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3. Morphological, cellular, and molecular basis of brain infection in COVID-19 patients

Author

Crunfli, Fernanda, Carregari, Victor C., Veras, Flavio P., Silva, Lucas S., Nogueira, Mateus Henrique, Antunes, André Saraiva Leão Marcelo, Vendramini, Pedro Henrique, Valença, Aline Gazzola Fragnani, Brandão-Teles, Caroline, da Silva Zuccoli, Giuliana, Reis-de-Oliveira, Guilherme, Silva-Costa, Lícia C., Saia-Cereda, Verônica Monteiro, Smith, Bradley J., Codo, Ana Campos, de Souza, Gabriela F., Muraro, Stéfanie P., Parise, Pierina Lorencini, Toledo-Teixeira, Daniel A., de Castro, Ícaro Maia Santos, Melo, Bruno Marcel, Almeida, Glaucia M., Firmino, Egidi Mayara Silva, Paiva, Isadora Marques, Silva, Bruna Manuella Souza, Guimarães, Rafaela Mano, Mendes, Niele D., Ludwig, Raíssa L., Ruiz, Gabriel P., Knittel, Thiago L., Davanzo, Gustavo G., Gerhardt, Jaqueline Aline, Rodrigues, Patrícia Brito, Forato, Julia, Amorim, Mariene Ribeiro, Brunetti, Natália S., Martini, Matheus Cavalheiro, Benatti, Maíra Nilson, Batah, Sabrina S., Siyuan, Li, João, Rafael B., Aventurato, Ítalo K., de Brito, Mariana Rabelo, Mendes, Maria J., da Costa, Beatriz A., Alvim, Marina K. M., da Silva Júnior, José Roberto, Damião, Lívia L., de Sousa, Iêda Maria P., da Rocha, Elessandra D., Gonçalves, Solange M., da Silva, Luiz H. Lopes, Bettini, Vanessa, Campos, Brunno M., Ludwig, Guilherme, Tavares, Lucas Alves, Pontelli, Marjorie Cornejo, Viana, Rosa Maria Mendes, Martins, Ronaldo B., Vieira, Andre Schwambach, Alves-Filho, José Carlos, Arruda, Eurico, Podolsky-Gondim, Guilherme Gozzoli, Santos, Marcelo Volpon, Neder, Luciano, Damasio, André, Rehen, Stevens, Vinolo, Marco Aurélio Ramirez, Munhoz, Carolina Demarchi, Louzada-Junior, Paulo, Oliveira, Renê Donizeti, Cunha, Fernando Q., Nakaya, Helder I., Mauad, Thais, Duarte-Neto, Amaro Nunes, da Silva, Luiz Fernando Ferraz, Dolhnikoff, Marisa, Saldiva, Paulo Hilario Nascimento, Farias, Alessandro S., Cendes, Fernando, Moraes-Vieira, Pedro Manoel M., Fabro, Alexandre T., Sebollela, Adriano, Proença-Modena, José L., Yasuda, Clarissa L., Mori, Marcelo A., Cunha, Thiago M., and Martins-de-Souza, Daniel

Published
2022

4. SARS-CoV-2 infects adipose tissue in a fat depot- and viral lineage-dependent manner

Author

Saccon, Tatiana Dandolini, Mousovich-Neto, Felippe, Ludwig, Raissa Guimarães, Carregari, Victor Corasolla, dos Anjos Souza, Ana Beatriz, dos Passos, Amanda Stephane Cruz, Martini, Matheus Cavalheiro, Barbosa, Priscilla Paschoal, de Souza, Gabriela Fabiano, Muraro, Stéfanie Primon, Forato, Julia, Amorim, Mariene Ribeiro, Marques, Rafael Elias, Veras, Flavio Protasio, Barreto, Ester, Gonçalves, Tiago Tomazini, Paiva, Isadora Marques, Fazolini, Narayana P. B., Onodera, Carolina Mie Kawagosi, Martins Junior, Ronaldo Bragança, de Araújo, Paulo Henrique Cavalcanti, Batah, Sabrina Setembre, Viana, Rosa Maria Mendes, de Melo, Danilo Machado, Fabro, Alexandre Todorovic, Arruda, Eurico, Queiroz Cunha, Fernando, Cunha, Thiago Mattar, Pretti, Marco Antônio M., Smith, Bradley Joseph, Marques-Souza, Henrique, Knittel, Thiago L., Ruiz, Gabriel Palermo, Profeta, Gerson S., Fontes-Cal, Tereza Cristina Minto, Boroni, Mariana, Vinolo, Marco Aurélio Ramirez, Farias, Alessandro S., Moraes-Vieira, Pedro Manoel M., Bizzacchi, Joyce Maria Annichino, Teesalu, Tambet, Chaim, Felipe David Mendonça, Cazzo, Everton, Chaim, Elinton Adami, Proença-Módena, José Luiz, Martins-de-Souza, Daniel, Osako, Mariana Kiomy, Leiria, Luiz Osório, and Mori, Marcelo A.

