DUSP3 modulates IRES-dependent translation of mRNAs through dephosphorylation of the HNRNPC protein in cells under genotoxic stimulus.

BACKGROUND INFORMATION: The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. RESULTS: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. CONCLUSIONS AND SIGNIFICANCE: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.

Hsp90 and metal-binding J-protein family chaperones are not critically involved in cellular iron-sulfur protein assembly and iron regulation in yeast.

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast-localized DnaJ-like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5-DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron-related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5-DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions.

The ribosome assembly factor Nop53 has a structural role in the formation of nuclear pre-60S intermediates, affecting late maturation events.

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S 'foot'. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

Endoscopy infection control strategy during the COVID-19 pandemic: experience from a tertiary cancer center in Brazil.

OBJECTIVES: Strategic planning for coronavirus disease (COVID-19) care has dominated the agenda of medical services, which have been further restricted by the need for minimizing viral transmission. Risk is particularly relevant in relation to endoscopy procedures. This study aimed to describe a contingency plan for a tertiary academic cancer center, define a strategy to prioritize and postpone examinations, and evaluate the infection rate among healthcare workers (HCWs) in the endoscopy unit of the Cancer Institute of the State of São Paulo (ICESP). METHODS: We created a strategy to balance the risk of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to mitigate the effects of postponing endoscopic procedures in oncological patients. A retrospective analysis of prospectively collected data on all endoscopies between March and June 2020 compared with those during the same period in 2019 was carried out. All HCWs were interviewed to obtain clinical data and SARS-CoV-2 test results. RESULTS: During the COVID-19 outbreak, there was a reduction of 55% in endoscopy cases in total. Colonoscopy was the most affected modality. The total infection rate among all HCWs was 38%. None of the senior digestive endoscopists had COVID-19. However, all bronchoscopists had been infected. One of three fellows had a serological diagnosis of COVID-19. Two-thirds of all nurses were infected, whereas half of all technicians were infected. CONCLUSIONS: In this pandemic scenario, all endoscopy services must prioritize the procedures that will be performed. It was possible to maintain some endoscopic procedures, including those meant to provide nutritional access, tissue diagnosis, and endoscopic resection. Personal protective equipment (PPE) seems effective in preventing transmission of COVID-19 from patients to digestive endoscopists. These measures can be useful in planning, even for pandemics in the future.

The ribosome assembly factor Nop53 has a structural role in the formation of nuclear pre-60S intermediates, affecting late maturation events

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S ‘foot’. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation

Endoscopy infection control strategy during the COVID-19 pandemic: experience from a tertiary cancer center in Brazil

OBJECTIVES: Strategic planning for coronavirus disease (COVID-19) care has dominated the agenda of medical services, which have been further restricted by the need for minimizing viral transmission. Risk is particularly relevant in relation to endoscopy procedures. This study aimed to describe a contingency plan for a tertiary academic cancer center, define a strategy to prioritize and postpone examinations, and evaluate the infection rate among healthcare workers (HCWs) in the endoscopy unit of the Cancer Institute of the State of São Paulo (ICESP). METHODS: We created a strategy to balance the risk of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and to mitigate the effects of postponing endoscopic procedures in oncological patients. A retrospective analysis of prospectively collected data on all endoscopies between March and June 2020 compared with those during the same period in 2019 was carried out. All HCWs were interviewed to obtain clinical data and SARS-CoV-2 test results. RESULTS: During the COVID-19 outbreak, there was a reduction of 55% in endoscopy cases in total. Colonoscopy was the most affected modality. The total infection rate among all HCWs was 38%. None of the senior digestive endoscopists had COVID-19. However, all bronchoscopists had been infected. One of three fellows had a serological diagnosis of COVID-19. Two-thirds of all nurses were infected, whereas half of all technicians were infected. CONCLUSIONS: In this pandemic scenario, all endoscopy services must prioritize the procedures that will be performed. It was possible to maintain some endoscopic procedures, including those meant to provide nutritional access, tissue diagnosis, and endoscopic resection. Personal protective equipment (PPE) seems effective in preventing transmission of COVID-19 from patients to digestive endoscopists. These measures can be useful in planning, even for pandemics in the future.

