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Orientador: Marcelo Alves da Silva Mori Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: RNAs não codificantes como miRNAs e siRNAs possuem importante papel de regulação gênica em diferentes tipos celulares, contribuindo para a saúde do organismo. Além de seu papel como regulador da função celular autônoma, RNAs não codificantes também são secretados e regulam a função de células alvo distantes. Entretanto, os mecanismos de transporte destes RNAs e as consequências fisiológicas desta regulação ainda não estão completamente compreendidos. Em C. elegans, a família de proteínas SID (do inglês Systemic RNAi Defective) foi relacionada à secreção e incorporação de RNAs extracelulares. Em projeto prévio, avaliei vermes mutantes para as diferentes proteínas SID e observei alteração de diversos fenótipos como postura de ovos, tempo de desenvolvimento, resistência ao estresse e envelhecimento, sobretudo para os mutantes para SID-1 e SID-3, proteínas fundamentais para a internalização de RNAs extracelulares. Na presente tese expandi esta avaliação, utilizando novos alelos mutantes para sid-1 e sid-3, assim como modelos de expressão transgênica tecido-específica de sid-1. Confirmei os efeitos deletérios da mutação para sid-3 (ok973) com o alelo sid-3 (tm342). Por outro lado, os alelos de sid-1 (qt2) e sid-1(qt9) não promoveram os defeitos observados previamente com sid-1 (pk3321), que se revelou decorrente de mutações off-target no verme utilizado. Entretanto, observei que a expressão tecido específica de SID-1 em intestino, músculo ou neurônio afeta o tempo de vida do nematoide. Essa modulação do tempo de vida depende da dieta do animal e das vias de RNAi sistêmico e de miRNAs. Por fim, a expressão neuronal de SID-1 induz o fenótipo locomotor coil nos vermes, sugerindo alteração da função neuronal. Coletivamente, os dados indicam que alterar a função da maquinaria de transporte de RNAs tem efeitos profundos e diversos na fisiologia de C. elegans. Estes efeitos sugerem que os RNAs extracelulares são usados para regular diferentes funções biológicas e que a atividade de transporte de RNAs deve ser mantida sob controle fino a fim de evitar disfunção celular Abstract: Non-coding RNAs such as miRNAs and siRNAs have an important role in gene regulation in different cell types, contributing to the health of the organism. In addition to their role as an autonomous regulator of cellular function, non-coding RNAs are also secreted and regulate the function of distant target cells. However, the transport mechanisms of these RNAs and the physiological consequences of this regulation are not yet fully understood. In C. elegans, the Systemic RNAi Defective (SID) protein family was related to the secretion and incorporation of extracellular RNAs. In a previous project, we evaluated mutant worms for the different SID proteins and observed changes in several phenotypes such as egg laying, development time, resistance to stress and aging. These were more evident in mutants for SID-1 and SID-3, fundamental proteins for internalization of extracellular RNAs. In the present thesis I have expanded this evaluation, using new mutant alleles for sid-1 and sid-3, as well as models of transgenic tissue-specific expression of sid-1. I confirmed the deleterious effects of the sid-3 (ok973) mutation with the sid-3 (tm342) allele. On the other hand, the sid-1 (qt2) and sid-1 (qt9) alleles did not show the defects previously observed with sid-1 (pk3321), which was revealed to result from off-target mutations in the worm used. However, I observed that the tissue-specific expression of SID-1 in the intestine, muscle or neuron affects the nematode's lifespan. This modulation of lifespan depends on the animal's diet and with the systemic RNAi and miRNA pathways. Finally, the neuronal expression of SID-1 induces the locomotor coil phenotype in worms, suggesting changes in neuronal function. Collectively, the data indicate that changing the function of RNA transport machinery has profound and diverse effects on the physiology of C. elegans. These effects suggest that extracellular RNAs are used to regulate different biological functions and that RNA transport activity must be kept under fine control in order to avoid cellular dysfunction Doutorado Genética Animal e Evolução Doutor em Genética e Biologia Molecular FAPESP 2017/01339-2 CAPES 0

Impt-1 expression in impt-1 +/– mutants and worms exposed to impt-1 RNAi. (a,b) Impt-1 mRNA expression in day 0 N2 (n = 3 pools of 150 worms) and impt-1 +/– (ok3233) (n = 3 pools of 150 worms) worms (a) or N2 worms treated with L4440 (control) (n = 3 pools of 150 worms) or impt-1 RNAi (n = 3 pools of 150 worms) from eggs (b). Data are presented as mean ± SEM and compared using unpaired Student t test, **P = 0.0097 in comparison to N2, *P = 0.036 in comparison to L4440. The experiments were performed once in triplicate. Raw data in Additional file 11: Table S2. (PDF 296 kb)

