Protein Ligands to Hur Modulate Its Interaction with Target Mrnas in Vivo

Abstract

AU-rich elements (AREs) present in the 3′ untranslated regions of many protooncogene, cytokine, and lymphokine messages target them for rapid degradation. HuR, a ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds AREs and stabilizes ARE-containing mRNAs in transiently transfected cells. Here, we identify four mammalian proteins that bind regions of HuR known to be essential for its ability to shuttle between the nucleus and the cytoplasm and to stabilize mRNA: SETα, SETβ, pp32, and acidic protein rich in leucine (APRIL). Three have been reported to be protein phosphatase 2A inhibitors. All four ligands contain long, acidic COOH-terminal tails, while pp32 and APRIL share a second motif: rev-like leucine-rich repeats in their NH2-terminal regions. We show that pp32 and APRIL are nucleocytoplasmic shuttling proteins that interact with the nuclear export factor CRM1 (chromosomal region maintenance protein 1). The inhibition of CRM1 by leptomycin B leads to the nuclear retention of pp32 and APRIL, their increased association with HuR, and an increase in HuR's association with nuclear poly(A)+ RNA. Furthermore, transcripts from the ARE-containing c-fos gene are selectively retained in the nucleus, while the cytoplasmic distribution of total poly(A)+ RNA is not altered. These data provide evidence that interaction of its ligands with HuR modulate HuR's ability to bind its target mRNAs in vivo and suggest that CRM1 is instrumental in the export of at least some cellular mRNAs under certain conditions. We discuss the possible role of these ligands upstream of HuR in pathways that govern the stability of ARE-containing mRNAs.

Phosphorylation of the HuR ligand APRIL by casein kinase 2 regulates CD83 expression

“…Generally, the nuclear export of bulk poly(A) 1 mRNA is mediated in metazoans by the transport receptor NXF1/Tap, together with its small cofactor Nxt1/p15 (reviewed in detail in [30,[53][54][55]). However, it has also been reported that, for example, c-fos mRNA, or the message encoding interferon-a1, is translocated from the nucleus to the cytoplasm by a completely unrelated transport pathway [34,56]. This pathway is characterized by the export receptor : 70 mM) for 3 h. After 1 h, the respective cultures were activated with PMA/ ionomycin as before.…”

“…Instead, this interaction induces the CRM1-dependent cytoplasmic accumulation of CD83 mRNA [32]. In this respect, it is important to note that the interaction of HuR and CRM1 is indirect [34] and is, particularly in case of CD83 mRNA export, accomplished by the HuR protein ligand APRIL (ANP32B) [35]. APRIL belongs to the growing family of ANP32 proteins (ANP32a-h), a group of leucine-rich phosphoproteins that have been linked to various cellular activities such as, for example, gene expression, signaling, adhesion and apoptosis [36].…”

Isolation and functional identification of a novel human hepatic growth factor: Hepatopoietin Cn

“…LANP proteins typically contain leucine-rich repeat motifs followed by an acidic carboxyterminal tail and are heat-stable, and are involved in a variety of biological pathways, including signaling, protein degradation, cytoskeletal dynamics, and morphogenesis, presumably based on the ability of the leucine-rich repeat (LRR) domains to serve as adapter sites for protein-protein interactions. 23,24 Previous biochemical studies suggested that human LANP (pp32/I 1 PP2A/ mapmodulin/HPPCn) has been implicated in a number of cellular processes in both nucleus and cytoplasm, including proliferation, 24 differentiation, 23 caspase-dependent and caspase-independent apoptosis, 25 suppression of transformation in vivo, 26 inhibition of protein phosphatase 2A, 27 regulation of mRNA trafficking and stability in association with HuR, 28 and inhibition of acetyltransferases as part of the INHAT complex. 29 In this report, we found that rhHPPCn may stimulate the DNA synthesis and proliferation of hepatocytes and hepatoma cell lines and activate kinase pathways including SPK, ERK1/2, and Stat3.…”

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

“…28 HNS is mainly responsible for nuclear/cytoplasmic shuttling upon binding adaptor proteins for nuclear export such as pp32/PHAP-I and APRIL 29,30 and with import factors transportin-1, -2 and importin a: [31][32][33] Previous Electrophoretic Mobility Shift Assay (EMSA)-based report 34 suggests that the RRM23 linker, together with the RRM3 domain, could also have an additional role in stabilizing HuR-AREs complexes. This enhancement of ARE-binding activity was greater than that observed in Surface Plasmon Resonance (SPR) experiments with HuD, a highly homolog protein to HuR, 35 where the RRM23 linker showed a negligible effect in RNA binding.…”