Vergleichende Analysen der nukleär/zytoplasmatischen Exporteigenschaften von SR-ähnlichen Proteinen und ihre regulatorische Funktion im mRNA Spleißing-Prozess

Eukaryontische 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 („me...

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Bibliographic Details
Main Author: Hackmann, Alexandra
Contributors: Krebber, Heike (Prof.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2010
Online Access:PDF Full Text
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Table of Contents: 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.