Published
2022
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5. Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise

Author

Brandão, Bruna B., Madsen, Søren, Rabiee, Atefeh, Oliverio, Matteo, Ruiz, Gabriel P., Ferrucci, Danilo L., Branquinho, Jéssica L., Razolli, Daniela, Pinto, Silas, Nielsen, Thomas S., Festuccia, William T., Martins, Adriano S., Guerra, Beatriz A., Knittel, Thiago L., Søgaard, Ditte, Larsen, Steen, Helge, Jørn W., Brandauer, Josef, Velloso, Lício A., Emanuelli, Brice, Kornfeld, Jan-Wilhelm, Kahn, Ronald, Vienberg, Sara G., Zierath, Juleen R., Treebak, Jonas T., and Mori, Marcelo A.

Published
2020

7. SARS-CoV-2 uses CD4 to infect T helper lymphocytes

Author

Brunetti, Natalia S, primary, Davanzo, Gustavo G, primary, de Moraes, Diogo, primary, Ferrari, Allan JR, primary, Souza, Gabriela F, primary, Muraro, Stéfanie Primon, primary, Knittel, Thiago L, primary, Boldrini, Vinicius O, primary, Monteiro, Lauar B, primary, Virgílio-da-Silva, João Victor, primary, Profeta, Gerson S, primary, Wassano, Natália S, primary, Nunes Santos, Luana, primary, Carregari, Victor C, primary, Dias, Artur HS, primary, Veras, Flavio P, additional, Tavares, Lucas A, additional, Forato, Julia, additional, Castro, Icaro MS, additional, Silva-Costa, Lícia C, additional, Palma, André C, additional, Mansour, Eli, additional, Ulaf, Raisa G, additional, Bernardes, Ana F, additional, Nunes, Thyago A, additional, Ribeiro, Luciana C, additional, Agrela, Marcus V, additional, Moretti, Maria Luiza, additional, Buscaratti, Lucas I, additional, Crunfli, Fernanda, additional, Ludwig, Raissa G, additional, Gerhardt, Jaqueline A, additional, Munhoz-Alves, Natália, additional, Marques, Ana Maria, additional, Sesti-Costa, Renata, additional, Amorim, Mariene R, additional, Toledo-Teixeira, Daniel A, additional, Parise, Pierina Lorencini, additional, Martini, Matheus Cavalheiro, additional, Bispos-dos-Santos, Karina, additional, Simeoni, Camila L, additional, Granja, Fabiana, additional, Silvestrini, Virgínia C, additional, de Oliveira, Eduardo B, additional, Faca, Vitor M, additional, Carvalho, Murilo, additional, Castelucci, Bianca G, additional, Pereira, Alexandre B, additional, Coimbra, Laís D, additional, Dias, Marieli MG, additional, Rodrigues, Patricia B, additional, Gomes, Arilson Bernardo SP, additional, Pereira, Fabricio B, additional, Santos, Leonilda MB, additional, Bloyet, Louis-Marie, additional, Stumpf, Spencer, additional, Pontelli, Marjorie C, additional, Whelan, Sean, additional, Sposito, Andrei C, additional, Carvalho, Robson F, additional, Vieira, André S, additional, Vinolo, Marco AR, additional, Damasio, André, additional, Velloso, Licio, additional, Figueira, Ana Carolina M, additional, da Silva, Luis LP, additional, Cunha, Thiago Mattar, additional, Nakaya, Helder I, additional, Marques-Souza, Henrique, additional, Marques, Rafael E, additional, Martins-de-Souza, Daniel, additional, Skaf, Munir S, additional, Proenca-Modena, Jose Luiz, additional, Moraes-Vieira, Pedro MM, additional, Mori, Marcelo A, additional, and Farias, Alessandro S, additional

Published
2023
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8. Author response: SARS-CoV-2 uses CD4 to infect T helper lymphocytes

