Structural basis for recognition of AU-rich element RNA by the HuD protein

Hu proteins bind to adenosine-uridine (AU)-rich elements (AREs) in the 3' untranslated regions of many short-lived mRNAs, thereby stabilizing them. Here we report the crystal structures of the first two RNA recognition motif (RRM) domains of the HuD protein in complex with an 11-nucleotide fragment of a class I ARE (the c-fos ARE; to 1.8 A), and with an 11-nucleotide fragment of a class II ARE (the tumor necrosis factor alpha ARE; to 2.3 A). These structures reveal a consensus RNA recognition sequence that suggests a preference for pyrimidine-rich sequences and a requirement for a central uracil residue in the clustered AUUUA repeats found in class II AREs. Comparison to structures of other RRM domain-nucleic acid complexes reveals two base recognition pockets in all the structures that interact with bases using residues in conserved ribonucleoprotein motifs and at the C-terminal ends of RRM domains. Different conformations of nucleic acid can be bound by RRM domains by using different combinations of base recognition pockets and multiple RRM domains.     

RNA-binding protein HuD controls insulin translation

Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic β cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic β cells.     

Functional and direct interaction between the RNA binding protein HuD and active Akt1

The RNA binding protein HuD plays essential roles in neuronal development and plasticity. We have previously shown that HuD stimulates translation. Key for this enhancer function is the linker region and the poly(A) binding domain of HuD that are also critical for its function in neurite outgrowth. Here, we further explored the underlying molecular interactions and found that HuD but not the ubiquitously expressed HuR interacts directly with active Akt1. We identify that the linker region of HuD is required for this interaction. We also show by using chimeric mutants of HuD and HuR, which contain the reciprocal linker between RNA-binding domain 2 (RBD2) and RBD3, respectively, and by overexpressing a dominant negative mutant of Akt1 that the HuD-Akt1 interaction is functionally important, as it is required for the induction of neurite outgrowth in PC12 cells. These results suggest the model whereby RNA-bound HuD functions as an adapter to recruit Akt1 to trigger neurite outgrowth. These data might also help to explain how HuD enhances translation of mRNAs that encode proteins involved in neuronal development.     

Human PABP binds AU-rich RNA via RNA-binding domains 3 and 4.

Poly(A) binding protein (PABP) binds mRNA poly(A) tails and affects mRNA stability and translation. We show here that there is little free PABP in NIH3T3 cells, with the vast majority complexed with RNA. We found that PABP in NIH3T3 cytoplasmic lysates and recombinant human PABP can bind to AU-rich RNA with high affinity. Human PABP bound an AU-rich RNA with Kd in the nm range, which was only sixfold weaker than the affinity for oligo(A) RNA. Truncated PABP containing RNA recognition motif domains 3 and 4 retained binding to both AU-rich and oligo(A) RNA, whereas a truncated PABP containing RNA recognition motif domains 1 and 2 was highly selective for oligo(A) RNA. The inducible PABP, iPABP, was found to be even less discriminating than PABP in RNA binding, with affinities for AU-rich and oligo(A) RNAs differing by only twofold. These data suggest that iPABP and PABP may in some situations interact with other RNA regions in addition to the poly(A) tail.     

Alternatively expressed domains of AU-rich element RNA-binding protein 1 (AUF1) regulate RNA-binding affinity, RNA-induced protein oligomerization, and the local conformation of bound RNA ligands

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3'-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.     

Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1

Interleukin-6 mRNA is unstable and degraded with a half-life of 30 min. Instability determinants can entirely be attributed to the 3' untranslated region. By grafting segments of this region to stable green fluorescent protein mRNA and subsequent scanning mutagenesis, we have identified two conserved elements, which together account for most of the instability. The first corresponds to a short noncanonical AU-rich element. The other, 80 nucleotides further 5', comprises a sequence predicted to form a stem-loop structure. Neither element alone was sufficient to confer full instability, suggesting that they might cooperate. Overexpression of myc-tagged AUF1 p37 and p42 isoforms as well as suppression of endogenous AUF1 by RNA interference stabilized interleukin-6 mRNA. Both effects required the AU-rich instability element. Similarly, the proteasome inhibitor MG132 stabilized interleukin-6 mRNA probably through an increase of AUF1 levels. The mRNA coimmunoprecipitated specifically with myc-tagged AUF1 p37 and p42 in cell extracts but only when the AU-rich instability element was present. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes IL-6 mRNA degradation.     

Poly(A) tail length-dependent stabilization of GAP-43 mRNA by the RNA-binding protein HuD

The neuronal ELAV-like RNA-binding protein HuD binds to a regulatory element in the 3'-untranslated region of the growth-associated protein-43 (GAP-43) mRNA. Here we report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail. Using a polysome-based in vitro mRNA decay assay, we found that addition of recombinant HuD protein to the system increased the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect was caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization was specific for GAP-43 mRNA containing the HuD binding element in the 3'-untranslated region and a poly(A) tail of at least 150 A nucleotides. In correlation with the effect of HuD on GAP-43 mRNA stability, we found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). We conclude that HuD stabilizes the GAP-43 mRNA through a mechanism that is dependent on the length of the poly(A) tail and involves changes in its affinity for the mRNA.     

HuD binds to three AU-rich sequences in the 3'-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein

Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3′-untranslated region (3′-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3′-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3′-UTR, which prolongs the mRNA lifetime and increases protein level.     

AU-rich RNA-binding induces changes in the quaternary structure of AUH

The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.     

NMR investigation of the interaction between the neuronal protein tau and the microtubules

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.     

Two different RNA binding activities for the AU-rich element and the poly(A) sequence of the mouse neuronal protein mHuC.

HuC is one of the RNA binding proteins which are suggested to play important roles in neuronal differentiation and maintenance. We cloned and sequenced cDNAs encoding a mouse protein which is homologous to human HuC (hHuC). The longest cDNA encodes a 367 amino acid protein with three RNA recognition motifs (RRMs) and displays 96% identity to hHuC. Northern blot analysis showed that two different mRNAs, of 5.3 and 4.3 kb, for mouse HuC (mHuC) are expressed specifically in brain tissue. Comparison of cDNA sequences with the corresponding genomic sequence revealed that alternative 3' splice site selection generates two closely related mHuC isoforms. Iterative in vitro RNA selection and binding analyses showed that both HuC isoforms can bind with almost identical specificity to sequences similar to the AU-rich element (ARE), which is involved in the regulation of mRNA stability. Functional domain mapping using mHuC deletion mutants showed that the first RRM binds to ARE, that the second RRM has no RNA binding activity by itself, but facilitates ARE binding by the first RRM and that the third RRM has specific binding activity for the poly(A) sequence.