Nucleolar localization of the yeast RNA exosome subunit Rrp44 hints at early pre-rRNA processing as its main function.

The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.

The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.

Eukaryotic ribosomal biogenesis is a high-energy-demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S-processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. The yeast pre-60S assembly factor Nop53 has previously been shown to associate with the nucleoplasmic pre-60S in a region containing the "foot" structure assembled around the 3' end of the 7S pre-rRNA. Nop53 interacts with 25S rRNA and with several 60S assembly factors, including the RNA exosome, specifically, with its catalytic subunit Rrp6 and with the exosome-associated RNA helicase Mtr4. Nop53 is therefore considered the adaptor responsible for recruiting the exosome complex for 7S processing. Here, using proteomics-based approaches in budding yeast to analyze the effects of Nop53 on the exosome interactome, we found that the exosome binds pre-ribosomal complexes early during the ribosome maturation pathway. We also identified interactions through which Nop53 modulates exosome activity in the context of 60S maturation and provide evidence that in addition to recruiting the exosome, Nop53 may also be important for positioning the exosome during 7S processing. On the basis of these findings, we propose that the exosome is recruited much earlier during ribosome assembly than previously thought, suggesting the existence of additional interactions that remain to be described.

Human RNF113A participates of pre-mRNA splicing in vitro.

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Human RNF113A participates of pre-mRNA splicing in vitro

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

Ocorrência e fatores de risco associados à infecção por lentivírus de pequenos ruminantes no Estado de Sergipe / Occorrence and risk factors associated with small ruminant lentivirus infection in the State of Sergipe, Brazil

Os lentiviros de pequenos ruminantes (LVPR) são responsáveis por enfermidades infecciosas e multissistêmicas causadas pelo Vírus da Artrite Encefalite Caprina (CAEV) e o Vírus da Maedi-Visna (MVV), e se apresentam sob as formas clínicas: articular, mamária, respiratória e nervosa. Desta forma esse trabalho objetivou determinar a ocorrência e avaliar os fatores de risco associados à infecção por LVPR no Estado de Sergipe, Brasil. Foram coletadas amostras sanguíneas de 1200 ovinos e 675 caprinos oriundos respectivamente de 60 e 41 propriedades localizadas em 25 municípios sergipanos no período de 2011 a 2014. Os diagnósticos dos LVPR foram determinados pela técnica sorológica de Imunodifusão em Gel Ágar (IDGA) usando o kit comercial da marca Biovetech®. Os dados das variáveis associadas aos fatores de risco foram obtidos a partir de questionários aplicados aos proprietários dos rebanhos e analisados estatisticamente. As frequências absolutas e relativas foram determinadas por análise estatística descritiva e os fatores de risco por análise univariada das variáveis de interesse pelo Teste de Qui-quadrado de Pearson e Exato de Fisher, quando necessário, e em seguida submetidos à análise de regressão logística. Foi evidenciada uma soropositividade de 5,03% (34/675) em caprinos e 1,50% em ovinos com 26,82% (11/41) e 28,33% (17/60) das propriedades apresentando ao menos um animal positivo respectivamente. Na análise dos fatores de risco, não foram observadas diferenças significantes para os ovinos, enquanto que, para os caprinos, rebanhos acima de 100 animais, que pastejam em áreas comuns com outros rebanhos, em uma distância ≤500 metros entre as propriedades, que adotam medidas biotecnológicas da reprodução e não utilizam agulhas estéreis, são mais susceptíveis à infecção por LVPR. Sendo assim, conclui-se que, há a presença dos LVPR em rebanhos sergipanos, e mesmo que em baixas frequências faz-se necessário a implementação de medidas profiláticas devido a possibilidade de expansão e desenvolvimento da caprinocultura do estado, e o alto padrão genético da raça Santa Inês.(AU)

