Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

Structure of the RNA Helicase MLE Reveals the Molecular Mechanisms for Uridine Specificity and RNA-ATP Coupling.

The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. We identified a stable MLE core comprising the DExH helicase module and two auxiliary domains: a dsRBD and an OB-like fold. MLEcore is an unusual DExH helicase that can unwind blunt-ended RNA duplexes and has specificity for uridine nucleotides. We determined the 2.1 Å resolution structure of MLEcore bound to a U10 RNA and ADP-AlF4. The OB-like and dsRBD folds bind the DExH module and contribute to form the entrance of the helicase channel. Four uridine nucleotides engage in base-specific interactions, rationalizing the conservation of uridine-rich sequences in critical roX substrates. roX2 binding is orchestrated by MLE's auxiliary domains, which is prerequisite for MLE localization to the male X chromosome. The structure visualizes a transition-state mimic of the reaction and suggests how eukaryotic DEAH/RHA helicases couple ATP hydrolysis to RNA translocation.

Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5-SMG7 and SMG6.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7. To understand the molecular mechanisms, we recapitulated these steps of NMD in vitro using purified components. We find that a short C-terminal segment of phosphorylated UPF1 containing the last two Ser-Gln motifs is recognized by the heterodimer of SMG5 and SMG7 14-3-3-like proteins. In contrast, the SMG6 14-3-3-like domain is a monomer. The crystal structure indicates that the phosphoserine binding site of the SMG6 14-3-3-like domain is similar to that of SMG5 and can mediate a weak phospho-dependent interaction with UPF1. The dominant SMG6-UPF1 interaction is mediated by a low-complexity region bordering the 14-3-3-like domain of SMG6 and by the helicase domain and C-terminal tail of UPF1. This interaction is phosphorylation independent. Our study demonstrates that SMG5-SMG7 and SMG6 exhibit different and non-overlapping modes of UPF1 recognition, thus pointing at distinguished roles in integrating the complex NMD interaction network.

Crystal structure of the human eIF4AIII-CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain.

DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22-eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins.

Helix 8 plays a crucial role in bradykinin B(2) receptor trafficking and signaling.

Upon activation the human bradykinin B(2) receptor (B(2)R) acts as guanine nucleotide exchange factor for the G proteins G(q/11) and G(i). Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits ß-arrestins, which block further G protein activation and promote B(2)R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B(2)R. We show here that disruption of helix 8 in the B(2)R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B(2)R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B(2)R with GRK2/3 and ß-arrestin2 as shown by co-immunoprecipitation. Recruitment of ß-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B(2)R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B(2)R with GRK2/3 and ß-arrestins. Additional data obtained with chimera of B(2)R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.

The role of helix 8 and of the cytosolic C-termini in the internalization and signal transduction of B(1) and B(2) bradykinin receptors.

Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B(2) bradykinin receptor (B(2)wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B(1) bradykinin receptor (B(1)wt) containing these B(2)wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B(1)wt C-terminus with corresponding B(2)wt regions either at C330(7.71) following putative helix 8 (B(1)CB(2)) or at the preceding Y312(7.53) in the NPXXY sequence (B(1)YB(2)) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B(1)KB(2)). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B(1)wt to serine (as in B(2)wt) as being responsible for this effect. The slowly internalizing chimera as well as a B(1)wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B(1)wt or the other chimeras. The slow internalization of B(1)KB(2) was also accompanied by a lack of agonist-induced phosphorylation, that in contrast was observed for B(1)YB(2) and B(1)CB(2), suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.

The structure of the Pan2-Pan3 core complex reveals cross-talk between deadenylase and pseudokinase.

Pan2-Pan3 is a conserved complex involved in the shortening of mRNA poly(A) tails, the initial step in eukaryotic mRNA turnover. We show that recombinant Saccharomyces cerevisiae Pan2-Pan3 can deadenylate RNAs in vitro without needing the poly(A)-binding protein Pab1. The crystal structure of an active ~200-kDa core complex reveals that Pan2 and Pan3 interact with an unusual 1:2 stoichiometry imparted by the asymmetric nature of the Pan3 homodimer. An extended region of Pan2 wraps around Pan3 and provides a major anchoring point for complex assembly. A Pan2 module formed by the pseudoubiquitin-hydrolase and RNase domains latches onto the Pan3 pseudokinase with intertwined interactions that orient the deadenylase active site toward the A-binding site of the interacting Pan3. The molecular architecture of Pan2-Pan3 suggests how the nuclease and its pseudokinase regulator act in synergy to promote deadenylation.

Alanine screening of the intracellular loops of the human bradykinin B receptor--effects on receptor maintenance, G protein activation and internalization.

The bradykinin B(2) receptor is coupled to G protein G(q/11) and becomes sequestered into intracellular compartments after activation. To more closely define the receptor sequences involved in these processes and their functions, we systematically mutated all three intracellular loops (ICLs), either as point mutations or in groups of three to five amino acids to Ala, obtaining a total of 14 mutants. All constructs were stably expressed in HEK 293 cells and, with the exception of triple mutant DRY --> AAA, retained the ability to specifically bind [(3)H]bradykinin. The binding affinities at 4 or 37 degrees C of several mutants differed considerably from those determined for the wild-type receptor, indicating an allosteric connection between the conformation of the binding site and that of the ICLs. Mutations in ICL-1 strongly reduced surface expression without affecting G protein signaling or [(3)H]bradykinin internalization. Two cluster mutants in the middle of ICL-2 containing basic residues displayed considerably reduced potencies, whereas two mutations in ICL-3 resulted in receptor conformations that were considered to be semi-active, based on the observation that they responded with phosphoinositide hydrolysis to compounds normally considered to be antagonists. This, and the fact that a cluster mutant at the C-terminal end of ICL-3 was signaling incompetent, hint at the involvement of ICL-2 and ICL-3 in G(q/11) activation, albeit with different functions. None of the mutants displayed reduced ligand-induced receptor internalization, indicating that the loops are not essential for this process. No conclusion could be drawn, however, with regard to the role of the DRY sequence, as the corresponding triplet mutation lacked binding capability.

C-terminal fusion of eGFP to the bradykinin B2 receptor strongly affects down-regulation but not receptor internalization or signaling.

A functional comparison was made between the wild-type bradykinin B2 receptor (B2wt) and the chimera B2eGFP (enhanced green-fluorescent protein fused to the C-terminus of B2wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 microM bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B2wt, but not in B2eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B2wt(low) to less than 20% within 1 h, whereas the chimera B2eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B2wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B2eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B2wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.

Mutation of tyrosine in the conserved NPXXY sequence leads to constitutive phosphorylation and internalization, but not signaling, of the human B2 bradykinin receptor.

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/beta-adrenergic-like receptors. Exchange of Tyr(305) in the corresponding NPLVY sequence of the bradykinin B(2) receptor (B(2)R) for Ala resulted in a mutant, termed Y305A, that internalized [(3)H]bradykinin (BK) almost as rapidly as the wild-type (wt) B(2)R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B(2)R. Confocal fluorescence microscopy revealed that, in contrast to the B(2)R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B(2)R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G(q/11) without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B(2)R. We conclude, therefore, that the Y305A mutation of B(2)R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G(q/11), thereby limiting the extent of ligand-independent receptor internalization.