ABSTRACT.Our initial understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the "central dogma of molecular biology", was eventually amended to account for the information back-flow from RNA to DNA (reverse transcription), and for its "side-flow" from RNA to RNA (RNA-dependent RNA synthesis, RdRs). These processes, both potentially leading to protein production, were described only in viral systems, and although putative RNA-dependent RNA polymerase (RdRp) was shown to be present, and RdRs to occur, in most, if not all, mammalian cells, its function was presumed to be restricted to regulatory. Here we report the occurrence of protein-encoding RNA to RNA information transfer in mammalian cells. We describe below the detection, by next generation sequencing (NGS), of a chimeric doublestranded/pinhead intermediate containing both sense and antisense globin RNA strands covalently joined in a predicted and uniquely defined manner, whose cleavage at the pinhead would result in the generation of an endproduct containing the intact coding region of the original mRNA. We also describe the identification of the putative end product of RNA-dependent globin mRNA amplification. It is heavily modified, uniformly truncated at both untranslated regions (UTRs), terminates with the OH group at the 5' end, consistent with a cleavagegenerated 5' terminus, and its massive cellular amount is unprecedented for a conventional mRNA transcription product. It also translates in a cell-free system into polypeptides indistinguishable from the translation product of conventional globin mRNA. The physiological significance of the mammalian mRNA amplification, which might operate during terminal differentiation and in the production of highly abundant rapidly generated proteins such as some collagens or other components of extracellular matrix, with every genome-originated mRNA molecule acting as a potential template, as well as possible implications, including physiologically occurring intracellular PCR process, iPCR, are discussed in the paper.All rights reserved. No reuse allowed without permission.

The aim of the experiments described in this paper was to test for the presence of antisense globin RNA in mouse erythroid tissues and, if found, to characterize these molecules. The present study made use of a multistep procedure in which a molecular tag is attached to cellular RNA by ligation with a defined ribooligonucleotide. The act of ligation preserves the termini of RNA molecules, which become the junctions between cellular RNAs and the ligated ribooligonucleotide. It also unambiguously preserves the identity of cellular RNA as a sense or antisense molecule through all subsequent manipulations. Using this approach, we identified and characterized antisense 8-globin RNA in erythroid spleen cells and reticulocytes from anemic mice. We show in this paper that the antisense globin RNA is fully complementary to spliced globin mRNA, indicative of the template/transcript relationship. It terminates at the 5' end with a uridylate stretch, reflecting the presence of poly(A) at the 3' end of the sense globin mRNA. With respect to the structure of their 3' termini, antisense globin RNA can be divided into three categories: full-size molecules corresponding precisely to globin mRNA, truncated molecules lacking predominantly 14 3'-terminal nucleotides, and extended antisense RNA containing 17 additional 3'-terminal nucleotides. The full-size antisense globin RNA contains two 14-nt-long complementary sequences within its 3'-terminal segment corresponding to the 5'-untranslated region of globin mRNA. This, together with the nature of the predominant truncation, suggests a mechanism by which antisense RNA might give rise to new sensestrand globin mRNA.Earlier, we reported the detection and partial characterization of antisense globin RNA in murine erythroleukemia (MEL) cells (1). This RNA appeared to be a complement of the corresponding sense globin mRNA. Indeed, antisense globin RNA had an electrophoretic mobility similar to that of globin mRNA, and it hybridized with probes corresponding to the 5'-and 3'-terminal segments of globin mRNA, as well as with probes corresponding to the entire globin mRNA. Experiments with whole cells (1) and with cytoplasts obtained by enucleation of MEL cells (2) indicated that antisense globin RNA, as well as its sense counterpart, is synthesized in the cytoplasm and suggested that it may serve as an intermediate in cytoplasmic globin mRNA synthesis by RNA-dependent RNA polymerase, an activity that has been detected in erythroleukemia cells (1), as well as in normal reticulocytes (3). If such a process indeed occurred physiologically, one would expect (i) to find the antisense globin RNA molecule not only in MEL cells but also in animal erythroid tissues, and (ii) that these molecules are precise complements of their sense counterparts. Accordingly, the present study was undertaken to test for the occurrence of antisense globin RNA in erythroid cells from mouse tissues and to analyze its primary structure, The publication costs of this article were defrayed in part by page charge...

Harmful conditions including heat shock, oxidative stress, UV, and so forth cause programmed cell death, whose triggering requires activation of the Jun N-terminal kinase, JNK. High levels of Hsp72, a heat-inducible member of Hsp70 family, protect cells against a variety of stresses by a mechanism that is unclear at present. Here we report that elevated levels of Hsp72 inhibit a signal transduction pathway leading to programmed cell death by preventing stress-induced activation of JNK. Stress-induced activation of another stress-kinase, p38 (HOG1), is also blocked when the level of Hsp72 is increased. Similarly, addition of a purified recombinant Hsp72 to a crude cell lysate reduced p38 kinase activation, while depletion of the whole family of Hsp70 proteins with a monoclonal antibody enhanced such activation. In addition, we have found that accumulation of abnormal proteins in cells upon incubation with amino acid analogs causes activation of JNK and p38 kinases, which can be prevented by overproduction of Hsp72. Taken together, these data suggest that, in regulation of JNK and p38 kinases, Hsp70 serves as a "sensor" of the build-up of abnormal proteins after heat shock and other stresses. The inhibitory effect of an increased level of Hsp70 on JNK appears to be a major contributor to acquired thermotolerance in mammalian cells.

The present review analyzes the results of recent clinical trials of β secretase inhibition in sporadic Alzheimer’s disease (SAD), considers the striking dichotomy between successes in tests of β-site Amyloid Precursor Protein-Cleaving Enzyme (BACE) inhibitors in healthy subjects and familial Alzheimer’s disease (FAD) models versus persistent failures of clinical trials and interprets it as a confirmation of key predictions for a mechanism of amyloid precursor protein (APP)-independent, β secretase inhibition-resistant production of β amyloid in SAD, previously proposed by us. In light of this concept, FAD and SAD should be regarded as distinctly different diseases as far as β-amyloid generation mechanisms are concerned, and whereas β secretase inhibition would be neither applicable nor effective in the treatment of SAD, the β-site APP-Cleaving Enzyme (BACE) inhibitor(s) deemed failed in SAD trials could be perfectly suitable for the treatment of FAD. Moreover, targeting the aspects of Alzheimer’s disease (AD) other than cleavages of the APP by β and α secretases should have analogous impacts in both FAD and SAD.