activation (Prp38p, Prp8p and Prp19p) as well as substrate mutations (weak branch point mutants). This evolutionary conserved protein had been found in both yeast and mammalian splicing complexes. However, its role in splicing had not been elucidated. This study focused on understanding the cellular role of Spp382p in splicing and particularly in the discard pathway. Spp382p was found to be essential for normal splicing and for efficient intron turnover. Since Spp382p binds Prp43p and is required for intron release in vitro, spp382 mediated suppression of splicing factor mutations might reflect lowered Prp43p activity. In agreement with this, we find that prp43 mutants also act as suppressors. This leads us to propose a model in which defects in spliceosome assembly, like those caused by prp38-1, prompt Spp382p-mediated disassembly of the defective complex via Prp43p Bolstering this theory, we find that Spp382p is specifically recruited to defective complexes lacking the 5 exon cleavage intermediate and spp382 mutants suppress other splicing defects. I show by stringent proteomic and two-hybrid analyses that Spp382p interacts with Cwc23p, a DnaJ-like protein present in the spliceosome and co-purified the Prp43p-DExD/H-box protein. In this study, I also show that Cwc23p is itself essential for splicing and normal intron turnover. Enhanced expression of another protein, Sqs1p, structurally related to Spp382p and also found associated with Prp43p is inhibitory to both growth and splicing. Synthetic lethal and dosage suppression studies bolster a functional linkage between Spp382p, Cwc23p, Sqs1p and Prp43p and together, the data support the existence of a Spp382p -dependent spliceosome integrity (SPIN) complex acting to remove defective spliceosomes.

In eukaryotes, regulation of translation and decay of messenger RNA are critical for fine-tuned control of gene expression. An important point of control is the key transition where mRNAs exit translation and assemble into a non-translating mRNP state that can accumulate in cytoplasmic granules such as P bodies and/or Stress granules. In the budding yeast Saccharomyces cerevisiae , the activators of decapping Dhh1 and Pat1 appear to promote the exit of mRNAs from translation. In my work, summarized below, I describe a new regulator of translation repression and mRNA degradation, Stm1, and its novel mode of action. First, I identified Stm1 as a novel regulator of translation repression and mRNA decay. Stm1 shows several genetic interactions with Pat1 and Dhh1, in a manner consistent with Stm1 promoting the function of Dhh1. This suggests that Stm1 has a role to play in translation repression and/or activation of mRNA decay. stm1 δ strains are defective in the degradation of a subset of mRNAs that include EDC1 and COX17 . These results strongly argue that Stm1 is a novel addition to the mRNA degradation machinery. Second, I have shown that Stm1, a known ribosome-associated protein, can bind and stall 80S ribosomes to repress translation and promote decay. Stm1 is able to repress translation and stall an 80S complex in vitro . Several mutations were identified in the protein, which link the in vitrophenotype to its biological functionin vivo. The analysis of different steps in translation reveals Stm1 functions in a novel manner to inhibit translation after the formation of an 80S complex. Since most of the regulation of translation is thought to happen at the stage of initiation, this study reveals a novel mode of translation regulation. These results also provide a direct and mechanistic link between ribosome function, inhibition of translation and the degradation of messenger RNAs.

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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.

The cannabinoid system is involved in many functions of mammalian brain, such as learning and memory, pain perception and 'locomotion. The "brain type" cannabinoid receptor CB 1 is one of the key elements of the cannabinoid system. In this Thesis, some aspects of the neurobiology of mouse CB 1 are described. CB 1 mRNA distribution was analysed by single and double in situ hybridization (ISH), revealing the expression of the receptor in specific neuronal subpopulations. This expression pattern suggests many putative functional cross-talks between the cannabinoid system and other signalling molecules in the brain, such as glutamate, GABA, cholecystokinin and nitric oxide (NO). The putative functional interactions of the cannabinoid system with the NO pathway was studied by pharmacological treatment of neuronal NO synthase (nNOS) mutant mice with the CBI agonist A9-tetrahydrocannabinol (A9-THC). The results showed that nNOS is necessary for some central effects of A9-THC. Moreover, ISH analysis revealed. that nNOS-deficient mice contain levels of CBI lower than normal in selected brain regions. A "conditional" targeting approach was developed to gain insights into the specific functions of CB 1 in mouse brain. By gene targeting experiments, two mutant lines were obtained. The "Flox CB 1" mouse line, containing the whole open reading frame of CB I flanked by two loxP sites will be the key tool for the generation of mouse mutants with a spatiotemporal-restricted deletion of CB I. The "CBN" mice, carrying a "null" mutation of CB 1, were used for a study aimed to clarify some aspects of the in vitro neuroprotective activity of cannabinoids and, in particular, the involvement of CB 1. In vitro oxidative stress assays were performed on cell lines and on primary neuronal cultures derived from homozygous CBN/CBN mice and wild type littermates. The results indicate a differential protective activity of cannabinoids on cell lines and primary cultures. However, CBI does not appear to be involved in the in vitro leuroprotective effects of cannabinoids.

DGCR8 (DiGeorge critical region 8) protein constitutes part of the Microprocessor complex together with Drosha, and is involved in the nuclear phase of microRNA (miRNA) biogenesis. DGCR8 recognises the hairpin RNA substrates of precursor miRNAs through two double-stranded RNA (dsRNA) binding motifs and acts as a molecular anchor to direct Drosha cleavage at the base of the pri-miRNA hairpin. Recent characterisation of the RNA targets of the Microprocessor by HITSCLIP of DGCR8 protein revealed that this complex also binds and regulates the stability of several types of transcripts, including mRNAs, lncRNAs and retrotransposons. Of particular interest is the binding of DGCR8 to mature small nucleolar RNA (snoRNA) transcripts, since the stability of these transcripts is dependent on DGCR8, but independent of Drosha. This raises the interesting possibility that there could be alternative DGCR8 complex/es using different nucleases to process a variety of cellular RNAs. We performed mass spectrometry experiments and revealed that DGCR8 copurifies with subunits of the nucleolar exosome, which contains the exonuclease RRP6. We demonstrated DGCR8 and the exosome form a nucleolar complex, which degrade the mature snoRNAs tested within this study. Interestingly, we also show that DGCR8/exosome complex controls the stability of the human telomerase RNA component (hTR/TERC), and absence of DGCR8 creates a concomitant telomere phenotype. In order to identify the RNA targets of the DGCR8/Exosome complex on a global scale we performed iCLIP of endogenous and overexpressed RRP6 (wild-type and a catalytically inactive form). Thus, intersection of CLIP datasets from DGCR8 and RRP6 identified common substrates; accordingly snoRNAs were the most represented. In addition, we identified the cellular RNA targets of the RRP6 associated human exosome. The use of a catalytically inactive form of RRP6 stabilised important in vivo interactions that are highly dynamic and transient and also highlighted the role of RRP6-mediated trimming of 3’flanks of immature non-coding RNAs. We will present a global view of the RNA-binding capacity of the RRP6-associated exosome. In sum, we identified a novel function for DGCR8, acting as an adaptor to recruit the exosome to structured RNAs and induce their degradation. Moreover, we have identified DGCR8-depenedent substrates of the exosome and have demonstrated the requirement of RRP6 for 3’ processing of ncRNAs.