The lentiviruses of small ruminants are infectious and multisystemic diseases caused by the Caprine Arthritis Encephalitis Virus (CAEV) and the Maedi-Visna Virus (MVV), and present the clinical forms: articular, mammary, respiratory and nervous. This work aimed to determine the occurrence and to evaluate the risk factors associated with lentivirus infection of small ruminants in the State of Sergipe, Brazil. Blood samples were collected from 1200 sheep and 675 goats from 60 and 41 farms respectively, located in 25 Sergipe municipalities from 2011 to 2014. The diagnosis of small ruminant lentiviruses (LVPR) was determined by the serological technique of Immunodiffusion in Gel Agar (IDGA) using the commercial kit of the brand Biovetech®. Data from the variables associated with risk factors were obtained from questionnaires applied to the owners of the herds and analyzed statistically. Absolute and relative frequencies were determined by descriptive statistical analysis and risk factors by univariate analysis of the variables of interest by Pearson's Chi-square test and Fisher's exact test, when necessary. A logistic regression analysis was used, considering as a dependent variable for LVPR infection the reactive or non-reactive result observed in the IDGA. A seropositivity of 5.03% (34/675) was observed in goats and 1.50% in sheep with 26.82% (11/41) and 28.33% (17/60) of the properties had at least one animal positive respectively. The analysis of the risk factors, no significant differences were observed for sheep, while for goats, herds above 100 animals grazing in common areas with other herds, at a distance ≤ 500 meters between the properties, that adopt Biotechnological measures of reproduction and do not use sterile needles, are more susceptible to LVPR infection. Therefore, it´s concluded there is presence of lentiviruses of small ruminants in sergipan herds, and even if at low frequencies it is necessary to implement prophylactic measures due to the possibility of expansion and development of goat breeding of the state and the high genetic standard of the Santa Inês breed.(AU)

Ocorrência e fatores de risco associados à infecção por lentivírus de pequenos ruminantes no Estado de Sergipe / Occorrence and risk factors associated with small ruminant lentivirus infection in the State of Sergipe, Brazil

Os lentiviros de pequenos ruminantes (LVPR) são responsáveis por enfermidades infecciosas e multissistêmicas causadas pelo Vírus da Artrite Encefalite Caprina (CAEV) e o Vírus da Maedi-Visna (MVV), e se apresentam sob as formas clínicas: articular, mamária, respiratória e nervosa. Desta forma esse trabalho objetivou determinar a ocorrência e avaliar os fatores de risco associados à infecção por LVPR no Estado de Sergipe, Brasil. Foram coletadas amostras sanguíneas de 1200 ovinos e 675 caprinos oriundos respectivamente de 60 e 41 propriedades localizadas em 25 municípios sergipanos no período de 2011 a 2014. Os diagnósticos dos LVPR foram determinados pela técnica sorológica de Imunodifusão em Gel Ágar (IDGA) usando o kit comercial da marca Biovetech®. Os dados das variáveis associadas aos fatores de risco foram obtidos a partir de questionários aplicados aos proprietários dos rebanhos e analisados estatisticamente. As frequências absolutas e relativas foram determinadas por análise estatística descritiva e os fatores de risco por análise univariada das variáveis de interesse pelo Teste de Qui-quadrado de Pearson e Exato de Fisher, quando necessário, e em seguida submetidos à análise de regressão logística. Foi evidenciada uma soropositividade de 5,03% (34/675) em caprinos e 1,50% em ovinos com 26,82% (11/41) e 28,33% (17/60) das propriedades apresentando ao menos um animal positivo respectivamente. Na análise dos fatores de risco, não foram observadas diferenças significantes para os ovinos, enquanto que, para os caprinos, rebanhos acima de 100 animais, que pastejam em áreas comuns com outros rebanhos, em uma distância ≤500 metros entre as propriedades, que adotam medidas biotecnológicas da reprodução e não utilizam agulhas estéreis, são mais susceptíveis à infecção por LVPR. Sendo assim, conclui-se que, há a presença dos LVPR em rebanhos sergipanos, e mesmo que em baixas frequências faz-se necessário a implementação de medidas profiláticas devido a possibilidade de expansão e desenvolvimento da caprinocultura do estado, e o alto padrão genético da raça Santa Inês.(AU)

