58 research outputs found
The mRNA export factor Npl3 mediates the nuclear export of large ribosomal subunits
No full textThe nuclear export of large ribonucleoparticles is complex and requires specific transport factors. Messenger RNAs are exported through the RNA-binding protein Npl3 and the interacting export receptor Mex67. Export of large ribosomal subunits also requires Mex67; however, in this case, Mex67 binds directly to the 5S ribosomal RNA (rRNA) and does not require the Npl3 adaptor. Here, we have discovered a new function of Npl3 in mediating the export of pre-60S ribosomal subunit independently of Mex67. Npl3 interacts with the 25S rRNA, ribosomal and ribosome-associated proteins, as well as with the nuclear pore complex. Mutations in NPL3 lead to export defects of the large subunit and genetic interactions with other pre-60S export factors.Deutsche Forschungsgemeinschaft; [SFB593]; [SFB860
Quality control of spliced mRNAs requires the shuttling SR proteins Gbp2 and Hrb1
No full textHackmann A, Wu H, Schneider U-M, Meyer K, Jung K, Krebber H. Quality control of spliced mRNAs requires the shuttling SR proteins Gbp2 and Hrb1. Nature Communications. 2014;5(1): 3123.Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature mRNAs associate with the export receptor Mex67/TAP and enter the cytoplasm. Here we show that the two shuttling serine/arginine (SR)-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP complex to spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR proteins in mRNA surveillance and nuclear mRNA quality control
Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.
No full textThe DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms
mRNA quality control is bypassed for immediate export of stress-responsive transcripts
No full textCells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated1. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67-Mtr2 (TAP-p15 in humans)(2). Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.Deutsche Forschungsgemeinschaft; [SFB860
Monosome Formation during Translation Initiation Requires the Serine/Arginine-Rich Protein Npl3
No full textThe yeast shuttling serine/arginine-rich protein Npl3 is required for the export of mRNAs and pre-60S ribosomal subunits from the nucleus to the cytoplasm. Here, we report a novel function of Npl3 in translation initiation. A mutation in its C terminus that prevents its dimerization (npl3 Delta 100) is lethal to cells and leads to translational defects, as shown by [S-35] methionine incorporation assays and a hypersensitivity to the translational inhibitor cycloheximide. Moreover, this Npl3 mutant shows halfmers in polysomal profiles that are indicative of defects in monosome formation. Strikingly, the loss of the ability of Npl3 to dimerize does not affect mRNA and pre-60S export. In fact, the mRNA and rRNA binding capacities of npl3 Delta 100 and wild-type Npl3 are similar. Intriguingly, overexpression of the dimerization domain of Npl3 disturbs dimer formation and results in a dominant-negative effect, reflected in growth defects and a halfmer formation phenotype. In addition, we found specific genetic interactions with the ribosomal subunit joining factors Rpl10 and eukaryotic translation initiation factor 5B/Fun12 and detected a substantially decreased binding of npl3 Delta 100 to the Rpl10-containing complex. These findings indicate an essential novel function for Npl3 in the cytoplasm, which supports monosome formation for translation initiation.Deutsche Forschungsgemeinschaft [SFB860
Comparative analysis of the nuclear/cytoplasmic export requirements of SR-like proteins and their regulatory function in mRNA splicing process
No full textEukaryontische Zellen zeichnen sich durch ihre Kompartimentierung in Zytoplasma und Zellkern aus, die eine regulierte Genexpression ermöglicht. Im Zellkern erfolgt die Transkription der Gene in prä-mRNAs, die nach extensiver Prozessierung zur Export kompetenten mRNA im Ribonukleoprotein-Komplex („messenger ribonucleoprotein particle“ = mRNP) heranreifen. Die SR-ähnlichen mRNA Bindeproteine Npl3p, Gbp2p und Hrb1p assoziieren während der Transkription mit der mRNA im Zellkern und Npl3p interagiert mit dem Export Rezeptor Mex67p-Mtr2p, der die Export-kompetente mRNA durch die Kernporen-Komplexe der Kernmembran in das Zytoplasma transportiert. Dort erfolgt an den Ribosomen die Proteintranslation anhand der mRNA kodierten genetischen Information. Npl3p ist in vielen Saccharomyces cerevisiae Stämmen ein essentielles Protein und Mutationen führen zu mRNA Export-Defekten. In einem weltweiten Deletionsprojekt wurde eine überlebensfähige NPL3-Deletion im Stammhintergund BY4741 hergestellt. Interessanterweise sind in npl3∆ keine signifikanten mRNA Export Defekte vorhanden, was auf eine weitere, bisher unbekannte Funktion von NPL3 hindeutet. In Lokalisationsstudien wurden jedoch nukleäre Export-Defekte der ribosomalen prä-60S, nicht aber der 40S Untereinheit in npl3∆-Zellen nachgewiesen. Npl3p interagiert physikalisch sowohl über das ribosomale Protein Rpl25p als auch über die 25S rRNA mit der prä-60S Untereinheit. Eine Funktion von Npl3p in der Prozessierung des ribosomalen Vorläufers ist aufgrund von nicht detektierten rRNA Prozessierungsdefekten und nukleolaren Mislokalisationen in npl3∆ unwahrscheinlich. Npl3p interagiert mit den Faktoren der Export kompetenten prä-60S Untereinheit Nmd3p und dem ebenfalls im prä-60S Export agierenden Export-Rezeptor Mex67p. Eine Deletion von NPL3 beeinflußt jedoch nicht die Rekrutierung dieser prä-60S Exportfaktoren zur ribosomalen Untereinheit. Da Npl3p die prä-60S Untereinheit physikalisch bindet und seine Deletion nukleäre Export-Defekte hervorruft, wirkt Npl3p als unabhängiger Exportadapter im prä-60S Transportprozess. Npl3p hat das Potential den Kontakt zwischen der prä-60S Untereinheit und den Kernporen-Komplexen über eine physikalische Interaktion mit dem Nup60p assoziierten Mlp1p zu vermitteln.
