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Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

a. Do ponto de vista metodológico dois problemas foram enfrentados neste trabalho: i) extração dos RNA\'s e ii) fracionamento das células das glândulas salivares de R. angelae. i) O processo prático elaborado para resolver o primeiro problema se constituiu numa variação da técnica clássica de extração com fenol. Jogando com a presença de detergente e variações de pH e de temperatura, foi possível se chegar a um método que garante a extração e um fracionamento parcial de, praticamente, todo RNA celular. Com extrações a baixa temperatura obtém-se preferencialmente rRNA e tRNA. O RNA refratário a extração com fenol a frio tem propriedades do RNA nuclear, mas o citoplasma, também parece conter classes de RNA somente extraíveis a quente. A dificuldade de extração do RNA nuclear com fenol frio, foi especulativamente interpretada como uma propriedade das RNP\'s que encerram esta classe de RNA. A grande vantagem deste método de extração está no fato de ter permitido isolar, com as extrações a alta temperatura, frações de RNA enriquecidas em produtos de transcrição específicos de determinados períodos do desenvolvimento larval. ii) O fracionamento celular das glândulas salivares não é alcançado pela aplicação das técnicas tradicionais de fracionamento de tecido. Em vista disso foi necessário procurar uma alternativa experimental para êste caso. Combinando um enzima proteolítico com a ação de detergentes não iônicos foi conseguido um método útil para o fracionamento das células das glândulas salivares. Êste método foi aplicado nesta tese para um estudo inicial das RNP\'s e da transferência do rRNA do núcleo para o citoplasma nas células das glândulas salivares. b. Foi possível verificar com êste trabalho que o rRNA é sintetizado inicialmente na forma de um precursor primário de 37s, o qual é processado no interior do núcleo para dar as espécies maduras de rRNA segundo o esquema: (Ver no arquivo) No 3º período do 4º estádio do desenvolvimento larval de R. angelae, a síntese de rRNA é intensa. Neste mesmo período observa-se claramente um nucleolo típico na base do cromossomo X. No período seguinte ocorre uma redução na síntese das formas maduras de rRNA. Esta queda de síntese é desencadeada por um bloqueio ao nível do processamento dos precursores nucleares de rRNA citoplasmático. Estudos citológicos tem revelado que neste período o nucleolo sofre uma aparente desagregação. c. Os resultados de incorporação de uridina-H3 mostraram que o 3º e 4º períodos tem uma velocidade de síntese de RNA muito à do 2º período. No 4º período quando a síntese de rRNA é inibida aumenta de importância a síntese de outras classes de RNA. Aparentemente é muito intensificada a síntese de RNA nuclear. Êstes resultados são interpretados como consequência da ativação de novos genes nesta época do desenvolvimento larval. Êstes fatos não são inesperados pois este estágio se caracteriza pelo desenvolvimento de \"puffs\" gigantes, os quais são específicos do tecido e do período de desenvolvimento. O 5º período parece representar uma fase de transição entre uma época de ativa síntese de RNA e outra onde a atividade transcritiva sofre uma redução drástica, provavelmente devido à histólise do tecido que vai ocorrer logo em seguida. d. Os dados apresentados nesta tese são considerados sob todos os aspectos significativos. Com isto faz-se uma tentativa de discussão crítica, no sentido de avaliar a importância do sistema glândulas salivares de R. angelae, como modelo de estudo para a genética molecular dos organismos superiores. / Not available