The lentiviruses of small ruminants are infectious and multisystemic diseases caused by the Caprine Arthritis Encephalitis Virus (CAEV) and the Maedi-Visna Virus (MVV), and present the clinical forms: articular, mammary, respiratory and nervous. This work aimed to determine the occurrence and to evaluate the risk factors associated with lentivirus infection of small ruminants in the State of Sergipe, Brazil. Blood samples were collected from 1200 sheep and 675 goats from 60 and 41 farms respectively, located in 25 Sergipe municipalities from 2011 to 2014. The diagnosis of small ruminant lentiviruses (LVPR) was determined by the serological technique of Immunodiffusion in Gel Agar (IDGA) using the commercial kit of the brand Biovetech®. Data from the variables associated with risk factors were obtained from questionnaires applied to the owners of the herds and analyzed statistically. Absolute and relative frequencies were determined by descriptive statistical analysis and risk factors by univariate analysis of the variables of interest by Pearson's Chi-square test and Fisher's exact test, when necessary. A logistic regression analysis was used, considering as a dependent variable for LVPR infection the reactive or non-reactive result observed in the IDGA. A seropositivity of 5.03% (34/675) was observed in goats and 1.50% in sheep with 26.82% (11/41) and 28.33% (17/60) of the properties had at least one animal positive respectively. The analysis of the risk factors, no significant differences were observed for sheep, while for goats, herds above 100 animals grazing in common areas with other herds, at a distance ≤ 500 meters between the properties, that adopt Biotechnological measures of reproduction and do not use sterile needles, are more susceptible to LVPR infection. Therefore, it´s concluded there is presence of lentiviruses of small ruminants in sergipan herds, and even if at low frequencies it is necessary to implement prophylactic measures due to the possibility of expansion and development of goat breeding of the state and the high genetic standard of the Santa Inês breed.(AU)

Ocorrência e fatores de risco associados à infecção por lentivírus de pequenos ruminantes no Estado de Sergipe

ABSTRACT: The lentiviruses of small ruminants are infectious and multisystemic diseases caused by the Caprine Arthritis Encephalitis Virus (CAEV) and the Maedi-Visna Virus (MVV), and present the clinical forms: articular, mammary, respiratory and nervous. This work aimed to determine the occurrence and to evaluate the risk factors associated with lentivirus infection of small ruminants in the State of Sergipe, Brazil. Blood samples were collected from 1200 sheep and 675 goats from 60 and 41 farms respectively, located in 25 Sergipe municipalities from 2011 to 2014. The diagnosis of small ruminant lentiviruses (LVPR) was determined by the serological technique of Immunodiffusion in Gel Agar (IDGA) using the commercial kit of the brand Biovetech®. Data from the variables associated with risk factors were obtained from questionnaires applied to the owners of the herds and analyzed statistically. Absolute and relative frequencies were determined by descriptive statistical analysis and risk factors by univariate analysis of the variables of interest by Pearsons Chi-square test and Fishers exact test, when necessary. A logistic regression analysis was used, considering as a dependent variable for LVPR infection the reactive or non-reactive result observed in the IDGA. A seropositivity of 5.03% (34/675) was observed in goats and 1.50% in sheep with 26.82% (11/41) and 28.33% (17/60) of the properties had at least one animal positive respectively. The analysis of the risk factors, no significant differences were observed for sheep, while for goats, herds above 100 animals grazing in common areas with other herds, at a distance 500 meters between the properties, that adopt Biotechnological measures of reproduction and do not use sterile needles, are more susceptible to LVPR infection. Therefore, it´s concluded there is presence of lentiviruses of small ruminants in sergipan herds, and even if at low frequencies it is necessary to implement prophylactic measures due to the possibility of expansion and development of goat breeding of the state and the high genetic standard of the Santa Inês breed.

