RNA editing.

Since its discovery, RNA editing in kinetoplastid mitochondria has proven a fascinating topic of study, and the last one and a half years have witnessed enormous advances in our understanding of this unprecedented form of RNA processing. The information flow in this RNA editing, once considered a candidate for defying the central dogma, is now known to conform to the DNA-to-RNA-to-protein paradigm, with the novel feature that the sequence of an edited region is not actually present in any DNA segment, but instead derives by a novel micro-interdigitating of information encoded in multiple DNA regions.     

RNA processing.

Significant progress has been made over the last year in our understanding of the roles that RNA-binding proteins play in pre-mRNA splicing, the components of the spliceosome and how these components relate to the mechanism of splicing. Of particular importance has been the sequence analysis of the first mammalian splicing factors and structural determination of an RNA-binding domain.     

Evidence that immune RNA is messenger RNA.

Exposure of rabbit spleen cell cultures to i-RNA isolated from T2 phage-exposed rabbit peritoneal exudate cells induces the synthesis of antigen and allotype specific 19S proteins even in the presence of actinomycin D. The same i-RNA directs the synthesis of proteins with comparable properties in cell-free extracts prepared from mouse L cells, indicating that i-RNA functions as mRNA and contains the information required to code for the synthesis of IgM antibodies.     

Interaction of RNA with transformed glucocorticoid receptor. II. Identification of the RNA as transfer RNA

An endogenous RNA (designated as PIVB RNA), which is capable of associating with the 4 S glucocorticoid receptor (GR) to generate the 6 S form, has been purified from AtT-20 cells (Ali, M., and Vedeckis, W. V. (1987) J. Biol. Chem., 262, 6771-6777). We describe here the physiochemical properties, GR-RNA interaction characteristics, and the chemical identification of PIVB RNA. 32P-Labeled PIVB RNA was similar to transfer RNA (tRNA) in its sedimentation coefficient (4 S) on sucrose gradients, electrophoretic mobility on formaldehyde-agarose gels, and receptor binding characteristics. The amino acid acceptor activity of PIVB RNA displayed a typical tRNA-dependent saturation curve and was 2-3-fold higher than that of homologous rabbit liver tRNA when tested using rabbit liver aminoacyl-tRNA synthetase. The purified [3H] aminoacyl-PIVB complex was also capable of binding to the 4 S GR to generate the 6 S form. The analysis of PIVB RNA on an acrylamide-urea sequencing gel revealed that it contained a major tRNA of 76 nucleotides and other minor tRNA species of 74 and 78 nucleotides. The identity of the tRNA present in the PIVB RNA was indirectly deduced by analyzing the 3H-amino acids, liberated from the [3H]aminoacyl-PIVB RNA (tRNA) complex, and subsequent analysis on an amino acid analyzer. PIVB RNA mainly contained tRNAArg (51.8%), tRNALys (17.1%), and tRNAHis (9.2%) which together accounted for 78% of the total PIVB tRNA. The remaining 22% of tRNA was contributed by threonine, valine, aspartic acid, alanine, and phenylalanine tRNAs. The GR displayed no species specificity, and tRNA samples from mouse, cow, rabbit, yeast, and Escherichia coli can bind to the mouse 4 S GR to generate the 6 S form. However, PIVB RNA did not affect the sedimentation profiles of albumin, chymotrypsinogen, and histone, indicating that PIVB RNA does not bind to all proteins. Thus, there may exist some specificity both at the level of protein (GR) and the selection of RNA (tRNA). The GR binding to PIVB RNA occurred at low (nM) receptor concentration, and PIVB RNA showed limited capacity to shift 4 S GR to the 6 S form. 22.4 X 10(-11) mol of PIVB RNA can completely shift 4.8 X 10(-13) mol of 4 S GR to 6 S. That is, PIVB RNA has to be in a 500-600-fold excess over the amounts of GR to observe a stable 6 S GR X RNA complex on sucrose gradients. These results conclusively demonstrate that the transformed GR specifically binds to endogenous tRNA.     

Small RNA database.

The small RNA database is a compilation of all the small size RNA sequences available to date, including nuclear, nucleolar, cytoplasmic and mitochondria small RNAs from eukaryotic organisms and small RNAs from prokaryotic cells as well as viruses. Currently, approximately 600 small RNA sequences are in our database. It also gives the sources of individual RNAs and their GenBank accession numbers. The small RNA database can be accessed through the WWW (World Wide Web). Our WWW URL address is: http://mbcr.bcm.tmc. edu/smallRNA/smallrna.html . The new small RNA sequences published since our last compilation are listed in this paper (Table 1).     

Anti-DNA.RNA sera

Summary The specificity of anti-nucleic acid antisera can immunocytochemically be evaluated with test systems which apply various nucleic acids immobilized to inert matrices. When using polyrA.polyrU as a model compoud for dsRNA, it is important to prevent the formation of the triple stranded form polyrA.(polyrU)₂.Anti-dsRNA antibodies which, when tested with the correct test system, proved to be present in an earlier described anti-DNA.RNA serum, could be removed by adsorption. By cytofluorometric comparison of the immunofluorescence signals obtained with the anti-dsRNA containing serum and the absorbed serum, it could be shown that the anti-dsRNA antibodies do not contribute to the specific signals measured after in situ hybridization.Repetitive incubations with the anti-DNA.RNA serum led to a considerable increase in immunofluorescence signal.This work was subsidized in part by Het Praeventiefonds, The Hague, The Netherlands     

Nuclear RNA processing.

Continued progress has been made during the past year in understanding the basic biochemical mechanisms involved in nuclear RNA processing. Of particular importance have been the advances made in purifying and characterizing protein factors involved in splicing and polyadenylation of pre-mRNAs.     

Small RNA database.

The small RNA database is a compilation of all the small size RNA sequences available to date from prokaryotic and eukaryotic organisms. About 500 small RNA sequences are in our database currently. The sources of individual RNAs and their GenBank accession numbers are also included. The small RNA database can be accessed through the World Wide Web(WWW). Our WWW URL is http://mbcr.bcm.tmc.edu/smallRNA/smallrna. html. The new small RNA sequences published since our last compilation are listed in this paper.     

How RNA folds.

We describe the RNA folding problem and contrast it with the much more difficult protein folding problem. RNA has four similar monomer units, whereas proteins have 20 very different residues. The folding of RNA is hierarchical in that secondary structure is much more stable than tertiary folding. In RNA the two levels of folding (secondary and tertiary) can be experimentally separated by the presence or absence of Mg2+. Secondary structure can be predicted successfully from experimental thermodynamic data on secondary structure elements: helices, loops, and bulges. Tertiary interactions can then be added without much distortion of the secondary structure. These observations suggest a folding algorithm to predict the structure of an RNA from its sequence. However, to solve the RNA folding problem one needs thermodynamic data on tertiary structure interactions, and identification and characterization of metal-ion binding sites. These data, together with force versus extension measurements on single RNA molecules, should provide the information necessary to test and refine the proposed algorithm.