A genome-scale shRNA resource for transgenic RNAi in Drosophila

Ni, Jian; Zhou, Rui; Czech, Benjamin; Liu, Lu Ping; Holderbaum, Laura; Yang‐Zhou, Donghui; Shim, Hye Seok; Tao, Rong; Handler, Dominik; Karpowicz, Phillip; Binari, Richard; Booker, Matthew A.; Brennecke, Julius; Perkins, Lizabeth A.; Hannon, Gregory J.; Perrimon, Norbert

doi:10.1038/nmeth.1592

Cited by 774 publications

(829 citation statements)

References 27 publications

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“…These RNAi lines target different regions of the tlk transcript and are designed to avoid off-target effect. 30 The quantitative RT-PCR verified that these RNAi lines indeed reduced the tlk transcripts (Supplementary Figure S1).…”

Section: Resultsmentioning

confidence: 75%

“…These RNAi lines are inserted into a defined genomic locus, thus eliminating the variation of positional effects compared with random P-element insertions. 30 We identified nine suppressor and enhancer lines ( Table 1). Most of the suppressors are components of the mediator family, which affected the transcription of tlk.…”

Section: Atg2 Functioned Downstream Of the Tlk-mediated Pcdmentioning

confidence: 99%

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Tousled-like kinase mediated a new type of cell death pathway in Drosophila

et al. 2015

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Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. We found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 was likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, our results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity.

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Section: Resultsmentioning

confidence: 75%

Section: Atg2 Functioned Downstream Of the Tlk-mediated Pcdmentioning

confidence: 99%

Tousled-like kinase mediated a new type of cell death pathway in Drosophila

et al. 2015

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“…We used Drosophila spermatogenesis (Fig. 1B) as a system to test the reproductive function of these genes by performing ubiquitous knockdown of each of the four candidate genes in adult flies using RNA interference (RNAi) (15).…”

Section: Resultsmentioning

confidence: 99%

Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans

Sun

Wen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Processing of pre-mRNA into mRNA is an important regulatory mechanism in eukaryotes that is mediated by the spliceosome, a huge and dynamic ribonucleoprotein complex. Splicing defects are implicated in a spectrum of human disease, but the underlying mechanistic links remain largely unresolved. Using a genome-wide association approach, we have recently identified single nucleotide polymorphisms in humans that associate with nonobstructive azoospermia (NOA), a common cause of male infertility. Here, using genetic manipulation of corresponding candidate loci in Drosophila, we show that the spliceosome component SNRPA1/U2A is essential for male fertility. Loss of U2A in germ cells of the Drosophila testis does not affect germline stem cells, but does result in the accumulation of mitotic spermatogonia that fail to differentiate into spermatocytes and mature sperm. Lack of U2A causes insufficient splicing of mRNAs required for the transition of germ cells from proliferation to differentiation. We show that germ cellspecific disruption of other components of the major spliceosome manifests with the same phenotype, demonstrating that mRNA processing is required for the differentiation of spermatogonia. This requirement is conserved, and expression of human SNRPA1 fully restores spermatogenesis in U2A mutant flies. We further report that several missense mutations in human SNRPA1 that inhibit the assembly of the major spliceosome dominantly disrupt spermatogonial differentiation in Drosophila. Collectively, our findings uncover a conserved and specific requirement for the major spliceosome during the transition from spermatogonial proliferation to differentiation in the male testis, suggesting that spliceosome defects affecting the differentiation of human spermatogonia contribute to NOA.

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“…40 A gene-specific 21-bp oligonucleotide (5 0 -GCACATTTGTGATGCACTACA-3 0 ) was selected and inserted into the vector.…”

Section: Methodsmentioning

confidence: 99%

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

et al. 2014

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Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neoepitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway. Cell Death and Differentiation (2014) 21, 1961-1970 doi:10.1038/cdd.2014 published online 22 August 2014 Morphogenesis in multicellular organisms is a process with a balanced control of cell proliferation and cell death. To maintain this homeostasis, superfluous or unwanted cells are usually removed promptly via programmed cell death or apoptosis. 1,2 Compelling evidence has shown that dysregulation of apoptosis results in a variety of diseases, such as cancer, neurodegenerative disorders and autoimmune diseases. [3][4][5][6] The apoptotic machinery is conserved from invertebrates to vertebrates. Drosophila has been used as an excellent model to study apoptosis because of its advantages in genetic manipulation. A crucial step in apoptosis is the cascade activation of initiator and effector caspases that eventually causes cell death. Under normal circumstances, the activities of caspases are kept in check by a conserved family of anti-apoptotic proteins termed inhibitor of apoptosis proteins (IAPs). The Drosophila genome encodes four IAPs, including Drosophila inhibitor of apoptosis protein 1 (DIAP1), DIAP2, DBruce and Deterin. 7-10 Among these four proteins, DIAP1 is stringently required to prevent caspase activation. 11,12 Although the requirement of DIAP1 in the apoptosis pathway is well documented, it is unclear how the activity of DIAP1 is regulated during development.The covalent attachment of ubiquitin to proteins is a crucial regulatory mechanism in many developmental and physiological processes. 13 Ubiquitination is a catalytic cascade involving ubiquitin-ac...

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A genome-scale shRNA resource for transgenic RNAi in Drosophila

Cited by 774 publications

References 27 publications

Tousled-like kinase mediated a new type of cell death pathway in Drosophila

Tousled-like kinase mediated a new type of cell death pathway in Drosophila

Major spliceosome defects cause male infertility and are associated with nonobstructive azoospermia in humans

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

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