Author

Brunetti, Natalia S, primary, Davanzo, Gustavo G, primary, de Moraes, Diogo, primary, Ferrari, Allan JR, primary, Souza, Gabriela F, primary, Muraro, Stéfanie Primon, primary, Knittel, Thiago L, primary, Boldrini, Vinicius O, primary, Monteiro, Lauar B, primary, Virgílio-da-Silva, João Victor, primary, Profeta, Gerson S, primary, Wassano, Natália S, primary, Nunes Santos, Luana, primary, Carregari, Victor C, primary, Dias, Artur HS, primary, Veras, Flavio P, additional, Tavares, Lucas A, additional, Forato, Julia, additional, Castro, Icaro MS, additional, Silva-Costa, Lícia C, additional, Palma, André C, additional, Mansour, Eli, additional, Ulaf, Raisa G, additional, Bernardes, Ana F, additional, Nunes, Thyago A, additional, Ribeiro, Luciana C, additional, Agrela, Marcus V, additional, Moretti, Maria Luiza, additional, Buscaratti, Lucas I, additional, Crunfli, Fernanda, additional, Ludwig, Raissa G, additional, Gerhardt, Jaqueline A, additional, Munhoz-Alves, Natália, additional, Marques, Ana Maria, additional, Sesti-Costa, Renata, additional, Amorim, Mariene R, additional, Toledo-Teixeira, Daniel A, additional, Parise, Pierina Lorencini, additional, Martini, Matheus Cavalheiro, additional, Bispos-dos-Santos, Karina, additional, Simeoni, Camila L, additional, Granja, Fabiana, additional, Silvestrini, Virgínia C, additional, de Oliveira, Eduardo B, additional, Faca, Vitor M, additional, Carvalho, Murilo, additional, Castelucci, Bianca G, additional, Pereira, Alexandre B, additional, Coimbra, Laís D, additional, Dias, Marieli MG, additional, Rodrigues, Patricia B, additional, Gomes, Arilson Bernardo SP, additional, Pereira, Fabricio B, additional, Santos, Leonilda MB, additional, Bloyet, Louis-Marie, additional, Stumpf, Spencer, additional, Pontelli, Marjorie C, additional, Whelan, Sean, additional, Sposito, Andrei C, additional, Carvalho, Robson F, additional, Vieira, André S, additional, Vinolo, Marco AR, additional, Damasio, André, additional, Velloso, Licio, additional, Figueira, Ana Carolina M, additional, da Silva, Luis LP, additional, Cunha, Thiago Mattar, additional, Nakaya, Helder I, additional, Marques-Souza, Henrique, additional, Marques, Rafael E, additional, Martins-de-Souza, Daniel, additional, Skaf, Munir S, additional, Proenca-Modena, Jose Luiz, additional, Moraes-Vieira, Pedro MM, additional, Mori, Marcelo A, additional, and Farias, Alessandro S, additional

Published
2023
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9. SARS-CoV-2 uses CD4 to infect T helper lymphocytes

