Protein-Encoding RNA to RNA Information Transfer in Mammalian Cells: RNA-dependent mRNA Amplification. Identification of Chimeric RNA Intermediates and Putative RNA End Products

Abstract

Our initial unidirectional understanding of the flow of protein-encoding genetic information, DNA to RNA to protein, a process defined as the "Central Dogma of Molecular Biology" and usually depicted as a downward arrow, was eventually amended to account for the "vertical" information back-flow from RNA to DNA, reverse transcription, and for its "horizontal" side-flow from RNA to RNA, RNA-dependent RNA synthesis, RdRs. These processes, both potentially leading to protein production, were assumed to be strictly virus-specific. However, whereas this presumption might be true for the former, it became apparent that the cellular enzymatic machinery for the later, a conventional RNA-dependent RNA polymerase activity, RdRp, is ubiquitously present and RdRs regularly occurs in eukaryotes. The strongest evidence for the occurrence and functionality of RdRp activity in mammalian cells comes from viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the host. Eventually, it became clear that RdRp activity, apparently in a non-conventional form, is constitutively present in most, if not in all, mammalian cells. Because such activity was shown to produce short transcripts, because of its apparent involvement in RNA interference phenomena, and because double-stranded RNA is known to trigger cellular responses leading to its degradation, it was generally assumed that its role in mammalian cells is restricted to a regulatory function. However, at the same time, an enzymatic activity capable of generating complete antisense RNA complements of mRNAs was discovered in mammalian cells undergoing terminal differentiation. Moreover, observations of widespread synthesis of antisense RNA initiating at the 3'poly(A) of mRNAs in human cells suggested an extensive cellular utilization of mammalian RdRp. These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA. Here, we report the in vivo detection in cells undergoing terminal erythroid differentiation of the major model-predicted identifiers of such a process, a chimeric double-stranded/pinhead-structured intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner. We also report the identification of the putative chimeric RNA end product of mRNA amplification. It is heavily modified, uniformly truncated, yet retains the intact coding region, and terminates with the OH group at both ends; its massive cellular amount is unprecedented for a conventional mRNA transcription product and it translates into polypeptides indistinguishable from the translation product of conventional mRNA. Moreover, we describe the occurrence of the second Tier of mammalian RNA-dependent mRNA amplification, a physiologically occurring, RdRp-driven intracellular PCR process, "iPCR", and report the detection of its distinct RNA end products. Whether mammalian mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation in cells programmed for only a short survival span or a general physiological phenomenon was answered in the companion article Volloch et al. Ann Integr Mol Med. 2019;1(1):1004. by the detection of major identifiers of this process for mRNA encoding α1, β1, and γ1 chains of laminin, a major extracellular matrix protein abundantly produced throughout the tissue and organ development and homeostasis and an exceptionally revealing indicator of the range and scope of this phenomenon. The results obtained introduce the occurrence of RNA-dependent mRNA amplification as a new mode of genomic protein-encoding information transfer in mammalian cells and establish it as a general physiological phenomenon.