In einem zweiten Kapitel dieser Arbeit erfolgte die vergleichende Analyse der Exporteigenschaften von Gbp2p, Hrb1p und Npl3, welche erstmalig eine Verbindung zum Spleißing-Prozess aufzeigt. In einem „Screen“ wurden die Spleißingfaktor-Mutanten prp8-908/988 und prp17-Q336* identifiziert, die starke nukleäre Export-Defekte von Gbp2p und Hrb1, nicht aber von Npl3p hervorrufen. GBP2 und HRB1 Deletionen interagieren genetisch mit diesen Spleißingfaktor-Mutanten der Spleißing Spätphase, während eine NPL3 Deletion weder genetisch noch physikalisch mit diesen Faktoren interagiert. Gbp2p und Hrb1p zeigen Protein-Protein Wechselwirkungen mit Prp17p und mit Prp43p des postspleißosomalen Komplexes. Die nukleären Mislokalisationen von Gbp2p und Hrb1p in Spleißing-faktor Mutanten der Spleißing Spätphase beruhen auf eine starke Störung der mRNA Bindung, die in RNA-Co IP Studien ermittelt wurden. Für Gbp2p und Hrb1p wurde in RIP-Chip und qRT-PCR-Studien eine präferentielle Bindung für mRNAs identifiziert, die Intronsequenzen enthalten. Marginale Spleißing Defekte, aber signifikante Proteinexpressionsabnahmen Intron-haltiger Gene wurden vor allem in der Doppel-Deletion gbp2∆ hrb1∆ nachgewiesen. Gbp2p und Hrb1p interagieren mit dem Export-Rezeptor Mex67p und sind somit als neue mRNA Export Adapter identifiziert worden. In Spleißingfaktor-Mutanten sind diese Adapter-Rezeptor Bindungen gestört, wodurch der Export von ungespleißten mRNAs vermutlich zurückgehalten wird. Gbp2p und Hrb1p zeigen eine Verbindung zum Kernporen-Komplex assoziierten Mlp1p. Dieser Faktor hat eine Funktion in der finalen Qualitätskontrolle von gespleißten mRNAs bevor diese den Zellkern verlassen können. Daraus ergibt sich folgendes Modell: Gbp2p und Hrb1p werden Spleißing-abhängig zur mRNA rekrutiert und unterstützen den Export von gespleißten mRNAs über eine Interaktion mit dem Export-Rezeptor Mex67p. Im Gegensatz dazu agiert Npl3p als universeller Export-Adapter, der unabhängig vom Intron-Status der mRNA Mex67p bindet. Bekannterweise wird Npl3p über die RNA Polymerase II frühzeitig auf alle mRNAs geladen, während Gbp2p und Hrb1p in der Elongations- phase zur mRNA gelangen. Eine stabile Assoziation von Gbp2p und Hrb1p mit der mRNA erfolgt erst durch die Assemblierung des Spleißosom, mit dessen Komponenten der Spleißing-Spätphase sie physikalisch interagieren. Diese Wechselwirkungen unterstützen Spleißing und ermöglichen über die sich anschließende Adapter-Rezeptor Interaktion einen effizienten und kontrollierten Export von gespleißten mRNAs.In eukaryotic cells the separation into a nuclear and cytoplasmic compartment leads to an advantage for regulation of an efficient gene expression. Gene expression starts in the nucleus with the transcription of DNA to premature mRNAs. These transcripts are extensively processed and mature to the export competent mRNA within the messenger ribonucleoprotein particle (mRNP). Three mRNA binding SR-like proteins Npl3p, Gbp2p, and Hrb1p are loaded co-transcriptionally onto the mRNA and Npl3p interacts with the export receptor Mex67p-Mtr2p which translocates the processed and export competent mRNA through the nuclear pore complex into the cytoplasm. Once in the cytoplasm the mRNA encoded genetic information is translated into protein at the ribosoms. In general, NPL3 is essential in most yeast strains and npl3 mutants lead to mRNA export defects. However, in a worldwide deletion project a viable npl3∆ strain was created in the BY4741 background. Interestingly, in npl3∆ no mRNA export defects were detected, suggesting a further yet unknown function for NPL3. However GFP-localization studies of representing reporter proteins revealed a nuclear export defect for the ribosomal pre-60S but not for the 40S subunit in npl3∆. Npl3p physically interacts with the ribosomal protein Rpl25p and binds to the 25S rRNA of the pre-60S subunit. Nevertheless export defects unlikely refer to preribosome processing defects as in npl3∆ a nucleolar mislocalization was not observed and rRNA processing defects were not detected. In fact, Npl3p physically interacts with pre-60S export adapter Nmd3p and the export receptor Mex67p which marks for the export competent pre-60S subunit. Npl3p is not acting as an adapter for these factors as npl3∆ does not influence the binding of these factors to the pre-60S subunit. Here, we propose a novel function for Npl3p as an independent adapter for the export of the pre-60S subunit as it interacts directly with the pre-60S subunit and a deletion of NPL3 leads to export defects of this ribosomal subunit in the nucleus. Furthermore Npl3p has the potential to mediate the contact of these macromolecules to the nuclear pore complex via an interaction with the nucleoporin Nup60p associated Mlp1p.
