A carregar...
Publicação
CG9925 and CG9684 are homologues of the TDRD1 piRNA protein family and occupy different subcellular compartments in the Drosophila germline
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 3.58 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
A via dos piRNA silencia a expressão de elementos repetitivos nas gónadas de invertebrados e vertebrados. Os piRNAs são pequenos RNA de interferência que se ligam a membros da subfamília PIWI pertencente à família de Argonautas conhecida pelo seu papel na supressão da expressão de genes através de RNAi. Proteínas da família PIWI localizam-se tanto no núcleo como no citoplasma, mas a maior parte encontra-se na região perinuclear chamada de nuage, onde estas proteínas formam complexos ribonucleoproteicos que participam na formação de piRNAs e na degradação de elementos repetitivos. Para além destas, outras classes de proteínas são também necessárias para uma via de piRNA funcional. Entre elas encontram-se proteínas que contêm domínios TUDOR (TDRD). TDRDs são proteínas importantes que agem como um andaime molecular e que interagem com proteínas da família PIWI, promovendo a sua estabilidade e facilitando a sua ação contra elementos repetitivos.
A proteína TDRD1 é um componente da região da nuage da linha germinal e é necessária para a espermatogénese em vertebrados. A análise dos domínios que esta proteína apresenta sugere que o genoma de Drosophila Melanogaster codifica para três genes estruturalmente relacionados (Vreteno, CG9925 e CG9684). O Vreteno foi recentemente relacionado com a regulação de retrotransposões dependente de piRNAs, mas no entanto os restantes dois continuam sem ser caracterizados.
Através de microscopia confocal foi possível demonstrar que CG9925 e CG9684 ocupam localizações complementares mas não se localizam no mesmo local. Nos ovários, enquanto que a CG9925 está localizada na nuage das células “nurse”, a CG9684 situa-se em redor do oócito. Nos testículos ambos situam-se na nuage e encontram-se principalmente numa estrutura conhecida como piNG body, mas a sua localização não se sobrepõe. Ensaios de PCR quantitativa, usando novos mutantes contendo deleções para os genes de interesse, mostram que pelo menos a CG9925 é necessária para a fertilidade da mosca e para a repressão da expressão de Stellate nos testículos, sugerindo que tem um papel funcional na via dos piRNAs. Por último, a descoberta de que cada TDRD1 da Drosophila localiza-se numa zona celular diferente na linha germinal sugere que estas proteínas tenham um papel não redundante na via de piRNAs.
The piRNA-pathway silences repetitive element expression in the gonad of invertebrates and vertebrates. piRNAs are small, 26-31 nt-long interfering RNAs that bind members of the PIWI subfamily of the larger Argonaute family of small RNAi-related pathway effector proteins. PIWI-like proteins can be nuclear or cytoplasmic, but are most typically found at the perinuclear nuage, where they assemble ribonucleoprotein complexes that participate in piRNA formation and complementary target transcript degradation. Apart from the PIWI-like effectors, other proteins are necessary for a functional piRNA-pathway. Amongst these, many TUDOR domain-containing (TDRD) proteins have been shown to be required for a functional piRNA-pathway. TDRDs are considered to be important scaffold proteins that interact with PIWI proteins and promote their stability, thereby potentiating an efficient response against repetitive elements. The Tudor domain-containing 1 (TDRD1)/cancer-testis antigen is a perinuclear nuage component of the germline that is required for spermatogenesis in vertebrates. Domain architecture analyses suggest that the Drosophila genome encodes three structurally related TDRD1-like genes (Vreteno, CG9925 and CG9684). Vreteno has been recently shown to be required for piRNA-based transposon regulation, whereas the two other dTDRD1-like genes remain uncharacterized. Confocal imaging using new specific antibodies and tagged transgenes show that CG9925 and CG9684 occupy complementary but non-overlapping subcellular locations in the Drosophila germline. In the ovary, while CG9925 is enriched in the perinuclear nuage of developing nurse cells, CG9684 localizes preferentially to the developing oocyte. In testes, both decorate the nuage and are enriched around a piNG-like structure in developing spermatocytes, always non-overlapping. Quantitative PCR assays using new deletion mutations showed that at least CG9925 is required for fertility and is necessary for the repression of Stellate repeats in the testis, suggesting that it is a functional TDRD1 homologue working in the Drosophila piRNA pathway. Finally, the finding that the Drosophila TDRD1-like homologues localize to distinct sub-cellular compartments in the germline supports a scenario where each member plays a non-redundant role in the piRNA pathway.
The piRNA-pathway silences repetitive element expression in the gonad of invertebrates and vertebrates. piRNAs are small, 26-31 nt-long interfering RNAs that bind members of the PIWI subfamily of the larger Argonaute family of small RNAi-related pathway effector proteins. PIWI-like proteins can be nuclear or cytoplasmic, but are most typically found at the perinuclear nuage, where they assemble ribonucleoprotein complexes that participate in piRNA formation and complementary target transcript degradation. Apart from the PIWI-like effectors, other proteins are necessary for a functional piRNA-pathway. Amongst these, many TUDOR domain-containing (TDRD) proteins have been shown to be required for a functional piRNA-pathway. TDRDs are considered to be important scaffold proteins that interact with PIWI proteins and promote their stability, thereby potentiating an efficient response against repetitive elements. The Tudor domain-containing 1 (TDRD1)/cancer-testis antigen is a perinuclear nuage component of the germline that is required for spermatogenesis in vertebrates. Domain architecture analyses suggest that the Drosophila genome encodes three structurally related TDRD1-like genes (Vreteno, CG9925 and CG9684). Vreteno has been recently shown to be required for piRNA-based transposon regulation, whereas the two other dTDRD1-like genes remain uncharacterized. Confocal imaging using new specific antibodies and tagged transgenes show that CG9925 and CG9684 occupy complementary but non-overlapping subcellular locations in the Drosophila germline. In the ovary, while CG9925 is enriched in the perinuclear nuage of developing nurse cells, CG9684 localizes preferentially to the developing oocyte. In testes, both decorate the nuage and are enriched around a piNG-like structure in developing spermatocytes, always non-overlapping. Quantitative PCR assays using new deletion mutations showed that at least CG9925 is required for fertility and is necessary for the repression of Stellate repeats in the testis, suggesting that it is a functional TDRD1 homologue working in the Drosophila piRNA pathway. Finally, the finding that the Drosophila TDRD1-like homologues localize to distinct sub-cellular compartments in the germline supports a scenario where each member plays a non-redundant role in the piRNA pathway.
Descrição
Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2013
Palavras-chave
Bioquímica médica Teses de mestrado - 2013