Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5′-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5′-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3′-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.

A host-specific function is required for ligation of a wide variety of ribozyme-processed RNAs

Hepatitis δ virus (HDV) replicates its circular RNA genome via a rolling circle mechanism. During this process, cis-acting ribozymes cleave adjacent upstream sequences and thereby resolve replication intermediates to unit-length RNA. The subsequent ligation of these 5′OH and 2′,3′-cyclic phosphate termini to form circular RNA is an essential step in the life cycle of the virus. Here we present evidence for the involvement of a host activity in the ligation of HDV RNA. We used both HDV and hammerhead ribozymes to generate a panel of HDV and non-HDV RNA substrates that bear 5′ hydroxyl and 2′,3′- cyclic phosphate termini. We found that ligation of these substrates occurred in host cells, but not in vitro or in Escherichia coli. The host-specific ligation activity was capable of joining RNA in both bimolecular and intramolecular reactions and functioned in a sequence-independent manner. We conclude that mammalian cells contain a default pathway that efficiently circularizes ribozyme processed RNAs. This pathway could be exploited in the delivery of stable antisense and decoy RNA to the nucleus.

Identification and modulation of a naturally processed T cell epitope from the diabetes-associated autoantigen human glutamic acid decarboxylase 65 (hGAD65)

T cell recognition of autoantigens is critical to progressive immune-mediated destruction of islet cells, which leads to autoimmune diabetes. We identified a naturally presented autoantigen from the human islet antigen glutamic acid decarboxylase, 65-kDa isoform (GAD65), by using a combination of chromatography and mass spectrometry of peptides bound by the type I diabetes (insulin-dependent diabetes mellitus, IDDM)-associated HLA-DR4 molecule. Peptides encompassing this epitope-stimulated GAD65-specific T cells from diabetic patients and a DR4-positive individual at high risk for developing IDDM. T cell responses were antagonized by altered peptide ligands containing single amino acid modifications. This direct identification and manipulation of GAD65 epitope recognition provides an approach toward dissection of the complex CD4+ T cell response in IDDM.

Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.

High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation.

We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze-drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.

Expression of a single-chain HLA class I molecule in a human cell line: presentation of exogenous peptide and processed antigen to cytotoxic T lymphocytes.

We have synthesized a recombinant gene encoding a single-chain HLA-A2/beta 2-microglobulin (beta 2m) molecule by linking beta 2m through its carboxyl terminus via a short peptide spacer to HLA-A2 (A*0201). This gene has been expressed in the beta 2m-deficient colorectal tumor cell line DLD-1. Transfection of this cell with the single-chain construct was associated with conformationally correct cell surface expression of a class I molecule of appropriate molecular mass. The single-chain HLA class I molecule presented either exogenously added peptide or (after interferon-gamma treatment) endogenously processed antigen to an influenza A matrix-specific, HLA-A2-restricted cytotoxic T-lymphocyte line. The need for interferon gamma for the processing and presentation of endogenous antigen suggests that DLD-1 has an antigen-processing defect that can be up-regulated, a feature that may be found in other carcinomas. Our data indicate that single-chain HLA class I constructs can form functional class I molecules capable of presenting endogenously processed antigens. Such molecules should be of use for functional studies, as well as providing potential anticancer immunotherapeutic agents or vaccines.

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. The nature of the influences exerted by the sequences flanking CTL epitopes on these processing events remains controversial. Here we show that each epitope within an artificial polyepitope protein containing nine minimal CD8+ CTL epitopes in sequence was processed and presented to appropriate CTL clones. Natural flanking sequences were thus not required to direct class I proteolytic processing. In addition, unnatural flanking sequences containing other CTL epitopes did not interfere with processing. The ability of every CTL epitope to be effectively processed from a protein containing only CTL epitopes is likely to find application in the construction of recombinant polyepitope CTL vaccines.