A riboswitch separated from its ribosome-binding site still regulates translation.

Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10-G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch-akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.

Structure and function analysis of a type III preQ1-I riboswitch from Escherichia coli reveals direct metabolite sensing by the Shine-Dalgarno sequence.

Riboswitches are small noncoding RNAs found primarily in the 5' leader regions of bacterial messenger RNAs where they regulate expression of downstream genes in response to binding one or more cellular metabolites. Such noncoding RNAs are often regulated at the translation level, which is thought to be mediated by the accessibility of the Shine-Dalgarno sequence (SDS) ribosome-binding site. Three classes (I-III) of prequeuosine1 (preQ1)-sensing riboswitches are known that control translation. Class I is divided into three subtypes (types I-III) that have diverse mechanisms of sensing preQ1, which is involved in queuosine biosynthesis. To provide insight into translation control, we determined a 2.30 Å-resolution cocrystal structure of a class I type III preQ1-sensing riboswitch identified in Escherichia coli (Eco) by bioinformatic searches. The Eco riboswitch structure differs from previous preQ1 riboswitch structures because it has the smallest naturally occurring aptamer and the SDS directly contacts the preQ1 metabolite. We validated structural observations using surface plasmon resonance and in vivo gene-expression assays, which showed strong switching in live E. coli. Our results demonstrate that the Eco riboswitch is relatively sensitive to mutations that disrupt noncanonical interactions that form the pseudoknot. In contrast to type II preQ1 riboswitches, a kinetic analysis showed that the type III Eco riboswitch strongly prefers preQ1 over the chemically similar metabolic precursor preQ0. Our results reveal the importance of noncanonical interactions in riboswitch-driven gene regulation and the versatility of the class I preQ1 riboswitch pseudoknot as a metabolite-sensing platform that supports SDS sequestration.

Pre-mRNA splicing factor U2AF2 recognizes distinct conformations of nucleotide variants at the center of the pre-mRNA splice site signal.

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced UV-crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites, with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

Full-Length NAD+-I Riboswitches Bind a Single Cofactor but Cannot Discriminate against Adenosine Triphosphate.

Bacterial riboswitches are structured RNAs that bind small metabolites to control downstream gene expression. Two riboswitch classes have been reported to sense nicotinamide adenine dinucleotide (NAD+), which plays a key redox role in cellular metabolism. The NAD+-I (class I) riboswitch stands out because it comprises two homologous, tandemly arranged domains. However, previous studies examined the isolated domains rather than the full-length riboswitch. Crystallography and ligand binding analyses led to the hypothesis that each domain senses NAD+ but with disparate equilibrium binding constants (KD) of 127 µM (domain I) and 3.4 mM (domain II). Here, we analyzed individual domains and the full-length riboswitch by isothermal titration calorimetry to quantify the cofactor affinity and specificity. Domain I senses NAD+ with a KD of 24.6 ± 8.4 µM but with a reduced ligand-to-receptor stoichiometry, consistent with nonproductive domain self-association observed by gel-filtration chromatography; domain II revealed no detectable binding. By contrast, the full-length riboswitch binds a single NAD+ with a KD of 31.5 ± 1.5 µM; dinucleotides NADH and AP2-ribavirin also bind with one-to-one stoichiometry. Unexpectedly, the full-length riboswitch also binds a single ATP equivalent (KD = 11.0 ± 3.5 µM). The affinity trend of the full-length riboswitch is ADP = ATP > NAD+ = AP2-ribavirin > NADH. Although our results support riboswitch sensing of a single NAD+ at concentrations significantly below the intracellular levels of this cofactor, our findings do not support the level of specificity expected for a riboswitch that exclusively senses NAD+. Gene regulatory implications and future challenges are discussed.

A UHM-ULM interface with unusual structural features contributes to U2AF2 and SF3B1 association for pre-mRNA splicing.

During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process.

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation.

Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1-exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1-sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.

Representative cancer-associated U2AF2 mutations alter RNA interactions and splicing.

High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors.

RNA-protein interfaces control key replication events during the HIV-1 life cycle. The viral trans-activator of transcription (Tat) protein uses an archetypal arginine-rich motif (ARM) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific ß2-ß3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the ß2-ß3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding ß2-ß3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 and 3.0 ± 0.3 nm). Our efforts yielded low-molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6 to 22 µm). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction.

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.