RESUMO: Os lentiviros de pequenos ruminantes (LVPR) são responsáveis por enfermidades infecciosas e multissistêmicas causadas pelo Vírus da Artrite Encefalite Caprina (CAEV) e o Vírus da Maedi-Visna (MVV), e se apresentam sob as formas clínicas: articular, mamária, respiratória e nervosa. Desta forma esse trabalho objetivou determinar a ocorrência e avaliar os fatores de risco associados à infecção por LVPR no Estado de Sergipe, Brasil. Foram coletadas amostras sanguíneas de 1200 ovinos e 675 caprinos oriundos respectivamente de 60 e 41 propriedades localizadas em 25 municípios sergipanos no período de 2011 a 2014. Os diagnósticos dos LVPR foram determinados pela técnica sorológica de Imunodifusão em Gel Ágar (IDGA) usando o kit comercial da marca Biovetech®. Os dados das variáveis associadas aos fatores de risco foram obtidos a partir de questionários aplicados aos proprietários dos rebanhos e analisados estatisticamente. As frequências absolutas e relativas foram determinadas por análise estatística descritiva e os fatores de risco por análise univariada das variáveis de interesse pelo Teste de Qui-quadrado de Pearson e Exato de Fisher, quando necessário, e em seguida submetidos à análise de regressão logística. Foi evidenciada uma soropositividade de 5,03% (34/675) em caprinos e 1,50% em ovinos com 26,82% (11/41) e 28,33% (17/60) das propriedades apresentando ao menos um animal positivo respectivamente. Na análise dos fatores de risco, não foram observadas diferenças significantes para os ovinos, enquanto que, para os caprinos, rebanhos acima de 100 animais, que pastejam em áreas comuns com outros rebanhos, em uma distância 500 metros entre as propriedades, que adotam medidas biotecnológicas da reprodução e não utilizam agulhas estéreis, são mais susceptíveis à infecção por LVPR. Sendo assim, conclui-se que, há a presença dos LVPR em rebanhos sergipanos, e mesmo que em baixas frequências faz-se necessário a implementação de medidas profiláticas devido a possibilidade de expansão e desenvolvimento da caprinocultura do estado, e o alto padrão genético da raça Santa Inês.

Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex.

BACKGROUND: Box C/D snoRNPs are responsible for rRNA methylation and processing, and are formed by snoRNAs and four conserved proteins, Nop1, Nop56, Nop58 and Snu13. The snoRNP assembly is a stepwise process, involving other protein complexes, among which the R(2)TP and Hsp90 chaperone. Nop17, also known as Pih1, has been shown to be a constituent of the R(2)TP (Rvb1, Rvb2, Tah1, Pih1) and to participate in box C/D snoRNP assembly by its interaction with Nop58. The molecular function of Nop17, however, has not yet been described. RESULTS: To shed light on the role played by Nop17 in the maturation of snoRNP, here we analyzed the interactions domains of Nop58 - Nop17 - Tah1 and the importance of ATP to the interaction between Nop17 and the ATPase Rvb1/2. CONCLUSIONS: Based on the results shown here, we propose a model for the assembly of box C/D snoRNP, according to which R(2)TP complex is important for reducing the affinity of Nop58 for snoRNA, and for the binding of the other snoRNP subunits.

Proteomic analysis of yeast mutant RNA exosome complexes.

