Identification and functional characterization of vegetally localized RNAs in Xenopus laevis oocytes

Die subzelluläre Anreicherung von mRNA Molekülen stellt einen wichtigen Mechanismus der Genregulation in eukaryotischen Zellen dar. In der Xenopus laevis Oozyte üben lokalisierte mRNAs wichtige Funktionen bei der frühembryonalen Entwicklung und Determination von Zellschicksalen aus. Mit dem Ziel, neue in der Xenopus Oozyte vegetal lokalisierte RNA-Moleküle zu isolieren, wurde in der vorliegenden Arbeit eine cDNA Bibliothek erstellt, die angereichert vegetal-kortikal lokalisierte RNAs enthält. Diese wurde in einer whole mount in situ-Hybridisierungs-basierten Durchmusterung auf vegetal lokalisierte Transkripte durchsucht und so konnten neben der Reisolierung bereits bekannter lokalisierter Transkripte auch vier neue lokalisierte RNAs identifiziert werden, die sich aufgrund ihres charakteristischen Verteilungsmusters den sogenannten frühen oder späten Sortierungswegen zuordnen lassen. Ein weiteres Transkript lokalisiert in einer bisher nicht beschriebenen Weise am äquatorialen Kortex der Oozyte und ist somit einem neuen Lokalisationstyp zuzuordnen. In Mikroinjektionsexperimenten wurden die Lokalisationselemente drei dieser RNAs, die den vegetalen Transport einer Reporter-RNA entlang des frühen bzw. des späten Verteilungsweges vermitteln können, auf 75 bis 300 Nukleotide lange Bereiche in den 3´- bzw. 5´-untranslatierten Regionen eingegrenzt. Diese cis-agierenden Elemente weisen keine charakteristischen Konsensussequenzen auf, sondern bilden vermutlich spezifische Sekundärstrukturelemente aus, die dann von zellulären Transportfaktoren erkannt werden. In UV-Quervernetzung- und Koimmunopräzipitationsexperimenten konnte weiterhin nachgewiesen werden, daß die Lokalisationselemente von RNAs unterschiedlicher Sortierungswege ein differentielles Bindungsverhalten an Proteine aus Xenopus Oozyten Extrakten, sowie an bekannte Lokalisationselement-bindende Proteine (Vg1RBP und Prrp) aufweisen

Functional Characterization of Drosophila Translin and Trax

The vertebrate RNA and ssDNA-binding protein Translin has been suggested to function in a variety of cellular processes, including DNA damage response, RNA transport, and translational control. The Translin-associated factor X (Trax) interacts with Translin, and Trax protein stability depends on the presence of Translin. To determine the function of the Drosophila Translin and Trax, we generated a translin null mutant and isolated a trax nonsense mutation. translin and trax single and double mutants are viable, fertile, and phenotypically normal. Meiotic recombination rates and chromosome segregation are also not affected in translin and trax mutants. In addition, we found no evidence for an increased sensitivity for DNA double-strand damage in embryos and developing larvae. Together with the lack of evidence for their involvement in DNA double-strand break checkpoints, this argues against a critical role for Translin and Trax in sensing or repairing such DNA damage. However, Drosophila translin is essential for stabilizing the Translin interaction partner Trax, a function that is surprisingly conserved throughout evolution. Conversely, trax is not essential for Translin stability as trax mutants exhibit normal levels of Translin protein

Global analysis of asymmetric RNA enrichment in oocytes reveals low conservation between closely related Xenopus species

RNAs that localize to the vegetal cortex during Xenopus laevis oogenesis have been reported to function in germ layer patterning, axis determination, and development of the primordial germ cells. Here we report on the genome-wide, comparative analysis of differentially localizing RNAs in Xenopus laevis and Xenopus tropicalis oocytes, revealing a surprisingly weak degree of conservation in respect to the identity of animally as well as vegetally enriched transcripts in these closely related species. Heterologous RNA injections and protein binding studies indicate that the different RNA localization patterns in these two species are due to gain/loss of cis-acting localization signals rather than to differences in the RNA-localizing machinery.ISSN:1939-4586ISSN:1059-152

Nuclear localization of the pre-mRNA associating protein THOC7 depends upon its direct interaction with Fms tyrosine kinase interacting protein (FMIP)

AbstractTHOC7 and Fms-interacting protein (FMIP) are members of the THO complex that associate with the mRNA export apparatus. FMIP is a nucleocytoplasmic shuttling protein with a nuclear localization signal (NLS), whereas THOC7 does not contain a typical NLS motif. We show here that THOC7 (50–137, amino acid numbers) binds to the N-terminal portion (1–199) of FMIP directly. FMIP is detected mainly in the nucleus. In the absence of exogenous FMIP, THOC7 resides mainly in the cytoplasm, while in the presence of FMIP, THOC7 is transported into the nucleus with FMIP. Furthermore, THOC7 lacking the FMIP binding site does not co-localize with FMIP, indicating that THOC7/FMIP interaction is required for nuclear localization of THOC7.Structured summaryMINT-6799962, MINT-6799973, MINT-6800005: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with THOC5 (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800108: FMIP (uniprotkb:Q13769) and THOC7 (uniprotkb: Q6I9Y2) co-localize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800052: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC1 (uniprotkb: Q96FV9) by anti tag coimmunoprecipitation (MI:0007)MINT-6800022: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with FMIP (uniprotkb:Q6DFL5) by pull down (MI:0096)MINT-6799989: THOC7 (uniprotkb:Q6I9Y2) binds (MI:0407) to FMIP (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800071, MINT-6800089: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC7 (uniprotkb:Q6I9Y2) and THOC1 (uniprotkb:Q96FV9) by anti tag coimmunoprecipitation (MI:0007

Global analysis of asymmetric RNA enrichment in oocytes reveals low conservation between closely related Xenopus

RNAs that localize to the vegetal cortex during Xenopus laevis oogenesis have been reported to function in germ layer patterning, axis determination, and development of the primordial germ cells. Here we report on the genome-wide, comparative analysis of differentially localizing RNAs in Xenopus laevis and Xenopus tropicalis oocytes, revealing a surprisingly weak degree of conservation in respect to the identity of animally as well as vegetally enriched transcripts in these closely related species. Heterologous RNA injections and protein binding studies indicate that the different RNA localization patterns in these two species are due to gain/loss of cis-acting localization signals rather than to differences in the RNA-localizing machinery