a. Do ponto de vista metodológico dois problemas foram enfrentados neste trabalho: i) extração dos RNA\'s e ii) fracionamento das células das glândulas salivares de R. angelae. i) O processo prático elaborado para resolver o primeiro problema se constituiu numa variação da técnica clássica de extração com fenol. Jogando com a presença de detergente e variações de pH e de temperatura, foi possível se chegar a um método que garante a extração e um fracionamento parcial de, praticamente, todo RNA celular. Com extrações a baixa temperatura obtém-se preferencialmente rRNA e tRNA. O RNA refratário a extração com fenol a frio tem propriedades do RNA nuclear, mas o citoplasma, também parece conter classes de RNA somente extraíveis a quente. A dificuldade de extração do RNA nuclear com fenol frio, foi especulativamente interpretada como uma propriedade das RNP\'s que encerram esta classe de RNA. A grande vantagem deste método de extração está no fato de ter permitido isolar, com as extrações a alta temperatura, frações de RNA enriquecidas em produtos de transcrição específicos de determinados períodos do desenvolvimento larval. ii) O fracionamento celular das glândulas salivares não é alcançado pela aplicação das técnicas tradicionais de fracionamento de tecido. Em vista disso foi necessário procurar uma alternativa experimental para êste caso. Combinando um enzima proteolítico com a ação de detergentes não iônicos foi conseguido um método útil para o fracionamento das células das glândulas salivares. Êste método foi aplicado nesta tese para um estudo inicial das RNP\'s e da transferência do rRNA do núcleo para o citoplasma nas células das glândulas salivares. b. Foi possível verificar com êste trabalho que o rRNA é sintetizado inicialmente na forma de um precursor primário de 37s, o qual é processado no interior do núcleo para dar as espécies maduras de rRNA segundo o esquema: (Ver no arquivo) No 3º período do 4º estádio do desenvolvimento larval de R. angelae, a síntese de rRNA é intensa. Neste mesmo período observa-se claramente um nucleolo típico na base do cromossomo X. No período seguinte ocorre uma redução na síntese das formas maduras de rRNA. Esta queda de síntese é desencadeada por um bloqueio ao nível do processamento dos precursores nucleares de rRNA citoplasmático. Estudos citológicos tem revelado que neste período o nucleolo sofre uma aparente desagregação. c. Os resultados de incorporação de uridina-H3 mostraram que o 3º e 4º períodos tem uma velocidade de síntese de RNA muito à do 2º período. No 4º período quando a síntese de rRNA é inibida aumenta de importância a síntese de outras classes de RNA. Aparentemente é muito intensificada a síntese de RNA nuclear. Êstes resultados são interpretados como consequência da ativação de novos genes nesta época do desenvolvimento larval. Êstes fatos não são inesperados pois este estágio se caracteriza pelo desenvolvimento de \"puffs\" gigantes, os quais são específicos do tecido e do período de desenvolvimento. O 5º período parece representar uma fase de transição entre uma época de ativa síntese de RNA e outra onde a atividade transcritiva sofre uma redução drástica, provavelmente devido à histólise do tecido que vai ocorrer logo em seguida. d. Os dados apresentados nesta tese são considerados sob todos os aspectos significativos. Com isto faz-se uma tentativa de discussão crítica, no sentido de avaliar a importância do sistema glândulas salivares de R. angelae, como modelo de estudo para a genética molecular dos organismos superiores. / Not available