Author

Brunetti, Natalia S, Davanzo, Gustavo G, de Moraes, Diogo, Ferrari, Allan JR, Souza, Gabriela F, Muraro, Stéfanie Primon, Knittel, Thiago L, Boldrini, Vinicius O, Monteiro, Lauar B, Virgílio-da-Silva, João Victor, Profeta, Gerson S, Wassano, Natália S, Nunes Santos, Luana, Carregari, Victor C, Dias, Artur HS, Veras, Flavio P, Tavares, Lucas A, Forato, Julia, Castro, Icaro MS, Silva-Costa, Lícia C, Palma, André C, Mansour, Eli, Ulaf, Raisa G, Bernardes, Ana F, Nunes, Thyago A, Ribeiro, Luciana C, Agrela, Marcus V, Moretti, Maria Luiza, Buscaratti, Lucas I, Crunfli, Fernanda, Ludwig, Raissa G, Gerhardt, Jaqueline A, Munhoz-Alves, Natália, Marques, Ana Maria, Sesti-Costa, Renata, Amorim, Mariene R, Toledo-Teixeira, Daniel A, Parise, Pierina Lorencini, Martini, Matheus Cavalheiro, Bispos-dos-Santos, Karina, Simeoni, Camila L, Granja, Fabiana, Silvestrini, Virgínia C, de Oliveira, Eduardo B, Faca, Vitor M, Carvalho, Murilo, Castelucci, Bianca G, Pereira, Alexandre B, Coimbra, Laís D, Dias, Marieli MG, Rodrigues, Patricia B, Gomes, Arilson Bernardo SP, Pereira, Fabricio B, Santos, Leonilda MB, Bloyet, Louis-Marie, Stumpf, Spencer, Pontelli, Marjorie C, Whelan, Sean, Sposito, Andrei C, Carvalho, Robson F, Vieira, André S, Vinolo, Marco AR, Damasio, André, Velloso, Licio, Figueira, Ana Carolina M, da Silva, Luis LP, Cunha, Thiago Mattar, Nakaya, Helder I, Marques-Souza, Henrique, Marques, Rafael E, Martins-de-Souza, Daniel, Skaf, Munir S, Proenca-Modena, Jose Luiz, Moraes-Vieira, Pedro MM, Mori, Marcelo A, Farias, Alessandro S, Brunetti, Natalia S, Davanzo, Gustavo G, de Moraes, Diogo, Ferrari, Allan JR, Souza, Gabriela F, Muraro, Stéfanie Primon, Knittel, Thiago L, Boldrini, Vinicius O, Monteiro, Lauar B, Virgílio-da-Silva, João Victor, Profeta, Gerson S, Wassano, Natália S, Nunes Santos, Luana, Carregari, Victor C, Dias, Artur HS, Veras, Flavio P, Tavares, Lucas A, Forato, Julia, Castro, Icaro MS, Silva-Costa, Lícia C, Palma, André C, Mansour, Eli, Ulaf, Raisa G, Bernardes, Ana F, Nunes, Thyago A, Ribeiro, Luciana C, Agrela, Marcus V, Moretti, Maria Luiza, Buscaratti, Lucas I, Crunfli, Fernanda, Ludwig, Raissa G, Gerhardt, Jaqueline A, Munhoz-Alves, Natália, Marques, Ana Maria, Sesti-Costa, Renata, Amorim, Mariene R, Toledo-Teixeira, Daniel A, Parise, Pierina Lorencini, Martini, Matheus Cavalheiro, Bispos-dos-Santos, Karina, Simeoni, Camila L, Granja, Fabiana, Silvestrini, Virgínia C, de Oliveira, Eduardo B, Faca, Vitor M, Carvalho, Murilo, Castelucci, Bianca G, Pereira, Alexandre B, Coimbra, Laís D, Dias, Marieli MG, Rodrigues, Patricia B, Gomes, Arilson Bernardo SP, Pereira, Fabricio B, Santos, Leonilda MB, Bloyet, Louis-Marie, Stumpf, Spencer, Pontelli, Marjorie C, Whelan, Sean, Sposito, Andrei C, Carvalho, Robson F, Vieira, André S, Vinolo, Marco AR, Damasio, André, Velloso, Licio, Figueira, Ana Carolina M, da Silva, Luis LP, Cunha, Thiago Mattar, Nakaya, Helder I, Marques-Souza, Henrique, Marques, Rafael E, Martins-de-Souza, Daniel, Skaf, Munir S, Proenca-Modena, Jose Luiz, Moraes-Vieira, Pedro MM, Mori, Marcelo A, and Farias, Alessandro S

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Published
2023

10. A low-abundance class of Dicer-dependent siRNAs produced from a variety of features in C. elegans

Author

Knittel, Thiago L., Montgomery, Brooke E., Tate, Alex J., Deihl, Ennis W., Nawrocki, Anastasia S., Hoerndli, Frederic J., and Montgomery, Taiowa A.

Abstract

Canonical small interfering RNAs (siRNAs) are processed from double-stranded RNA (dsRNA) by Dicer and associate with Argonautes to direct RNA silencing. In Caenorhabditis elegans, 22G-RNAs and 26G-RNAs are often referred to as siRNAs but display distinct characteristics. For example, 22G-RNAs do not originate from dsRNA and do not depend on Dicer, whereas 26G-RNAs require Dicer but derive from an atypical RNA duplex and are produced exclusively antisense to their messenger RNA (mRNA) templates. To identify canonical siRNAs in C. elegans, we first characterized the siRNAs produced via the exogenous RNA interference (RNAi) pathway. During RNAi, dsRNA is processed into ∼23 nt duplexes with ∼2 nt, 3′-overhangs, ultimately yielding siRNAs devoid of 5′G-containing sequences that bind with high affinity to the Argonaute RDE-1, but also to the microRNA (miRNA) pathway Argonaute, ALG-1. Using these characteristics, we searched for their endogenous counterparts and identified thousands of endogenous loci representing dozens of unique elements that give rise to mostly low to moderate levels of siRNAs, called 23H-RNAs. These loci include repetitive elements, putative coding genes, pseudogenes, noncoding RNAs, and unannotated features, many of which adopt hairpin (hp) structures reminiscent of the hpRNA/RNAi pathway in flies and mice. RDE-1 competes with other Argonautes for binding to 23H-RNAs. When RDE-1 is depleted, these siRNAs are enriched in ALG-1 and ALG-2 complexes. Our results expand the known repertoire of C. eleganssmall RNAs and their Argonaute interactors, and demonstrate that key features of the endogenous siRNA pathway are relatively unchanged in animals.

