Transcription activation of some genes is paralleled by their repositioning to

Transcription activation of some genes is paralleled by their repositioning to the nuclear periphery but the mechanism underlying gene anchoring is poorly defined. is also paralleled by its repositioning to the periphery and this relocation contributes to optimal gene expression (3). Importantly recent studies pointed to a direct physical link between Sus1p a component of the SAGA histone deacetylase coactivator complex and the Sac3-Thp1 complex which is usually part of the mRNA export machinery associated with pores (31). These data together suggested that transcription regulators could control the recruitment of genes to the nuclear periphery possibly linking gene repositioning to optimal activation. However KOS953 a rigid and systematic dependence of gene expression on peripheral positioning has not been exhibited. More generally the molecular basis of transcription-induced gene repositioning is usually poorly comprehended and whether it is KOS953 the cause or result of transcription activation is still unclear. Several observations indicated a possible role for the nascent messenger ribonucleoprotein (mRNP) Rabbit polyclonal to ITLN2. in stabilizing the association of a gene with the nuclear periphery. First mRNP components actually interact with the NPC-associated KOS953 Mlp1p and Mlp2p proteins (11 17 43 and the results of chromatin immunoprecipitation (ChIP) experiments suggest that Mlp1p associates with transcribing genes in an RNA-dependent manner (5). These observations raised the possibility that Mlp proteins contribute to gene anchoring by interacting with nascent transcripts. Second several mRNA export factors bind mRNA cotranscriptionally (28 38 45 consistent with a potential role for growing mRNPs in bridging active genes to the NPC. Moreover we recently showed that this mRNA export receptor Mex67p which promotes the translocation of mRNP complexes through the NPC (35) is also recruited cotranscriptionally (19). The association of Mex67p with transcribing genes and its ability to interact with various pore components raised the possibility that mRNP-bound Mex67p helps the anchoring of transcribing loci to the nuclear periphery. To test the potential functions of Mlp1p and Mex67p in gene anchoring we compared the localization of inducible genes in wild-type (WT) and or mutant cells (35). The results indicate that both Mlp1p and KOS953 Mex67p are required for efficient anchoring of the galactose-inducible and stress-inducible genes; however gene anchoring appears to be not essential for the transcription of these two genes. Notably loss of gene anchoring in the mutant correlates with the inability of the mutant protein to associate with the transcribing genes. Moreover we find that transcription-induced NPC anchoring of the gene does not require the mRNA-coding region suggesting that nascent mRNP may not be essential for bridging an conversation between an active gene and the NPC. These data and the observation that this cotranscriptional binding of Mex67p is usually RNA independent suggest that Mex67p may contribute to gene anchoring by interacting with activated chromatin rather than nascent RNA. MATERIALS AND METHODS Plasmid constructions. To place LacO repeats downstream of the genes the 3′ untranslated region (3′UTR) region of each gene was cloned in front of the LacO repeats carried by the integrating plasmid pAFS52 (CEN) isolated in a synthetic lethal screen and transporting the gene as the only complete open reading frame (D. Zenklusen and F. Stutz unpublished data). Yeast strains. The yeast strains used in this study are outlined in Table ?Table1.1. The KOS953 strain contains an integrated mutant gene (26). Wild-type and genes were genomically tagged with green fluorescent protein (GFP)-Kanr KOS953 by homologous recombination (29). GA1320-and GA1320-strains were obtained by crossing strain GA1320 (LacI-GFP-HIS3 Nup49-GFP) with the (26) strains. The and loci were subsequently tagged with LacO repeats in the GA1320 GA1320-strains by transformation of linearized pFS2913 and pFS3013 respectively followed by selection on Trp? plates. Insertions were confirmed by PCR on genomic DNA. The strain was obtained by transformation and homologous recombination of a PCR-generated cassette (18) transporting ends complementary to the 5′ and 3′ ends of the and strains were constructed using the same strategy with the forward primers GAL2-loxP-F1 (OFS1071) and GAL2-3′UTR-loxP-F1 (OFS1113) (5′TTACAACATG ACGACAAACC GTGGTACAAG GCCATGCTAG AATAACAGCT GAAGCTTCGT ACGC3′).