Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system and are able to sensitize even naïve T cells. Mature DC are characterized by expression of CD83, a surface molecule that is proposed to be involved in efficient T cell activation. It has been recently shown that CD83 mRNA is transported from the nucleus to the cytoplasm in a HuR-and CRM1-dependent manner. Therefore we here investigated the impact of two known protein ligands of HuR, pp32 and APRIL, on CD83 expression. Both pp32 (ANP32A) and APRIL (ANP32B) are shuttle proteins, and it has been reported earlier that these HuR ligands can act as adaptors that link HuR and the CRM1-specific nuclear export pathway. By employing RNA interference (RNAi) technology we demonstrate that pp32 is dispensable for CD83 expression, whereas APRIL contributes to the nuclear export and subsequent translation of CD83 mRNA. Furthermore, we have determined the nuclear import signal (NLS) as well as the nuclear export signal (NES) of human APRIL. Moreover, we analyzed the status of phosphorylation of endogenous APRIL and identified threonine 244 to be an as yet unrecognized phosphate acceptor. Finally, we were able to show that phosphorylation of this specific amino acid residue regulates the nuclear export of APRIL. In sum, we report here the signal sequences in APRIL that mediate its intracellular trafficking and provide evidence that this protein ligand of HuR is an important player in the post-transcriptional regulation of CD83 expression by affecting the nucleocytoplasmic translocation of CD83 mRNA.

Ribosomal protein L5 is part of the 60 S ribosomal subunit and localizes in both the cytoplasm and the nucleus of eukaryotic cells, accumulating particularly in the nucleoli. L5 is known to bind specifically to 5 S rRNA and is involved in nucleocytoplasmic transport of this rRNA. Here, we report a detailed analysis of the domain organization of the human ribosomal protein L5. We show that a signal that mediates nuclear import and nucleolar localization maps to amino acids 21-37 within the 297-amino acid L5 protein. Furthermore, carboxyl-terminal residues at positions 255-297 serve as an additional nuclear/nucleolar targeting signal. Domains involved in 5 S rRNA binding are located at both the amino terminus and the carboxyl terminus of L5. Microinjection studies in somatic cells demonstrate that a nuclear export signal (NES) that maps to amino acids 101-111 resides in the central region of L5. This NES is characterized by a pronounced clustering of critical leucine residues, which creates a peptide motif not previously observed in other leucine-rich NESs. Finally, we present a refined model of the multidomain structure of human ribosomal protein L5.The biogenesis of eukaryotic ribosomes occurs at a specific subnuclear compartment, the nucleolus, and requires the coordinated assembly of four different rRNAs and approximately 80 ribosomal proteins (1, 2). The 5.8, 18, and 28 S rRNAs are synthesized by RNA polymerase I in the nucleolus, whereas, in contrast, 5 S rRNA is transcribed by RNA polymerase III in the nucleoplasm. The ribosomal proteins are encoded by mRNAs that are synthesized by RNA polymerase II. After translation, these proteins are imported from the cytoplasm into the nucleolus for assembly of the 40 S and 60 S ribosomal subunits. These, in turn, are then exported to the cytoplasm. Thus, multiple intracellular transport activities between the nucleus and cytoplasm are required for de novo ribosome synthesis.In eukaryotic cells, the 5 S rRNA is part of the 60 S ribosomal subunit. In addition, a significant amount of 5 S rRNA is complexed with various proteins to form nonribosome-associated ribonucleoprotein particles. After transcription, 5 S rRNA is able to transiently bind the La antigen, a 50-kDa protein that acts in the termination of polymerase III transcripts, in the nucleus (3, 4). Furthermore, in the nucleus, 5 S rRNA also binds either its own transcription factor IIIA or ribosomal protein L5, forming 7 or 5 S ribonucleoprotein particles, respectively (reviewed in Ref. 5). In particular, it has been suggested that the 5 S rRNA⅐L5 complex (5 S ribonucleoprotein particle) acts as a precursor to ribosome assembly by delivering 5 S rRNA from the nucleoplasm to the nucleolar assembly site of 60 S ribosomal subunits (6). Studies in Xenopus oocytes have shown that 5 S rRNA can be exported from the nucleus to the cytoplasm for subsequent accumulation at distinct cytoplasmic storage sites by either transcription factor IIIA or L5 (7,8). As a consequence of increased ribosomal subunit synthesis, stored 5...

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