RNA-Dependent Synthesis of Mammalian mRNA: Identification of Chimeric Intermediate and Putative End Product

Abstract

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.

RNA-Dependent Amplification of Mammalian mRNA Encoding Extracellular Matrix Components: Identification of Chimeric RNA Intermediates for α1, β1, and γ1 Chains of Laminin

“…Likewise is their origin: these molecules are clearly produced by the extension of the 3'terminus of antisense RNA self-primed in a sequence-specific manner. These results can be interpreted within the framework of the mRNA amplification model developed and postulated in more detailed previous studies (1,2,(11)(12)(13)(14)(15)(16)(17)(18) of terminally differentiating cells where the putative mRNA end product of the amplification process accumulates to extraordinary high levels, constituting nearly a tenth of total ribosomal RNA (2). The findings of the present study are fully consistent with the postulated mechanism of mRNA amplification.…”

“…The findings of the present study are fully consistent with the postulated mechanism of mRNA amplification. Moreover, the possible occurrence of chimeric molecules in cells overproducing a specific protein, such as our study model, was predicted by the postulated amplification mechanism (1,2,18). The results of the present study constitute a confirmation of this prediction and validate the proposed mechanism.…”

“…The vast majority of mammalian mRNA species contains 3'-terminal poly(A) segments. The notion that many, or possibly most, of them could be eligible templates for RdRp was suggested in our previous studies (1,2). Subsequent observations by Kapranov et all showed a widespread synthesis of antisense RNA initiating, apparently indiscriminately, at the 3' poly(A) of mRNA in human cells (19).…”

“…Generation of a complete antisense transcript requires the presence of an eligible RNA template and a compatible polymerase activity. The only major prerequisite for a potential RNA template appears to be the presence of the poly(A) segment at its 3' terminus (1,2,19). The compatible polymerase activity is the RNA-dependent RNA polymerase, RdRp (20,21).…”

“…Subsequent observations by Kapranov et all showed a widespread synthesis of antisense RNA initiating, apparently indiscriminately, at the 3' poly(A) of mRNA in human cells (19). This, apparently undiscerning, RdRp template eligibility of the bulk of mammalian mRNA species raises questions with regard to mechanisms underlying the manifestly stringent specificity of the mRNA amplification process as seen, for example, in erythropoietic differentiation (1,2). The specificity of mRNA amplification appears to be determined at the 3' terminus of an antisense transcript by its ability or inability to support production of a complementary sense strand molecule.…”