Together, our data show that REF proteins play a direct role in the export of mRNAs, most likely by recruiting TAP to mRNP export complexes. Materials and Methods DNA Constructs. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm. The REF Rabbit polyclonal to CREB.This gene encodes a transcription factor that is a member of the leucine zipper family of DNA binding proteins.This protein binds as a homodimer to the cAMP-responsive element, an octameric palindrome. proteins belong to a superfamily of RNA binding proteins made up of ribonucleoprotein (RNP)-type RNA binding domains (RBD, ref. 1). The distinguishing feature of the REF family is the presence of two highly conserved motifs in their N and C termini: REF-N and REF-C boxes. Between the conserved motifs and the RBD, REF proteins have regions of variable length (N-vr and C-vr), which are related to the RGG boxes present in many heterogeneous RNP (hnRNP) proteins (refs. 1 and 2; see also Fig. ?Fig.22(2). RBD with the conserved RNP1 and RNP2 motifs; REF-N and REF-C, conserved N- and C-terminal motifs; N-vr and C-vr represent the N- and C-terminal variable regions specific to each member of the family. Numbers indicate the position in the amino acid sequence. (in rabbit RWJ-67657 reticulocyte lysates or lysates, respectively. Five-microliter aliquots were incubated with glutathione agarose beads precoated RWJ-67657 with the recombinant proteins indicated above the lanes. One-tenth of the input and one-quarter of the bound fractions were analyzed by SDS/PAGE followed by Coomassie stain and fluorography. Yra1p, a member of the REF family, is an essential nuclear protein first recognized from its RNA-annealing activity (3). More recently, Yra1p was shown to be involved in the export of mRNA from your nucleus in yeast cells (2, 4). In mouse, REFs are encoded by at least three different genes (1C3) and differ at multiple positions in the variable regions because of deletions and/or amino acid changes (2). In contrast, all murine REF proteins are 98% identical in the RBD and 100% in the conserved boxes (2). The complexity of the murine subfamily is usually further increased by the expression of multiple splice variants (2). Murine REF1-II is usually generated by option splicing of REF1-I (also named Aly; observe ref. 5) and lacks the N-terminal variable region (observe Fig. ?Fig.22oocytes. This export inhibition is usually observed whether or not the mRNAs have been generated by splicing. We show that microinjection of recombinant REFs stimulates directly the export of mRNAs that are normally exported inefficiently. Together, our data indicate that REF proteins play a direct role in the export of mRNAs, most likely by recruiting TAP to mRNP export complexes. Materials and Methods DNA Constructs. Most TAP and REF constructs used in this study have been explained (2). REF cDNA fragments were cloned by PCR using the Expand high-fidelity PCR system (Boehringer) with murine REF1-II and REF2-II cDNAs as themes. PCR fragments were cloned into the as a glutathione RNA Binding Assays. Gel retardation and GST pull-down assays were performed as explained (2). 35S-labeled proteins were synthesized by using the combined transcription/translation (TnT) RWJ-67657 kit from Promega. GST fusions were expressed in BL21(DE3) pLysS and purified as explained (15). The amount of recombinant proteins used in each binding reaction is usually indicated in the physique legends. Oocyte Microinjections. All DNA themes for synthesis of labeled RNAs have been explained. These were pBSAd1 and Ad-CTE pre-mRNAs (17); dihydrofolate reductase (DHFR) mRNA; histone H4 mRNA; U1Sm, U5Sm, and U6ss snRNAs; and human initiator methionyl RWJ-67657 tRNA (16, 17). AdHML81, Fushi tarazu (Ftz), and -globin pre-mRNAs have been explained (8, 14). Ftz-218 and -globin-247 cDNAs were kindly provided by Herv Le Hir (Brandeis University or college, Waltham, MA) [FluorImager (Fuji, FLA-2000)]. Oocyte injections and analysis of microinjected RNA by denaturing gel electrophoresis and autoradiography were performed as explained (17). Quantitation was carried out by PhosphorImager. The concentrations of antibodies and recombinant proteins in the injected samples are indicated in the physique legends. Immunofluorescence and Heterokaryon Assays. HeLa cells were transfected with pEGFP-C1 constructs by using FuGENE6 (Roche). Approximately 20 h after transfection, cells were fixed with 3.7% formaldehyde.