Published
2024
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12. SARS-CoV-2 Uses CD4 to Infect T Helper Lymphocytes

Author

Brunetti, Natália S., primary, Davanzo, Gustavo G., additional, de Moraes, Diogo, additional, Ferrari, Allan J. R., additional, de Souza, Gabriela F., additional, Muraro, Stefanie P., additional, Knittel, Thiago L., additional, Boldrini, Vinícius O., additional, Monteiro, Lauar B., additional, Virgilio-da-Silva, João Victor, additional, Profeta, Gerson S., additional, Wassano, Natália S., additional, Santos, Luana N., additional, Carregari, Victor C., additional, Dias, Artur H. S., additional, Veras, Flavio P., additional, Tavares, Lucas A., additional, Forato, Julia, additional, Castro, Ícaro, additional, Silva-Costa, Lícia C., additional, Palma, Andre, additional, Mansour, Eli, additional, Ulaf, Raisa G., additional, Bernardes, Ana F., additional, Nunes, Thyago A., additional, Ribeiro, Luciana C., additional, Agrela, Marcus V., additional, Moretti, Maria Luiza, additional, Buscaratti, Lucas I., additional, Crunfli, Fernanda, additional, Ludwig, Raissa G., additional, Gerhardt, Jaqueline A., additional, Munhoz-Alves, Natália, additional, Marques, Ana M., additional, Sesti-Costa, Renata, additional, Amorim, Mariene R., additional, Texeira, Daniel A. T., additional, Parise, Pierina L., additional, Martini, Matheus C., additional, Bispo-dos-Santos, Karina, additional, Simeoni, Camila L., additional, Granja, Fabiana, additional, Silvestrini, Virginia C., additional, de Oliveira, Eduardo B., additional, Faça, Vitor M., additional, Carvalho, Murilo, additional, Castelucci, Bianca G., additional, Pereira, Alexandre B., additional, Coimbra, Laís D., additional, Dias, Marieli M. G., additional, Rodrigues, Patricia B., additional, S. P. Gomes, Arilson Bernardo, additional, Pereira, Fabricio B., additional, Santos, Leonilda M. B., additional, Bloyet, Louis-Marie, additional, Stumpf, Spencer, additional, Pontelli, Marjorie C., additional, Whelan, Sean P. J., additional, Sposito, Andrei C., additional, Carvalho, Robson F., additional, Vieira, Andre S., additional, Vinolo, Marco A. R., additional, Damasio, André, additional, Velloso, Licio A., additional, M. Figueira, Ana Carolina, additional, da Silva, Luis L. P., additional, Cunha, Thiago M., additional, Nakaya, Helder I., additional, Marques-Souza, Henrique, additional, Marques, Rafael E., additional, Martins-de-Souza, Daniel, additional, Skaf, Munir S., additional, Proença-Modena, José Luiz, additional, Moraes-Vieira, Pedro M., additional, Mori, Marcelo A., additional, and Farias, Alessandro S., additional

Published
2020
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13. Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise

Author

Brandão, Bruna B, Madsen, Søren, Rabiee, Atefeh, Oliverio, Matteo, Ruiz, Gabriel P, Ferrucci, Danilo L, Branquinho, Jéssica L, Razolli, Daniela, Pinto, Silas, Nielsen, Thomas S, Festuccia, William T, Martins, Adriano S, Guerra, Beatriz A, Knittel, Thiago L, Søgaard, Ditte, Larsen, Steen, Helge, Jørn W, Brandauer, Josef, Velloso, Lício A, Emanuelli, Brice, Kornfeld, Jan-Wilhelm, Kahn, C Ronald, Vienberg, Sara G, Zierath, Juleen R, Treebak, Jonas T, Mori, Marcelo A, Brandão, Bruna B, Madsen, Søren, Rabiee, Atefeh, Oliverio, Matteo, Ruiz, Gabriel P, Ferrucci, Danilo L, Branquinho, Jéssica L, Razolli, Daniela, Pinto, Silas, Nielsen, Thomas S, Festuccia, William T, Martins, Adriano S, Guerra, Beatriz A, Knittel, Thiago L, Søgaard, Ditte, Larsen, Steen, Helge, Jørn W, Brandauer, Josef, Velloso, Lício A, Emanuelli, Brice, Kornfeld, Jan-Wilhelm, Kahn, C Ronald, Vienberg, Sara G, Zierath, Juleen R, Treebak, Jonas T, and Mori, Marcelo A

Abstract

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.

