Bovine adenovirus (BAdV)-3 is a non-enveloped icosahedral DNA virus, which replicates in the nucleus of infected cells, and is being developed as a vector for vaccination for humans and animals. The genome of BAdV-3 is organized into early, intermediate and late genes and it has thirty three predicted open reading frames (Reddy et al., 1998). The late region of BAdV-3 is divided into seven families (L1-L7) (Reddy et al., 1998). One of the proteins expressed in the L-6 region encodes a protein called pVIII, which is a minor capsid protein connecting the core with the inner surface of the capsid. The objective of the current study was to characterize pVIII protein of BAdV-3 and to examine its role in the life cycle of BAdV-3. Anti-pVIII serum detected a protein of 24 kDa at 12-48 hr post infection and an additional protein of 8 kDa at 24-48 hr post infection. While a 24 kDa protein is detected in empty capsids, only the C-terminal cleaved protein of 8 kDa is detected in the mature virion suggesting that amino acids 147-216 of conserved C- terminus of BAdV-3 pVIII are incorporated in mature virions. The pVIII protein predominantly localizes to the nucleus of BAdV-3 infected cells utilizing the classical importin α /β dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 57-72 of the conserved N-terminus bind to importin α-3 with high affinity and are required for the nuclear localization. Detection of hexon associated with both, precursor (24 kDa) and cleaved (8 kDa) form of pVIII suggests that the C-terminus of pVIII interacts with Hexon. Based on yeast II hybrid screening assay, we identified the cellular protein DDX3 as an interacting protein partner of pVIII. Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport (Yedavalli et al., 2004) and induction of interferon production (Schroder et al., 2008; Wang et al., 2009). Here, we provide evidence regarding the involvement of DDX3 in cap dependent cellular mRNA translation and show that targeting of DDX3 by the adenovirus pVIII protein abolishes cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacts with DDX3 in transfected and bovine adenovirus (BAdV-3) infected cells. pVIII inhibited capped mRNA translation in-vitro and in-vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in the cap binding complex. The total amount of eIFs appeared similar in uninfected or BAdV-3 infected cells. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E or PABP. These data indicate that interaction of pVIII with DDX3 depletes eIF3, eIF4E and PABP from the cap-binding complex. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA by excluding functional cap-binding complex from the capped cellular mRNA. BAdV-3 infection of DDX3 positive cells significantly inhibits cellular protein synthesis at late times post-infection. Interestingly, knockdown of DDX3 resulted in significant reduction in virus yield and expression of BAdV-3 late proteins at late times post-infection. Our results suggest that selective translation of BAdV-3 late mRNAs observed at late time post-infection of DDX3 positive cells is abrogated in DDX3 knock down cells. Moreover, the reduction in the extent of protein synthesis is evidenced by less functional 80S and polysomes in pVIII expressing plasmid transfected cells. Alternatively, DDX3 and pVIII binds to BAdV-3 tripartite leader (TPL) and the translation of mRNAs containing TPL at their 5’ ends is enhanced in the presence of pVIII and DDX3 proteins. From this observation, we concluded that pVIII and DDX-3 might promote the translation of late viral mRNAs by interacting with TPL.

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No description available.

To understand bovine oocytes meiotic maturation, developmental potential, gene expression is required. The gene expression studies in the preimplantation bovine oocytes has been difficult, because the procedures that are being employed for extracting total RNA are not specific for bovine oocytes and so far is not providing the required amount for further procedures. Quantification of genes generally requires large amounts of total RNA in order to overcome the problem of low amount of mRNA present, so a standardized specific protocol is recommended. These days most of the researchers are using commercial Kit protocols without knowing the significance of chemicals and how they are acting on cells. In present project a standardized protocol (modified trizol) was designed for bovine oocytes, which was specific and less expensive. The efficiency of this protocol compared with Pure Link (Kit Protocol), GNTC (Guanidinium thiocyanate) for extraction of total RNA from fresh oocytes, vitrified oocytes with PROH (1,2 propanediol) and DMSO (dimethylsulfoxide) cryoprotectans was much better. The RNA (absorbance 260/280) purity levels of the standardized protocol was ranging (1.50-2.10), whereas for GNTC protocol (1.05-1.36), Pure Link (kit protocol) (2.05-2.7). Amplification of housekeeping genes (SDHA and GAPDH gene) showed the specificity and efficiency of the standardized protocol over other protocols.

Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized. RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives. RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches.

Gene expression at the level of mRNA translation is critical for cells to respond to external signals; it allows changes in protein synthesis without triggering transcription of a new set of genes. Control of mRNA translation and stability is important in several cellular processes including cell growth and differentiation. Thus regulation of the cellular machinery involved in mRNA translation is crucial. Poly (A) binding protein (PABP1), eukaryotic elongation factor 1A (eEF1A) and ribosomal protein S6 (RPS6) are important members of the cellular mRNA translation machinery, the mRNAs that encode these proteins belong to the terminal oligo pyrimidine tract (TOP) containing family. Translation of the TOP mRNAs is regulated by growth signals and usually codes for several proteins involved in mRNA translation. Our laboratory has previously reported up regulation of PABP1 mRNA translation during recovery from heat shock. It was also shown that the terminal oligopyrmidine tract (TOP) cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation; however the mechanism for achieving this is unknown. In the studies reported here, we showed that translation of eEF1A and RPS6 expression was similarly enhanced during recovery from heat shock. Analyses of samples of in vivo cross linked RNA– protein complexes, immunoprecipitated by ZNF9 antibody, for the presence of specific mRNAs showed that the cellular nucleic acid binding protein ZNF9 binds not only to TOP mRNAs but also mRNA that lack the TOP element such as to β-actin mRNA. To elucidate the mechanism of activation of TOP mRNA translation, as a candidate trans acting factor, siRNA was used to deplete the cellular level of ZNF9 from heat shocked HeLa cells to examine its potential role in stimulation of TOP mRNA translation during recovery from heat shock. Results show that the knock down of ZNF9 disallowed the preferred stimulation of PABP1, eEF1A and RPS6 expression during recovery from heat shock. There was no detectable effect on the constitutive expression of either β-actin or PABP1, eEF1A and RPS6 in exponentially growing HeLa cells. These results suggest that binding of ZNF9 to TOP mRNAs per se does not inhibit translation, but more likely it acts as a general facilitator of mRNA translation. It is possible that modification of the interaction between ZNF9 with other unknown protein factors is responsible for its preferred effect on all three TOP mRNAs studied here. Additionally, results also suggest that a different TOP sequences amongst the observed TOP mRNAs responds similarly to ZNF9.

No description available.

Alternative pre-mRNA splicing is often controlled by cell signals (1-3). Membrane depolarization/calcium (Ca2+) signaling controls alternative splicing of a group of genes in neurons and endocrine cells (4-9), with important implications in memory formation or secretion of hormones and neurotransmitters (10-15). However, the underlying molecular basis remains largely unknown. In rat GH3 pituitary cells, BK potassium channels control cellular electrical firing, which is critical for the release of growth hormone and prolactin. Inclusion of the STREX exon of the Slo1 gene encoding the channel α subunit is repressed by the Ca2+/calmodulin-dependent kinase IV (CaMKIV) upon depolarization (4). We isolated CaMKIV-responsive RNA elements (CaRREs) from a library of 13-nucleotide random sequences through in vivo selection in HEK293T cells. Most elements are CA-rich or A-rich, with the heterogeneous nuclear ribonucleoprotein (hnRNP) L as a binding factor. This is consistent with the finding that CA-rich elements and hnRNP L are targeted by CaMKIV in the regulation of splicing (16). In further efforts to directly link the kinase with hnRNP L, we showed that hnRNP L is essential for the full repression of STREX by depolarization and that a highly conserved CaMKIV target serine (Ser513) of L is required. Ser513 phosphorylation enhanced L binding to the STREX CaRRE1, leading to reduced binding of the constitutive factor U2AF65 to the 3’ splice site of STREX. Mutation of Ser513 abolished both activities. Therefore, hnRNP L mediates the repression of STREX by depolarization through modulation of a key step in spliceosomal assembly. We further identified hnRNP L, L-like (LL) and PTB as repressors of STREX and other depolarization-regulated exons with differential effects. Moreover, a full response of STREX to depolarization is mediated by combinations of hnRNP L and LL or PTB. Another depolarization-responsive exon, the exon 18 of the neuregulin 1 gene, is also controlled in a similar way, with the hnRNP L Ser513 required as well. This work provides the first direct link between the Ca2+ signaling and a specific serine of a regulatory splicing factor. Elucidation of the underlying molecular mechanisms would likely help us understand the fine-tuning of hormone secretion and memory formation.

