TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/10431/
KW  - gene expression 
KW  -  mRNA export 
KW  -  gene expression
TI  - Role of the protein Sus1 and its interaction with the Sac3CID motif in transcription-coupled mRNA export
Y1  - 2010///
ID  - heidok10431
AV  - public
A1  - Klöckner, Christoph
N2  - The process of gene expression in the nucleus consists of gene transcription into mRNA, posttranscriptional modification of transcripts and finally export of mRNA through the nuclear pore complex into the cytoplasm before translation into proteins can occur.  Two of the protein complexes proposed to be involved in coupling of transcription and mRNA export are the SAGA complex (Spt-Ada-Gcn5-acetyltransferase) mediating transcription activation through histone acetylation and deubiquitination, and the TREX-2 (Sac3-Thp1-Cdc31-Sus1) complex, which interacts with the nuclear pore complex and is involved in the export of mRNA. Sac3, which is a central subunit of TREX-2, is suggested to integrate transcription and mRNA export through the CID (Cdc31-interacting domain) motif, which comprises ~80 amino acids in the C-terminal part of the protein. This motif recruits Cdc31, a calmodulin-like protein, and Sus1, a protein functioning in transcription as well as mRNA export. In addition, the CID motif is required for correct targeting of TREX-2 to nuclear pore complexes. The observation that Sac3CID acts as a crucial binding platform in TREX-2 and is also responsible for the interaction with nuclear pores makes it an attractive target to further unravel the genetic network of factors involved in transcription-coupled mRNA export. Mutants from a synthetic lethal (SL) screen with a sac3? allele have been analyzed for a genetic interaction with SAC3CID to identify factors that are specifically linked to transcription-coupled mRNA export. Among the SL strains that proved to be genetically interacting with SAC3CID, one strain was complemented by BUR6, the gene for a transcriptional regulator. Another strain could be complemented by SPE3, encoding the spermidine synthase. The small and basic molecule spermidine, produced by Spe3, was suggested to be involved in various nuclear processes like transcriptional repression and global chromatin organisation by interaction of spermidine with chromatin. These novel findings strengthen the connection of Sac3CID with transcriptional regulation. In addition to the search for novel genetic interactions, a complementary biochemical approach to study the link between transcription and export of mRNA was applied. Through a mutational analysis of Sus1, a factor interacting with Sac3CID inside TREX-2 as well as with the transcriptional coactivator SAGA, it was possible to identify conserved residues that are crucial for the interaction of Sus1 with its partner proteins. One of the engineered sus1 alleles showed significant loss of interaction with both SAGA and TREX-2. In contrast, two other alleles yielded highly reduced interaction with TREX-2 while interaction with SAGA remained undisturbed. Biochemical, cell biological and genetic experiments with these alleles substantiated that the binding of Sus1 to Sac3CID (and therefore TREX-2) was disrupted without affecting the interaction with the SAGA complex. A mutational analysis of the single amino acid mutations revealed their contribution to the defects of the combined alleles. These findings will help to define the selectivity and specificity of how Sus1 interacts with the complexes SAGA and TREX-2.  Taken together, the results of my PhD thesis have revealed novel interactions for the genetic network of the pivotal motif SAC3CID while the creation of alleles that selectively uncouple Sus1 from TREX-2 provides the basis for a detailed analysis of the functions of the versatile protein Sus1 in transcription and mRNA export. 
ER  -