An Allosteric Binding Site on Sortilin Regulates the Trafficking of VLDL, PCSK9, and LDLR in Hepatocytes.

ApoB lipoproteins (apo B-Lp) are produced in hepatocytes, and their secretion requires the cargo receptor sortilin. We examined the secretion of apo B-Lp-containing very low-density lipoprotein (VLDL), an LDL progenitor. Sortilin also regulates the trafficking of the subtilase PCSK9, which when secreted binds the LDL receptor (LDLR), resulting in its endocytosis and destruction at the lysosome. We show that the site 2 binding compound (cpd984) has multiple effects in hepatocytes, including (1) enhanced Apo-Lp secretion, (2) increased cellular PCSK9 retention, and (3) augmented levels of LDLR at the plasma membrane. We postulate that cpd984 enhances apo B-Lp secretion in part through binding the lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is present at higher levels on circulating VLDL form fed rats relative to after fasting. We attribute the enhanced VLDL secretion to its increased binding affinity for sortilin site 1 induced by cpd984 binding site 2. This hinders PCSK9 binding and secretion, which would subsequently prevent its binding to LDLR leading to its degradation. This suggests that site 2 is an allosteric regulator of site 1 binding. This effect is not limited to VLDL, as cpd984 augments binding of the neuropeptide neurotensin (NT) to sortilin site 1. Molecular dynamics simulations demonstrate that the C-terminus of NT (Ct-NT) stably binds site 1 through an electrostatic interaction. This was bolstered by the ability of Ct-NT to disrupt lower-affinity interactions between sortilin and the site 1 ligand PIP3. Together, these data show that binding cargo at sortilin site 1 is allosterically regulated through site 2 binding, with important ramifications for cellular lipid homeostasis involving proteins such as PCSK9 and LDLR.

The pre-mRNA splicing and transcription factor Tat-SF1 is a functional partner of the spliceosome SF3b1 subunit via a U2AF homology motif interface.

The transcription elongation and pre-mRNA splicing factor Tat-SF1 associates with the U2 small nuclear ribonucleoprotein (snRNP) of the spliceosome. However, the direct binding partner and underlying interactions mediating the Tat-SF1-U2 snRNP association remain unknown. Here, we identified SF3b1 as a Tat-SF1-interacting subunit of the U2 snRNP. Our 1.1 Å resolution crystal structure revealed that Tat-SF1 contains a U2AF homology motif (UHM) protein-protein interaction module. We demonstrated that Tat-SF1 preferentially and directly binds the SF3b1 subunit compared with other U2AF ligand motif (ULM)-containing splicing factors, and further established that SF3b1 association depends on the integrity of the Tat-SF1 UHM. We next compared the Tat-SF1-binding affinities for each of the five known SF3b1 ULMs and then determined the structures of representative high- and low-affinity SF3b1 ULM complexes with the Tat-SF1 UHM at 1.9 Å and 2.1 Å resolutions, respectively. These structures revealed a canonical UHM-ULM interface, comprising a Tat-SF1 binding pocket for a ULM tryptophan (SF3b1 Trp338) and electrostatic interactions with a basic ULM tail. Importantly, we found that SF3b1 regulates Tat-SF1 levels and that these two factors influence expression of overlapping representative transcripts, consistent with a functional partnership of Tat-SF1 and SF3b1. Altogether, these results define a new molecular interface of the Tat-SF1-U2 snRNP complex for gene regulation.

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers.

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures.

Somatic mutations of pre-mRNA splicing factors recur among patients with myelodysplastic syndrome (MDS) and related malignancies. Although these MDS-relevant mutations alter the splicing of a subset of transcripts, the mechanisms by which these single amino acid substitutions change gene expression remain controversial. New structures of spliceosome intermediates and associated protein complexes shed light on the molecular interactions mediated by 'hotspots' of the SF3B1 and U2AF1 pre-mRNA splicing factors. The frequently mutated SF3B1 residues contact the pre-mRNA splice site. Based on structural homology with other spliceosome subunits, and recent findings of altered RNA binding by mutant U2AF1 proteins, we suggest that affected U2AF1 residues also contact pre-mRNA. Altered pre-mRNA recognition emerges as a molecular theme among MDS-relevant mutations of pre-mRNA splicing factors.

Metalloriboswitches: RNA-based inorganic ion sensors that regulate genes.