Nuclear precursor of mature messenger RNA (pre-mRNA) splicing is one of the most highly regulated processes in eukaryotic organisms. In addition to its role in the removal of constitutive or alternative introns present in the pre-mRNA, splicing is also highly integrated into other layers of gene expression. This study investigates the potential role of the nuclear branchpoint binding protein (BBP) outside of the pre-mRNA splicing cycle. More specifically, we were interested in the biological relevance of its association with two cytoplasmic proteins Smy2 and Syh1. Smy2 and Syh1 belong to the GYF family of poly-proline binding proteins, and their roles in cell biology have not been well elucidated. Here we report that Smy2 and Syh1 act redundantly in: (i) limiting pre-mRNA accumulation when yeast cultures reach high cell density, potentially through promoting pre-mRNA decay in the cytoplasm; (ii) restricting Ty1 retrotransposition, apparently by limiting the Ty1 transcript abundance; (iii) limiting the accumulation of BBP-associated yet intronless TDA1 mRNA. With the presence of UACUAAC motif and BBP association as common features of these Smy2/Syh1 sensitive substrates, we tested if BBP interaction is required for Smy2/Syh1 function in RNA metabolism. Interestingly, we found that deletion of BBP C-terminal region (bbp∆C), which largely reduces or abolishes its association with Smy2, does not lead to similar phenotypes as observed in smy2∆ syh1∆ deletion mutant cells. In addition, mutagenesis of the TACTAAC BBP-binding site within the TDA1 coding region does not seem to affect TDA1 mRNA abundance or its sensitivity to the smy2∆ syh1∆ deletions. Therefore, we concluded that while the two BBP-binding proteins Smy2 and Syh1 impact the levels of certain cellular RNAs, this phenomenon is not strictly dependent upon BBP-Smy2 interaction and may be independent of BBP contribution. A model is proposed for Smy2 and Syh1 function in RNA metabolism based on our observations and interactions between these proteins with other factors implicated in RNA stability or translation.

There is a growing interest in assessing the neurotoxic potential and endocrine disrupting properties of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, pre- and post- natal development and behaviour. PFC exposure altered hormone levels (e.g. thyroid hormone, estrogen, and testosterone) and the expression of hormone-responsive genes in mammalian and aquatic species. Hormone-mediated events are critical in central nervous system development and function, especially those controlled by thyroid hormones (THs). The studies presented in this thesis are the first to assess the effects of PFCs on primary cultures of neuronal cells in two avian species; the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following TH-responsive genes were examined using real-time RT-PCR: type II iodothyronine 5’-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif binding factor (Oct-1), and myelin basic protein (MBP). Several PFCs were shown to alter mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. It was determined that short-chained PFCs (<8 carbons) altered the expression of TH-responsive genes to a greater extent than long-chained PFCs (≥8 carbons). Although several significant changes in mRNA expression were observed in TH-responsive genes following PFC exposure in chicken embryonic neuronal (CEN) cells (Chapter 2), there were fewer changes in herring gull embryonic neuronal (HGEN) cells (Chapter 3). The mRNA levels of D2, D3, TTR, and RC3 were altered following treatment with several short-chained PFCs in CEN cells. Oct-1 and RC3 expression were induced following treatment with several short-chained PFCs in HGEN cells. These studies are the first to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and provide insight into the possible mechanisms of action of PFCs in the avian brain.