Heterozygous mutations encoding abnormal forms of the death receptor Fas dominantly interfere with Fas-induced lymphocyte apoptosis in human autoimmune lymphoproliferative syndrome. This effect, rather than depending on ligand-induced receptor oligomerization, was found to stem from ligand- independent interaction of wild-type and mutant Fas receptors through a specific region in the extracellular domain. Preassociated Fas complexes were found in living cells by means of fluorescence resonance energy transfer between variants of green fluorescent protein. These results show that formation of preassociated receptor complexes is necessary for Fas signaling and dominant interference in human disease.

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigenbinding fragments (Fabs) targeting a large functional RNA. We have solved the crystal structure of the first Fab-RNA complex at 1.95 Å. Capability in phasing and crystal contact formation suggests that the Fab provides a potentially valuable crystal chaperone for RNA. The crystal structure reveals that the Fab achieves specific RNA binding on a shallow surface with complementaritydetermining region (CDR) sequence diversity, length variability, and main-chain conformational plasticity. The Fab-RNA interface also differs significantly from Fab-protein interfaces in amino acid composition and light-chain participation. These findings yield valuable insights for engineering of Fabs as RNA-binding modules and facilitate further development of Fabs as possible therapeutic drugs and biochemical tools to explore RNA biology.antigen-binding fragments ͉ x-ray crystallography A ntibodies are integral components of the immune system and represent a rapidly growing sector of the biotechnology industry (1, 2). Clinically, antibodies serve as diagnostic markers for disease antigens and play increasingly important roles as therapeutic agents for a wide range of diseases (3). Antibodies also provide invaluable biomedical research tools, serving to define the components and functions of macromolecular complexes, to establish cellular distributions of proteins, and to facilitate structural analysis as chaperones for crystallization of membrane proteins (4-6). Hybridoma and other technologies have yielded antibodies against a vast array of specific antigens (2). An enormous body of literature documents the molecular details of antibody interactions with a variety of antigens, including proteins (7), polysaccharides (8), and small haptens (9). However, much less information (and, in particular, no structural information) exists for antibody-RNA interactions.The relative absence of antibodies that bind RNA from the immunologic repository is striking, especially considering that recent genome-wide analyses of the metazoan transcriptome have revealed the presence of vast numbers of noncoding RNAs, including silencing RNAs, riboswitches, catalytic RNAs, and a multitude of other functional RNA moleucles (10,11). A large number of these RNAs adopt complex three-dimensional architectures that frequently act in complex with proteins to mediate their biological function (12, 13). Nevertheless, with the exception of a handful of examples, mostly isolated from the sera of autoimmune patients (14-17), we know little about anti-RNA antibodies and their recognition of nucleic acids. This dearth of information reflects our inability to elicit antibodies against RNA by using traditional approaches. RNA appears to lack immunogenic potency...

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756. 5′-phosphorothiolate | RNA catalysis | nucleolytic ribozymes | catalytic mechanism R ibozyme-mediated catalysis is important for both RNA splicing and translation (1), yet its chemical origins are incompletely understood. The nucleolytic ribozymes bring about the site-specific cleavage or ligation of RNA, with an acceleration of a millionfold or greater. The intensively studied protein RNase A catalyses an identical cleavage reaction, and much evidence supports the hypothesis that each of these phosphoryl transfer reactions is subject to general acid-base catalysis. This mechanism requires a general base to deprotonate the attacking nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1).The most common chemical entities implicated in RNA catalysis by the nucleolytic ribozymes are the nucleobases (2). Guanine appears to play a catalytic role in the hairpin, hammerhead, GlmS, and Varkud satellite (VS) ribozymes, adenine in the hairpin, and VS and cytosine in the hepatitis delta virus (HDV) ribozyme. Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5-8).In its simplest active form, the VS ribozyme comprises five helices (II through VI) organized by two three-way junctions, which acts in trans upon a substrate stem loop (helix I) with an internal loop that contains the scissile phosphate (Fig. 1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI. While no crystal structure of the VS ribozyme has yet been solved, a small-angle X-ray scattering-derived model plac...

Forty-two studies (1989-2012) specified the lymphoma subtypes for each diagnosis or indicated a rate at which the methods failed to provide a diagnosis. The median rate at which fine-needle aspiration cytology and core needle biopsies yielded a subtype-specific diagnosis of lymphoma was 74%. Strictly adhering to expert guidelines, which state that follicular lymphoma cannot be graded by these techniques, decreased the diagnostic yield further to 66%. Thus, 25% to 35% of fine-needle aspirates and/or core biopsies of nodes must be followed by an excisional lymph node biopsy to fully classify lymphoma.