The second part of this thesis leads to a characterization of the export requirements of Gbp2p, Hrb1p and Npl3p. For a first time a yet unknown link to late steps of the splicing process was observed for Gbp2p and Hrb1p but not for Npl3p. In a screen the splicing factor mutants prp8-908/988 and prp17-Q336* were identified, that lead to severe nuclear export defects for Gbp2p and Hrb1p but not for Npl3p. GBP2 and HRB1 deletion show genetic interactions with these splicing factor mutants in contrast to the deletion of NPL3 which showed neither physical nor genetic interactions. Gbp2p and Hrb1p interact with Prp17p and Prp43p, a factor of the postspliceosomal complex. The nuclear mislocalization of Gbp2p and Hrb1p are effect of substantially reduced mRNA binding levels in prp8-908/988, prp17∆ and prp43-S247A while the amount of bound mRNAs to Npl3p only little is affected. RIP-Chip and qRT-PCR analysis revealed a preference for Gbp2p and Hrb1p binding to mRNAs that contain introns. Especially the double deletion strain gbp2∆ hrb1∆ shows slight splicing defects and significant reduced protein expression levels of intron containing genes. Gbp2p and Hrb1p are novel identified mRNA export adapters for the interaction with the export receptor Mex67p. These adapter-receptor interactions are highly reduced in prp8-908/988 and prp17∆ that may deplete the export of unspliced mRNAs. Furthermore, Gbp2p and Hrb1p interact with the nuclear pore associated Mlp1p which performs a function in the quality control of spliced mRNAs before they leave into the cytoplasm and Gbp2p and Hrb1p may contribute to this final mRNA quality check at the nuclear pores. These findings lead to a following model: Gbp2p and Hrb1p in contrast to Npl3p are recruited to the mRNAs by the splicing machinery and the interaction of Gbp2p and Hrb1p with the export receptor Mex67p promotes the export of spliced mRNAs. Contrary, Npl3p is a general export adapter that interacts regardless of intron status with bulk mRNA and interacts with the mRNA export receptor Mex67p. It is well known that Npl3p is early recruited to the mRNA by action of RNA polymerase II while Gbp2p and Hrb1p are recruited to mRNA during transcription elongation. However, with the co-transcriptional association of the spliceosome, a direct interaction of Gbp2p and Hrb1p with splicing factors of the late splicing phase ascertains a strong binding to spliced mRNAs and allows for a recruitment of Mex67p molecules via physical interactions with Gbp2p and Hrb1p. In this way these factors promote an efficient and controlled transport of the spliced mRNAs into the cytoplasm
In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes
No full textEvotec International Gmb
Die symbolische Aneignung historischer Räume im östlichen Preußen. Nationale und regionale Strategien | The Symbolic Appropriation of Historical Spaces in East Prussia: National and Regional Strategies
No full textThe paper discusses different appropriation strategies applied to the same historical region of East Prussia. By dating the beginning of the symbolic appropriation to the early 19th century, the author reviews the strategies, first applied by Germans and Poles, and later also by Lithuanians and Russians, to make East Prussia or their respective part (Warmia and Masuria, Lithuania Minor, and the Kaliningrad Oblast) their own. This is demonstrated by several periods, starting with the situation before 1914, the First World War, the interwar period, and the Second World War, when East Prussia still existed; and finishing with the postwar period and the changes after 1989. A distinction is made between national and regional East Prussia appropriation strategies, as well as different levels of the process, i.e. publicistic (literary) and practical
- …