The yeast exosome is a conserved multiprotein complex essential for RNA processing and degradation. The complex is formed by a nine-subunit core that associates with two hydrolytic 3'-5' exoribonucleases. Although catalytically inert, the assembly of this nine-subunit core seems to be essential for the exosome activity, as mutations in regions that do not directly bind RNA or are not in the active sites of the exonucleases impair the function of the complex. Previously isolated mutations in the exosome core subunit Rrp43p have been shown to negatively affect the function of the complex. With the aim of investigating the effect of these mutations on the complex stability and activity, Rrp43p and its mutant forms were purified by means of the TAP method. Mass spectrometry analyses showed that lower amounts of the exosome subunits are copurified with the mutant Rrp43p proteins. Additionally, by decreasing the stability of the exosome, other nonspecific protein interactions are favored (the data have been deposited to the ProteomeXchange with identifier PXD000580). Exosome copurified with mutant Rrp43p exhibited increased exonuclease activity, suggesting higher dissociation constants for these mutant complexes. Therefore, data reported here indicate that complexes containing a mutant Rrp43p exhibit decreased stability and provide information on additional protein interactions.

Cwc24p is a general Saccharomyces cerevisiae splicing factor required for the stable U2 snRNP binding to primary transcripts.

Splicing of primary transcripts is an essential process for the control of gene expression. Specific conserved sequences in premature transcripts are important to recruit the spliceosome machinery. The Saccharomyces cerevisiae catalytic spliceosome is composed of about 60 proteins and 5 snRNAs (U1, U2, U4/U6 and U5). Among these proteins, there are core components and regulatory factors, which might stabilize or facilitate splicing of specific substrates. Assembly of a catalytic complex depends on the dynamics of interactions between these proteins and RNAs. Cwc24p is an essential S. cerevisiae protein, originally identified as a component of the NTC complex, and later shown to affect splicing in vivo. In this work, we show that Cwc24p also affects splicing in vitro. We show that Cwc24p is important for the U2 snRNP binding to primary transcripts, co-migrates with spliceosomes, and that it interacts with Brr2p. Additionally, we show that Cwc24p is important for the stable binding of Prp19p to the spliceosome. We propose a model in which Cwc24p is required for stabilizing the U2 association with primary transcripts, and therefore, especially important for splicing of RNAs containing non-consensus branchpoint sequences.

The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing.

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.

The essential nucleolar yeast protein Nop8p controls the exosome function during 60S ribosomal subunit maturation.

The yeast nucleolar protein Nop8p has previously been shown to interact with Nip7p and to be required for 60S ribosomal subunit formation. Although depletion of Nop8p in yeast cells leads to premature degradation of rRNAs, the biochemical mechanism responsible for this phenotype is still not known. In this work, we show that the Nop8p amino-terminal region mediates interaction with the 5.8S rRNA, while its carboxyl-terminal portion interacts with Nip7p and can partially complement the growth defect of the conditional mutant strain Δnop8/GAL::NOP8. Interestingly, Nop8p mediates association of Nip7p to pre-ribosomal particles. Nop8p also interacts with the exosome subunit Rrp6p and inhibits the complex activity in vitro, suggesting that the decrease in 60S ribosomal subunit levels detected upon depletion of Nop8p may result from degradation of pre-rRNAs by the exosome. These results strongly indicate that Nop8p may control the exosome function during pre-rRNA processing.

Utp25p, a nucleolar Saccharomyces cerevisiae protein, interacts with U3 snoRNP subunits and affects processing of the 35S pre-rRNA.

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. Two intermediate complexes, pre-40S and pre-60S, are formed at the early stages of 35S pre-rRNA processing and give rise to the mature ribosome subunits. Each of these complexes contains specific pre-rRNAs, some ribosomal proteins and processing factors. The novel yeast protein Utp25p has previously been identified in the nucleolus, an indication that this protein could be involved in ribosome biogenesis. Here we show that Utp25p interacts with the SSU processome proteins Sas10p and Mpp10p, and affects 18S rRNA maturation. Depletion of Utp25p leads to accumulation of the pre-rRNA 35S and the aberrant rRNA 23S, and to a severe reduction in 40S ribosomal subunit levels. Our results indicate that Utp25p is a novel SSU processome subunit involved in pre-40S maturation.

Identification of archaeal proteins that affect the exosome function in vitro.

BACKGROUND: The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. RESULTS: Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. CONCLUSIONS: Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.