The U11-48K Protein Contacts the 5′ Splice Site of U12-Type Introns and the U11-59K Protein▿ †

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5′ splice sites (5′ss). This interaction did not require branch point recognition and was sensitive to 5′ss mutations, suggesting that 48K interacts with the 5′ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5′ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5′ss, as well as functionally important interactions within the U11/U12 di-snRNP

Modulation of Pro-Inflammatory IL-6 Trans-Signaling Axis by Splice Switching Oligonucleotides as a Therapeutic Modality in Inflammation

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a crucial role in maintaining normal homeostatic processes under the pathogenesis of various inflammatory and autoimmune diseases. This context-dependent effect from a cytokine is due to two distinctive forms of signaling: cis-signaling and trans-signaling. IL-6 cis-signaling involves binding IL-6 to the membrane-bound IL-6 receptor and Glycoprotein 130 (GP130) signal-transducing subunit. By contrast, in IL-6 trans-signaling, complexes of IL-6 and the soluble form of the IL-6 receptor (sIL-6R) signal via membrane-bound GP130. Various strategies have been employed in the past decade to target the pro-inflammatory effect of IL-6 in numerous inflammatory disorders. However, their development has been hindered since these approaches generally target global IL-6 signaling, also affecting the anti-inflammatory effects of IL-6 signaling too. Therefore, novel strategies explicitly targeting the pro-inflammatory IL-6 trans-signaling without affecting the IL-6 cis-signaling are required and carry immense therapeutic potential. Here, we have developed a novel approach to specifically decoy IL-6-mediated trans-signaling by modulating alternative splicing in GP130, an IL-6 signal transducer, by employing splice switching oligonucleotides (SSO), to induce the expression of truncated soluble isoforms of the protein GP130. This isoform is devoid of signaling domains but allows for specifically sequestering the IL-6/sIL-6R receptor complex with high affinity in serum and thereby suppressing inflammation. Using the state-of-the-art Pip6a cell-penetrating peptide conjugated to PMO-based SSO targeting GP130 for efficient in vivo delivery, reduced disease phenotypes in two different inflammatory mouse models of systemic and intestinal inflammation were observed. Overall, this novel gene therapy platform holds great potential as a refined therapeutic intervention for chronic inflammatory diseases

Effects of human articular cartilage constituents on simultaneous diffusion of cationic and nonionic contrast agents

Contrast‐enhanced computed tomography is an emerging diagnostic technique for osteoarthritis. However, the effects of increased water content, as well as decreased collagen and proteoglycan concentrations due to cartilage degeneration, on the diffusion of cationic and nonionic agents, are not fully understood. We hypothesize that for a cationic agent, these variations increase the diffusion rate while decreasing partition, whereas, for a nonionic agent, these changes increase both the rate of diffusion and partition. Thus, we examine the diffusion of cationic and nonionic contrast agents within degraded tissue in time‐ and depth‐dependent manners. Osteochondral plugs (N = 15,\ua0d = 8 mm) were extracted from human cadaver knee joints, immersed in a mixture of cationic CA4+ and nonionic gadoteridol contrast agents, and imaged at multiple time‐points, using the dual‐contrast method. Water content, and collagen and proteoglycan concentrations were determined using lyophilization, infrared spectroscopy, and digital densitometry, respectively. Superficial to mid (0%‐60% depth) cartilage CA4+ partitions correlated with water content (R

Cationic contrast agent diffusion differs between cartilage and meniscus

Contrast enhanced computed tomography (CECT) is a non-destructive imaging technique used for the assessment of composition and structure of articular cartilage and meniscus. Due to structural and compositional differences between these tissues, diffusion and distribution of contrast agents may differ in cartilage and meniscus. The aim of this study is to determine the diffusion kinematics of a novel iodine based cationic contrast agent (CA) in cartilage and meniscus. Cylindrical cartilage and meniscus samples (d\ua0=\ua06\ua0mm, h\ua0≈\ua02\ua0mm) were harvested from healthy bovine knee joints (n\ua0=\ua010), immersed in isotonic cationic contrast agent (20\ua0mgI/mL), and imaged using a micro-CT scanner at 26 time points up to 48\ua0h. Subsequently, normalized X-ray attenuation and contrast agent diffusion flux, as well as water, collagen and proteoglycan (PG) contents in the tissues were determined. The contrast agent distributions within cartilage and meniscus were different. In addition, the normalized attenuation and diffusion flux were higher (p\ua

B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

<div><p>Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins.</p></div

Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing

Domain mapping of the MS/MS identified proteins.

<p>Table displays the domain architecture of the 446 proteins in the MS data. The domain architecture is shown as total domains in the 446 proteins and also divided into two parts; AKT motif containing proteins and non-motif containing proteins to identify the domain contents of each part. The table displays the highest number of domains in total, in AKT motif-containing proteins and in non-motif containing proteins.</p

Biological, physical and functional interaction predictions of the AKT-mTORC1/2 substrate (up-regulated) proteins.

<p>An interaction map was predicted to observe putative AKT-mTORC1/2 substrate interactions by using a web-based interface, String 9 tool for up-regulated proteins. Dense clusters representing high interaction between proteins can be seen for up-regulated proteins.</p

The Effect of MK-2206 and PP242 on RXRXXS/T phosphorylations.

<p>(A). Time course of the BCR-induced AKT activation and its effect on substrates in the presence or absence of MK-2206 (2 μM) for 3 h. Namalwa cells were starved and then stimulated with anti-IgM and followed for 1 h (1–60 min). Lysates were resolved on SDS-PAGE and immunoblotted with different phospho-specific antibodies for AKT and AKT-substrates. (B). Phosphorylation status 3 h after treatment of Namalwa cells with either DMSO or different pharmacological inhibitors, MK-2206, (2 μM), Rapamycin (150 nM), PP242 (1 μM), LY294002 (20 μM) or PD98059 (2 μM). The cells were serum-starved then stimulated with anti-IgM (20 μg/ml). Lysates were resolved on 4–12% SDS-PAGE and immunoblotted with different phospho-specific antibodies for pS473-AKT, RXRXXpS/T and pS660-PKC. Anti-pErk 44/42 antibody was used as control.</p