Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries

Benbahouche, Nour El Houda; Iliopoulos, Ioannis; Török, I.; Marhold, J.; Henri, Julien; Kajava, Andrey V.; Farkas, Róbert; Kempf, Tore; Schnölzer, Martina; Meyer, Philippe; Kiss, István; Bertrand, Édouard; Mechler, Bernard M.; Pradet‐Balade, Bérengère

doi:10.1074/jbc.m113.499608

Cited by 43 publications

(43 citation statements)

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“…Knockdown of Hsp83 (Hsp90 homolog), as well as of the R2TP cochaperone subunits Pontin (Rvb1 homolog) and Reptin (Rvb2 homolog), caused ‘GSC loss’ (Figure 2D, Table S1). In contrast, germline knockdown of the Drosophila Tah1 homolog Spaghetti (Spag; Figure S2; Benbahouche Nel et al, 2014), an exclusive component of the R2TP complex, led to the accumulation of spectrosome-containing undifferentiated germ cells (Figure 6F). This was also the case for the knockdown of CG16734, a dipteran-specific protein that directly binds to dTti1 (Glatter et al, 2011).…”

Section: Resultsmentioning

confidence: 99%

Regulation of Ribosome Biogenesis and Protein Synthesis Controls Germline Stem Cell Differentiation

et al. 2016

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SUMMARY Complex regulatory networks regulate stem cell behavior and contributions to tissue growth, repair, and homeostasis. A full understanding of the networks controlling stem cell self-renewal and differentiation, however, has not yet been realized. To systematically dissect these networks and identify their components, we performed an unbiased, transcriptome-wide in vivo RNAi screen in female Drosophila germline stem cells (GSCs). Based on characterized cellular defects, we classified 646 identified genes into phenotypic and functional groups, and unveiled a comprehensive set of networks regulating GSC maintenance, survival, and differentiation. This analysis revealed an unexpected role for ribosomal assembly factors in controlling stem cell cytokinesis. Moreover, our data show that the transition from self-renewal to differentiation relies on enhanced ribosome biogenesis accompanied by increased protein synthesis. Collectively, these results detail the extensive genetic networks that control stem cell homeostasis and highlights intricate regulation of protein synthesis during differentiation.

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Section: Resultsmentioning

confidence: 99%

Regulation of Ribosome Biogenesis and Protein Synthesis Controls Germline Stem Cell Differentiation

et al. 2016

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“…Firstly, the dual tetratricopeptide repeat (TPR) domains of RPAP3, a subunit of R2TP/PFDL, can bind to both Hsp70 and Hsp90 (refs 1, 2) in a manner akin to its closest paralog, STIP1/Hop. Also, and as the name implies, this complex contains a prefoldin-like module.…”

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confidence: 99%

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

et al. 2017

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The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1–TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.

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“…The cochaperone SPAG, which was identified in our screen of DBT regulators as one which strongly lengthens circadian period and is required for high DBT expression at ZT7 (23), is shown here to be required in Drosophila to maintain DBT in a mostly unphosphorylated state. SPAG contributes to assembly of HSP-90-dependent complexes such as RNA polymerase and ribonucleoprotein particles (24,35,36). It contains tetratricopeptide repeats (TPRs), as does BDBT, which is likewise essential for normal circadian rhythms, DBT phosphorylation, and DBT activity (15).…”

Section: Discussionmentioning

confidence: 99%

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis

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Bjes

et al. 2015

Molecular and Cellular Biology

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Drosophila DBT and vertebrate CKI/␦ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT C/ala ) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKI␦, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT C/ala did not affect circadian behavior differently from wild-type DBT (DBT WT ), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT WT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT C/ala did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.T he circadian clock produces daily changes in a wide range of physiological activities, as exemplified by the sleep-wake cycle, and is also essential for seasonal changes in response to changing photoperiods. It is found in single-cell organisms such as cyanobacteria as well as multicellular organisms such as humans (1). The disruption of the clock can cause many health problems, including metabolic disease, sleep disorders, or even cancers in humans (2), so it is important to understand its mechanism from both basic and clinical perspectives.Circadian clocks are the result of oscillations of several circadian clock proteins, including those of the period protein (PER) (3). In flies and humans, the casein kinase I ortholog (called the doubletime or dbt protein in flies) is essential for the oscillations of PER because it phosphorylates PER during the day and early evening to cause PER degradation (4-8). During the late night, DBT phosphorylation of PER is reduced, and PER accumulates in the nucleus as a consequence of its interaction with the timeless protein (TIM), which antagonizes phosphorylation of PER by DBT to confer rhythmic regulation of the per and tim genes (9, 10). Hence, the rhythmic phosphorylation of PER by of DBT is essential for the rhythmic accumulation of PER protein and transcriptional feedback that underlie the circadian clock (4-7).In such a mechanism, anything that confers temporal regulation to DBT activity would contribute to the oscillations. Clearly, one such regulator is the timeless protein (TIM), which is degraded in response to light (11-13) but whose accumulation at night leads to a PER/TIM heterodimer in which DBT activity ...

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Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries

Cited by 43 publications

References 41 publications

Regulation of Ribosome Biogenesis and Protein Synthesis Controls Germline Stem Cell Differentiation

Regulation of Ribosome Biogenesis and Protein Synthesis Controls Germline Stem Cell Differentiation

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis

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