Their function and regulation are largely mediated by RNA binding proteins (RBPs). RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA. Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). The nuclear tRNA receptor exportin-t is part of the VCC(N). Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. ![]() Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein.
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