Divalent ions fulfill essential cellular roles and are required for virulence by certain bacteria. Free intracellular Mg2+ can approach 5 mm, but at this level Mn2+, Ni2+, or Co2+ can be growth-inhibitory, and magnesium fluoride is toxic. To maintain ion homeostasis, many bacteria have evolved ion sensors embedded in the 5'-leader sequences of mRNAs encoding ion uptake or efflux channels. Here, we review current insights into these "metalloriboswitches," emphasizing ion-specific binding by structured RNA aptamers and associated conformational changes in downstream signal sequences. This riboswitch-effector interplay produces a layer of gene regulatory feedback that has elicited interest as an antibacterial target.

Cancer-Associated Mutations Mapped on High-Resolution Structures of the U2AF2 RNA Recognition Motifs.

Acquired point mutations of pre-mRNA splicing factors recur among cancers, leukemias, and related neoplasms. Several studies have established that somatic mutations of a U2AF1 subunit, which normally recognizes 3' splice site junctions, recur among myelodysplastic syndromes. The U2AF2 splicing factor recognizes polypyrimidine signals that precede most 3' splice sites as a heterodimer with U2AF1. In contrast with those of the well-studied U2AF1 subunit, descriptions of cancer-relevant U2AF2 mutations and their structural relationships are lacking. Here, we survey databases of cancer-associated mutations and identify recurring missense mutations in the U2AF2 gene. We determine ultra-high-resolution structures of the U2AF2 RNA recognition motifs (RRM1 and RRM2) at 1.1 Å resolution and map the structural locations of the mutated U2AF2 residues. Comparison with prior, lower-resolution structures of the tandem U2AF2 RRMs in the RNA-bound and apo states reveals clusters of cancer-associated mutations at the U2AF2 RRM-RNA or apo-RRM1-RRM2 interfaces. Although the role of U2AF2 mutations in malignant transformation remains uncertain, our results show that cancer-associated mutations correlate with functionally important surfaces of the U2AF2 splicing factor.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/ß by Akts inhibits their activity; nonphosphorylated GSK3ß inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/ß and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Structure-guided U2AF65 variant improves recognition and splicing of a defective pre-mRNA.

Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF(65) normally recognizes a Py tract consensus sequence preceding the major class of 3' splice sites. We found that neurofibromatosis- or retinitis pigmentosa-causing mutations in the 5' regions of Py tracts severely reduce U2AF(65) affinity. Conversely, we identified a preferred binding site of U2AF(65) for purine substitutions in the 3' regions of Py tracts. Based on a comparison of new U2AF(65) structures bound to either A- or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF(65) would specify U over other nucleotides. We found that the crystal structure of the U2AF(65)-D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF(65) affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF(65) binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF(65) variant is capable of manipulating gene expression in eukaryotic cells.

Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding.

Sortilin is a multi-ligand sorting receptor that interacts with B100-containing VLDL and LDL as well as other ligands including neurotensin (NT). The current study investigates the hypothesis that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated downstream of insulin action can directly bind to sortilin. NT binds to sortilin at a well characterized site via its carboxy terminus (C-term). Using a crystal structure of human sortilin (hsortilin), PIP3 is predicted to bind at this C-term site. Binding of PIP3 to hsortilin is demonstrated using surface plasmon resonance (SPR) flowing PIP3 nanodiscs over immobilized hsortilin. Studies were performed using SPR where dibutanoyl PIP3 is shown to compete with NT for sortilin binding. Rat VLDL and LDL were evaluated for PIP3 content immunologically using monoclonal antibodies directed against PIP3. Rat plasma VLDL contained three times more immunoreactive PIP3 than LDL per µg of protein. Because VLDL contains additional ligands that bind sortilin, to distinguish specific PIP3 binding, we used PIP3 liposomes. Liposome floatation assays were used to demonstrate PIP3 liposome binding to sortilin. Using SPR and immobilized hsortilin, the C-term NT tetrapeptide (P-Y-I-L) is shown to bind to hsortilin. A compound (cpd984) was identified with strong theoretical binding to the site on sortilin involved in NT N-terminal binding. When cpd984 is co-incubated with the tetrapeptide, the affinity of binding to sortilin is increased. Similarly, the affinity of PIP3 liposome binding increased in the presence of cpd984. Overall, results demonstrate that sortilin is a PIP3 binding protein with binding likely to occur at the C-term NT binding site. The presence of multiple ligands on B100-containing lipoproteins, VLDL and LDL, raises the interesting possibility for increased interaction with sortilin based on the presence of PIP3.