Non-protein coding eukaryotic genome sequences often referred to as junk DNA are estimated to encode several non-coding RNAs (ncRNAs) which may account for nearly 98% of all genomic output in humans. The output of such a wide spread transcription in eukaryotes consists of intronic, antisense and small RNAs. In addition to the classical ncRNAs such as rRNA, tRNA and small nucleolar RNAs, the eukaryotic genome encodes two distinct categories of ncRNAs, referred to as small ncRNAs and long mRNA–like ncRNAs (mlncRNAs). The long ncRNAs, which are transcribed by RNA Polymerase II, spliced and polyadenylated, are implicated in a number of regulatory processes such as imprinting, X-chromosome inactivation, DNA demethylation, transcription, RNA interference, chromatin structure dynamics and antisense mediated regulation. Expression of noncoding RNAs is altered during stress conditions and a large number of such transcripts have been identified of late. This study has identified a novel ncRNA whose expression is upregulated during viral infection of mouse brain. While we have named this RNA as VINC or virus inducible ncRNA, others have named it as NEAT1 (Hutchinson et al., 2007) and Men (Sunwoo et al., 2008). VINC/NEAT1/Men is associated with a distinct nuclear domain called paraspeckles Using a cell line as well as an animal model system we have investigated VINC in great detail and based on these studies we report that VINC is a nuclear ncRNA that localizes to paraspeckles and it interacts with the paraspeckle protein, P54nrb in both cell line model system as well as in animal tissues by a combination of in vitro and in vivo methods. We have also mapped the domains within VINC that are involved in P54nrb interactions. Till date, the only other RNA known to localise to paraspeckles is CTN-RNA. While CTN-RNA is a protein coding RNA, VINC does not code for a protein and thus VINC is the first ncRNA to be localized to paraspeckles. Further, the mechanism of nuclear retention of these two paraspeckle RNAs appears to be distinct. In case of CTN-RNA, it has been clearly shown that it is A-I edited and such hyperedited RNAs are retained by the p54/nrb mediated complex in nucleus (Zhang and Carmichael, 2001). However the mechanism by which VINC is retained in nucleus is not clear. There is apparently no A-I editing in VINC and hence VINC retention in the nucleus by binding to nuclear proteins such as p54/nrb might involve a different mechanism. It is well established of late that nuclear matrix retains RNAs and that there is a population of poly (A) RNA that is retained in nucleus (Huang et al.,1994 ; Carter et al.,1991). However the significance of such retention is not clear but it is believed that it might be important for some constitutive functions in nucleus (Nickerson et al., 1989). More investigations are needed to understand the exact functions of nuclear RNAs such as VINC in supporting the nuclear architecture. P54nrb is a multi functional nuclear protein that mediates most of its functions in association with PSF (Shav-Tal and Zipori, 2002). Phosphorylation status of P54nrb is a key determinant for its localisation to various nuclear regions. P54nrb is a multiphosphorylated protein during mitosis and its phosphorylation is mediated by PIN-1 at its C-terminus (Proteau et al., 2005). Tyrosine phosphorylation of P54nrb is essential for it to be retained in nuclear matrix (Otto et al., 2001). The N-terminal phosphorylation is speculated but not much has been investigated. The protein has two distinct RNA recognition motifs (RRMs) in its N-terminus that are responsible for its RNA binding activity. The significance of the p54/nrb-PSF heterodimer cannot be undermined as they have been shown to be important during HIV replication. The dimer is recruited by viral machinery and P54nrb has been shown to be exported to cytosol for binding to replicative complexes (Zolotukhin et al., 2003). During adenoviral replication in nucleus many SR proteins are recruited to viral replication foci and rearrangement of speckle components happen. It has been shown with respect to speckles that nuclear domains are highly dynamic and exchange of proteins depends upon the transcriptional status of cell (Lamond and Spector, 2003). Flaviviral replication complexes are hosted in nucleus and ~20% of this complex docks in nucleus and serves as an alternate site for viral replication. The presence of viral replicative complexes alters the nuclear organisation and hence modulation of gene expression is expected (Uchil et al., 2006). The up regulation of nuclear ncRNA such as VINC is definitively one of those events associated with viral replication and definitively one needs to study the various changes carefully to understand the role of VINC in virus life cycle and/or viral pathogenesis. VINC interaction with the multi-functional nuclear protein P54nrb raises interesting aspects related to function of P54nrb in JEV infection. Knockdown of P54nrb in human myeloid cell line results in abnormal size of paraspeckles and impairs chondrogenesis (Hata et al., 2008). PSF-P54nrb complex can divert many of HIV gag RNA complexes to paraspeckles thus trying to restrict viral replication. However the exact relationship between paraspeckles and its constituent proteins is not clear. The presence of ncRNA adds another new dimension to paraspeckles. It is unclear whether the ncRNA VINC is essential for paraspeckle structure but a recent study indicates that Men (VINC/NEATI) RNA may be essential for paraspeckle formation (Sunwoo et al., 2008). The exact function VINC in neuronal as well as non-neuronal cell nuclei remains elusive and more investigations are need to understand these aspects.

Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.

All organisms exist in some sort of symbiosis with their environment. The food we eat, air we breathe, and things we touch all have their own microbiota and we interact with these microbiota on a daily basis. As such, we employ a method of compartmentalization in order to keep foreign entities outside of the protected internal environments of the body. However, as other organisms seek to replicate themselves, they may invade our sterile compartments in order to do so. To protect ourselves from unfettered replication of pathogens or from cellular damage, we have developed a series of receptors and signaling pathways that detect foreign bodies as well as abnormal signals from our own perturbed cells. The downstream effector molecules that these signaling pathways initiate can be toxic and damaging to both pathogen and host, so special care is given to the regulation of these systems. One method of regulation is the production of endogenous small ribonucleic acids that can regulate the expression of various receptors and adaptors in the immune signaling pathways. In this dissertation, I present work that establishes an important protein in small ribonucleic acid regulation, Dicer, as an essential protein for regulating the innate immune response to immuno-stimulatory nucleic acids as well as regulating the productive infection of encephalomyocarditis virus. Depleting Dicer from murine embryonic fibroblasts renders a disparate type I interferon response where nucleic acid stimulation in the Dicer null cells fails to produce an appreciable interferon response while infection with the paramyxovirus, Sendai, induces a more robust interferon response than the wild-type control. Additionally, I show that Dicer plays a vital role in controlling infection by the picornavirus, encephalomyocarditis virus. Encephalomyocarditis virus fails to grow efficiently in Dicer null cells due to the inability for the virus to bind to the outside of the cell, suggesting that Dicer has a role in modulating viral infection by affecting host cellular protein levels. Together, this work identifies Dicer as a key protein in viral innate immunology by regulating both the growth of virus and also the immune response generated by exposure to pathogen associated molecular patterns. Understanding this regulation will be vital for future development of small molecule therapeutics that can either modulate the innate immune response or directly affect viral growth.

Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.