Lifespan increase induced by krs-1 RNAi is blocked in gcn-1 mutants. Lifespan assay of N2 worms and gcn-1(nc40) mutants treated with L4440 or krs-1 RNAi from day 0 adults. Values of median lifespan and statistics are reported in Additional file 10: Table S1. Survival curves were compared using the log-rank test. Data demonstrate the composite of experiments XXXVI and XXXVII. (PDF 146 kb)

SKN-1 and DAF-16 target gene expression upon impt-1 RNAi. (a) Average integrated density of GFP expression of the Pgst-4::GFP reporter at different larval stages [L1 (L4440, n = 19; impt-1, n = 20), L2 (L4440, n = 36; impt-1, n = 29), L4 (L4440, n = 23; impt-1, n = 31); Student t test). This is a composite of two independent experiments. (b) Representative images of Pgst-4::GFP expression in L2 larvae treated with L4440 (control) or impt-1 RNAi from L1. Dashed lines delimitate the body of the worm. Head and tail are indicated by arrows. (c) Average integrated density of GFP expression of the Psod-3::GFP reporter at different larval stages [L1 (L4440, n = 9; impt-1, n = 12), L2 (L4440, n = 18; impt-1, n = 24), L4 (L4440, n = 14; impt-1, n = 12); *P

Tissue-specific impt-1 RNAi in intestine, muscle, or neurons is not sufficient to increase lifespan. (a–c) Lifespan assays of worms with tissue-specific RNAi sensitivity in intestine (a), muscle (b), or neurons (c). The worms were treated with L4440 (control) or impt-1 RNAi from L1. Values of median lifespan and statistics are reported in Additional file 10: Table S1. Survival curves were compared using the long-rank test. All experiments were repeated three times. Data demonstrate the composite of experiments XXIII–XXV (a), XXVI–XXVIII (b), or XXIX–XXXI (c). (PDF 52 kb)

Impt-1 expression in response to DR or aging. (a) Impt-1 mRNA expression in N2 worms and worms carrying the daf-16(m26), skn-1(zu135), and hsf-1(sy441) mutations. These genes are necessary for some protocols of DR to extend lifespan in C. elegans. (b) Impt-1 mRNA expression in N2 worms and eat-2 (DR model) on days 0 and 10. n = 3 pools of at least 150 worms per group. Data are presented as mean ± SEM and compared using a one-way ANOVA with Dunnet post-hoc (a) or two-way ANOVA (b). The experiments were performed once in triplicate. *P

Impt-1 interactions with components of the DR pathway to control longevity. (a,b) Lifespan assays of aak-2(n48) and hsf-1(sy441) mutants treated with L4440 (control) or impt-1 RNAi from L1. (c–f) Lifespan assays of N2 worms and impt-1 +/– (ok3233) mutants treated with aak-2, let-363, hsf-1, or atf-5 RNAi from day 0 adults. Values of median lifespan and statistics are reported in Additional file 10: Table S1. Survival curves were compared using the log-rank test. All experiments were repeated at least twice. Data demonstrate the composite of experiments XI–XIII (a, b), XV–XVII (c), XIV–XVII (d), XIV, XV, XVII (e) and XVIII, XIX (f). (PDF 243 kb)

Phenotypic characterization of N2 worms treated with impt-1 RNAi from L1. (a) Developmental timing after egg laying of N2 and gcn-2(ok871) worms (N2 L4440 RNAi, n = 469; N2 impt-1 RNAi, n = 510; gcn-2 L4440 RNAi, n = 365; gcn-2 impt-1 RNAi, n = 411; P

Impt-1 knockdown promotes stress resistance. (a, b) Lifespan assays under heat stress (28 °C) of N2 worms and impt-1 +/– (ok3233) mutants (a) or N2 worms treated with L4440 (control) or impt-1 RNAi from L1 (b). Values of median lifespan and statistics are reported in Additional file 10: Table S1. Survival curves were compared by log-rank test. Experiment (a) was repeated three times and (b) was performed once. Data demonstrate the composite of experiments XXXII–XXXIV (a) or the experiment XXXV (b). (c) Fraction of dead N2, impt-1 +/– (ok3233) and gcn-2(ok871) worms when exposed to 5 mM or 7.5 mM sodium arsenite for 7 hours (N2, n = 44; impt-1 +/– , n = 46; gcn-2, n = 48; *P

Compilation of lifespan data. (DOCX 51 kb)