Published
2020

14. RNA interference may result in unexpected phenotypes in Caenorhabditis elegans

Author

De-Souza, Evandro A, primary, Camara, Henrique, additional, Salgueiro, Willian G, additional, Moro, Raíssa P, additional, Knittel, Thiago L, additional, Tonon, Guilherme, additional, Pinto, Silas, additional, Pinca, Ana Paula F, additional, Antebi, Adam, additional, Pasquinelli, Amy E, additional, Massirer, Katlin B, additional, and Mori, Marcelo A, additional

Published
2019
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15. RNA interference may result in unexpected phenotypes in Caenorhabditis elegans

Author

De-Souza, Evandro A., Camara, Henrique, Salgueiro, Willian G., Moro, Raissa P., Knittel, Thiago L., Tonon, Guilherme, Pinto, Silas, Pinca, Ana Paula F., Antebi, Adam, Pasquinelli, Amy E., Massirer, Katlin B., Mori, Marcelo A., De-Souza, Evandro A., Camara, Henrique, Salgueiro, Willian G., Moro, Raissa P., Knittel, Thiago L., Tonon, Guilherme, Pinto, Silas, Pinca, Ana Paula F., Antebi, Adam, Pasquinelli, Amy E., Massirer, Katlin B., and Mori, Marcelo A.

Abstract

RNA interference (RNAi) is a valuable technique to determine gene function. In Caenorhabditis elegans, RNAi can be achieved by feeding worms bacteria carrying a plasmid expressing double-stranded RNA (dsRNA) targeting a gene of interest. The most commonly used plasmid vector for this purpose is L4440. However, it has been noticed that sequences within L4440 may elicit unspecific effects. Here, we provide a comprehensive characterization of these effects and their mechanisms and describe new unexpected phenotypes uncovered by the administration of unspecific exogenous dsRNA. An example involves dsRNA produced by the multiple cloning site (MCS) of L4440, which shares complementary sequences with some widely used reporter vectors and induces partial transgene silencing via the canonical and antiviral RNAi pathway. Going beyond transgene silencing, we found that the reduced embryonic viability of mir-35-41(gk262) mutants is partially reversed by exogenous dsRNA via a mechanism that involves canonical RNAi. These results indicate cross-regulation between different small RNA pathways in C. elegans to regulate embryonic viability. Recognition of the possible unspecific effects elicited by RNAi vectors is important for rigorous interpretation of results from RNAi-based experiments.

Published
2019

16. Regulation of Microprocessor assembly and localization via Pasha's WW domain in C. elegans .

Author

Montgomery BE, Knittel TL, Reed KJ, Chong MC, Isolehto IJ, Cafferty ER, Smith MJ, Sprister RA, Magelky CN, Scherman H, Ketting RF, and Montgomery TA

Abstract

Primary microRNA (pri-miRNA) transcripts are processed by the Microprocessor, a protein complex that includes the ribonuclease Drosha and its RNA binding partner DGCR8/Pasha. We developed a live, whole animal, fluorescence-based sensor that reliably monitors pri-miRNA processing with high sensitivity in C. elegans . Through a forward genetic selection for alleles that desilence the sensor, we identified a mutation in the conserved G residue adjacent to the namesake W residue of Pasha's WW domain. Using genome editing we also mutated the W residue and reveal that both the G and W residue are required for dimerization of Pasha and proper assembly of the Microprocessor. Surprisingly, we find that the WW domain also facilitates nuclear localization of Pasha, which in turn promotes nuclear import or retention of Drosha. Furthermore, depletion of Pasha or Drosha causes both components of the Microprocessor to mislocalize to the cytoplasm. Thus, Pasha and Drosha mutually regulate each other's spatial expression in C. elegans ., Competing Interests: COMPETING INTERESTS The authors declare no competing interests.

Published
2024
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