Radioisotope uptake as a measure of synthesis of messenger RNA

Exogenously supplied radioactive uracil (or guanine) enters the intracellular pools of RNA precursors in Escherichia coli only as nucleotides are removed from these pools by net synthesis of RNA. Consequently uptake of uracil over a short period does not measure the sum of the synthesis of all forms of RNA, unstable and stable, as is often supposed. Uptake of uracil during changing conditions of growth may be influenced by changes in types of RNA's being made; under such conditions that no stable RNA is being made, the synthesis of unstable forms may be greatly underestimated.     

Translational potentiation of messenger RNA with secondary structure in Xenopus

Differential translation of messenger RNA (mRNA) with stable secondary structure in the 5' untranslated leader may contribute to the dramatic changes in protein synthetic patterns that occur during oogenesis and early development. Plasmids that contained the bacterial gene chloramphenicol acetyltransferase and which encoded mRNA with (hpCAT) or without (CAT) a stable hairpin secondary structure in the 5' noncoding region were transcribed in vitro, and the resulting mRNAs were injected into Xenopus oocytes, eggs, and early embryos. During early oogenesis, hpCAT mRNA was translated at less than 3 percent of the efficiency of CAT mRNA. The relative translational potential of hpCAT reached 100 percent in the newly fertilized egg and returned to approximately 3 percent after the midblastula transition.     

Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA

The human ferritin H chain messenger RNA contains a specific iron-responsive element (IRE) in its 5' untranslated region, which mediates regulation by iron of ferritin translation. An RNA gel retardation assay was used to demonstrate the affinity of a specific cytosolic binding protein for the IRE. A single-base deletion in the IRE eliminated both the interaction of the cytoplasmic protein with the IRE and translational regulation. Thus, the regulatory potential of the IRE correlates with its capacity to specifically interact with proteins. Titration curves of binding activity after treatment of cells with an iron chelator suggest that the factor acts as a repressor of ferritin translation.     

In vitro synthesis of an infectious mutant RNA with a normal RNA replicase

When purified Qbeta-RNA replicase is presented alternately with two genetically different Qbeta-RNA molecules, the RNA synthesized is identical to the initiating template. The results establish that the RNA is the instructive agent in the replicative process and hence that it satisfies the operational definition of a self-duplicating entity. The data also eliminate alternative explanations which do not involve self-propagation of the input RNA. An opportunity is now provided for studying the genetics and evolution of a self-duplicating nucleic acid molecule under conditions permitting detailed control of environmental parameters and chemical components.     

The intervening sequence RNA of Tetrahymena is an enzyme

A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro. The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1. The reaction mechanism resembles that of rRNA precursor self-splicing. With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid. Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template. At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.     

Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease

The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.     

Cellular localization of somatomedin (insulin-like growth factor) messenger RNA in the human fetus

The somatomedins or insulin-like growth factors (IGFs) are synthesized in many organs and tissues, but the specific cells that synthesize them in vivo have not been defined. By in situ hybridization histochemistry, IGF I (somatomedin C) and IGF II messenger RNAs were localized to connective tissues or cells of mesenchymal origin in 14 organs and tissues from human fetuses. IGF messenger RNAs were localized to perisinusoidal cells of liver, to perichondrium of cartilage, to sclera of eye, and to connective tissue layers, sheaths, septa, and capsules of each organ and tissue. All of the hybridizing regions are comprised predominantly of fibroblasts or other cells of mesenchymal origin. Because these cells are widely distributed and anatomically integrated into tissues and organs, they are ideally located for production of IGFs, which may exert paracrine effects on nearby target cells.     

RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme

A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C5 to generate chain lengths of 10 to 11 nucleotides, with longer products being generated at greatly reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template. The demonstration that an RNA enzyme can catalyze net elongation of an RNA primer supports theories of prebiotic RNA self-replication.     

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.