Dynamics, Degradation, and Chemical Modification of Non-Coding RNA

Our understanding of the function of RNA has grown significantly since the central dogma of molecular biology described RNA as a rudimentary conveyor of the genetic message. In the genomic era, we are beginning to learn the true depth and breadth of the non-coding (nc)RNA repertoire. A key attribute of RNA molecules is the conformational dynamics that they explore. Here, we have applied studies of structure, dynamics, and metal binding to models of helix 27 from 16S rRNA to elucidate the function of this important component of the ribosome. Our studies reveal the kinetic and thermodynamic framework within which this isolated helix undergoes secondary structure rearrangement. Both NMR and fluorescence techniques demonstrate millisecond exchange between the 885 and 888 conformations, defining an equilibrium constant close to one. Fluorescence studies also show that the antibiotic tetracycline interferes with this conformational exchange. Metal binding studies of helix 27 have confirmed data from ribosomal crystal structures and further correlated local and global metal binding features of this RNA. Another general attribute of RNA is its inherent lability. The great promise of a new class of therapeutics based on small interfering (si)RNA molecules and the RNA interference pathway has forced researchers to overcome the nucleolytic vulnerability of RNA molecules, primarily by introducing chemical modifications. Work in this thesis demonstrates that siRNA degradation in blood serum is asymmetric, where the guide strand is predisposed to efficient degradation due to differential stability of the terminal base pairs. We further show that a simple pattern of chemical modifications greatly stabilizes siRNAs in regions particularly susceptible to nuclease cleavage. We have shown that 21 and 24-nucleotide siRNA-like double-stranded RNAs are specifically protected in cell extract, a result which demonstrates intracellular siRNA stability and may help explain recent results suggesting that, once inside the cell, chemical modifications to siRNAs do not significantly increase the potency of the silencing effect. Taken together, the work presented in this thesis has helped to illuminate the function of important ncRNA molecules, furthering our understanding of how ncRNA contributes to the structural, catalytic, and regulatory landscape that defines the cellular lifecycle.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/58438/1/jhoerter_1.pd

Ligand-engaged TCR is triggered by Lck not associated with CD8 coreceptor

Producción CientíficaThe earliest molecular events in T-cell recognition have not yet been fully described, and the initial T-cell receptor (TCR)-triggering mechanism remains a subject of controversy. Here, using total internal reflection/Forster resonance energy transfer microscopy, we observe a two-stage interaction between TCR, CD8 and major histocompatibility complex (MHC)-peptide. There is an early (within seconds) interaction between CD3ζ and the coreceptor CD8 that is independent of the binding of CD8 to MHC, but that requires CD8 association with Lck. Later (several minutes) CD3ζ–CD8 interactions require CD8–MHC binding. Lck can be found free or bound to the coreceptor. This work indicates that the initial TCR-triggering event is induced by free Lck. The early signalling events that trigger initial T-cell receptor signalling are not clearly defined. Here the authors show that this occurs in two stages, the first between the CD8 coreceptor and CD3 requiring Lck association to CD8, while the second interaction requires binding of major histocompatibility molecules

Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide-MHC

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI.National Institutes of Health (U.S.). Pioneer Awar

siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time

Inhibition of the inositol kinase Itpkb augments calcium signaling in lymphocytes and reveals a novel strategy to treat autoimmune disease

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease

GRB2-mediated recruitment of THEMIS to LAT is essential for thymocyte development.

Thymocyte-expressed molecule involved in selection (THEMIS) is a recently identified regulator of thymocyte positive selection. THEMIS's mechanism of action is unknown, and whether it has a role in TCR-proximal signaling is controversial. In this article, we show that THEMIS and the adapter molecule growth factor receptor-bound protein 2 (GRB2) associate constitutively through binding of a conserved PxRPxK motif within the proline-rich region 1 of THEMIS to the C-terminal SH3-domain of GRB2. This association is indispensable for THEMIS recruitment to the immunological synapse via the transmembrane adapter linker for activation of T cells (LAT) and for THEMIS phosphorylation by Lck and ZAP-70. Two major sites of tyrosine phosphorylation were mapped to a YY-motif close to proline-rich region 1. The YY-motif was crucial for GRB2 binding, suggesting that this region of THEMIS might control local phosphorylation-dependent conformational changes important for THEMIS function. Finally, THEMIS binding to GRB2 was required for thymocyte development. Our data firmly assign THEMIS to the TCR-proximal signaling cascade as a participant in the LAT signalosome and suggest that the THEMIS-GRB2 complex might be involved in shaping the nature of Ras signaling, thereby governing thymic selection

Protein Kinase C η Is Required for T Cell Activation and Homeostatic Proliferation

Producción CientíficaProtein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη-/- T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation. In contrast, only PKCη-deficient T cells showed defective homeostatic proliferation, which requires self-antigen recognition. PKCη was dispensable for thymocyte development; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor development, indicating some redundancy between the PKC isoforms. Deficiency in PKCη or PKCθ had opposing effects on the relative numbers of CD4+ and CD8+ T cells. PKCη-/- mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ-/- mice had a lower ratio. Mice deficient in both isoforms exhibited normal cell ratios. Together, these data suggest that PKCη shares some redundant roles with PKCθ in T cell biology and also performs nonredundant functions that are required for T cell homeostasis and activation

Itpkb is required for T cell development.

<p><i>Itpkb</i>^<i>+/+</i> and <i>Itpkb</i>^<i>fl/fl</i> mice were treated with tamoxifen for 5 days followed by 2 days of rest. Thymocytes and splenocytes from tamoxifen-treated mice were compared to WT and <i>Itpkb</i>^<i>-/-</i> mice via flow cytometry. (A) Gating schemes used for thymocyte analysis with antibodies to CD4, CD8, CD3, and TCRb; numbers in the plots indicate the percentages of each gated population. (B) Total numbers of each thymocyte subset. (C) Gating scheme for analysis of the splenocytes stained with antibodies to CD4, CD8, CD3, and B220; numbers in the plots indicate the percentages of each gated population. (D) Total numbers of the indicated splenocyte populations. DP: double positive. Data from one representative experiment is shown. *, P < 0.05; **, P < 0.01.</p

Ins(1,3,4,5)P4 inhibits Orai1-mediated current.

<p>HEK293-Orai1-Stim1 cells in serum-free media were placed on the QPatch HT recording system. Following cell break-in (1), little current was measured. Orai1 currents were then activated in a time-dependent manner following passive store depletion with an EGTA/BAPTA solution to chelate intracellular Ca^<i>2+</i> (2), and then stabilized in an open state (3). Next, (4), Ins(1,3,4,5)P4 (A), a control Ins(1,4,5,6)P4 (B), or 0.1% DMSO (C) was applied for 15 minutes. At the end of each experiment, 2-APB (5) was applied to block any remaining Orai1 current. In (C), percent inhibition values are expressed as the difference in current amplitude before compound application and following 2-APB application (% inhibition = ((base pA–compound pA) / (base pA– 2-APB pA)) X 100. The numbers in parentheses indicate the number of cells tested for each condition on 2 separate experimental days. Data shown is representative of three independent experiments. *, P < 0.05; **, P < 0.01</p