RNA-dependent Amplification of Mammalian mRNA Encoding Extracellullar Matrix Proteins: Identification of Chimeric RNA Intermediates for α1, β1, and γ1 Chains of Laminin

Abstract

De novo production of RNA on RNA template, a process known as RNA-dependent RNA synthesis, RdRs, and the enzymatic activity conducting it, RNA-dependent RNA polymerase, RdRp, were initially considered to be exclusively virus-specific. Eventually, however, the occurrence of RdRs and the ubiquitous presence of conventional RdRp were demonstrated in numerous eukaryotic organisms. The evidence that the enzymatic machinery capable of RdRs is present in mammalian cells was derived from studies of viruses, such as hepatitis delta virus, HDV, that do not encode RdRp yet undergo a robust RNA replication once inside the mammalian host; thus firmly establishing its occurrence and functionality. Moreover, 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, given 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. Recent detection of the major model-predicted identifiers, chimeric RNA intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner, as well as the identification of a putative chimeric RNA end product of this process, validated the proposed model. The results corroborating mammalian RNA-dependent mRNA amplification were obtained in vivo with cells undergoing terminal erythroid differentiation and programmed for only a short survival span. This raises a question of whether mammalian RNA-dependent mRNA amplification is a specialized occurrence limited to extreme circumstances of terminal differentiation or a general physiological phenomenon. The present study addresses this question by testing for the occurrence of RNA-dependent amplification of mRNA encoding extracellular matrix proteins abundantly produced throughout the tissue and organ development and homeostasis, an exceptionally revealing indicator of the range and scope of this phenomenon. We report here the detection of major identifiers of RNA-dependent amplification of mRNA encoding α1, β1, and γ1 chains of laminin in mouse tissues producing large quantities of extracellular matrix proteins. The results obtained warrant reinterpretation of the mechanisms involved in ubiquitous and abundant production and deposition of extracellular matrix proteins, confir...

Precursor-Independent Overproduction of Beta-Amyloid in AD: Mitochondrial Dysfunction as Possible Initiator of Asymmetric RNA-Dependent βAPP mRNA Amplification. An Engine that Drives Alzheimer’s Disease

“…These results led to the development of a model of RdRp-facilitated and antisense RNA-mediated amplification of mammalian mRNA [47–49]. Recent detection of the major model-predicted identifiers, chimeric RNA intermediates containing both sense and antisense RNA strands covalently joined in a rigorously predicted and uniquely defined manner [49,50], as well as the identification of a putative chimeric RNA end product of this process [49], validated the proposed model.…”

“…The process of RNA-dependent amplification of mammalian mRNA is diagrammed in Figure 1 below and can be briefly summarized as follows. The amplification process occurs in the cytoplasm and starts with transcription of the antisense complement from a conventional spliced mRNA template, initiating at the 3’poly(A), possibly with the help of a uridilated protein, as seen in viral RdRs [51] (Figure 1, Step 1), and terminating at the 3’end with the “C”, a transcript of the capG of mRNA [48–50]. Generation of a complete antisense transcript requires the presence of an eligible RNA template and a compatible polymerase activity.…”

“…Subsequent studies identified full-length antisense transcripts of globin mRNA in erythroid cells [47–49]. It could be argued that the component responsible for the production of long antisense transcripts in mammalian cells is a processivity conferring co-factor of RdRp activity that is induced under special circumstances when overproduction of specific proteins is required [48–50]. The notion of a processivity co-factor is strongly supported by studies of HDV replication in “normal” (i.e.…”

“…Approximately 30 nucleotide-long 3’-terminal segment of the antisense strand of βAPP mRNA constitutes the TCE. Its counterpart, the ICE, is separated by nearly 2000 nucleotides, yet these elements are topologically compatible and the folding of the antisense molecule results in a self-priming configuration capable to accommodate an additional 3’C if the capG of βAPP mRNA is transcribed [48–50] (Figure 3a). The TCE serves as a primer and is extended; thus, generating the sense strand as shown in the Figure 3b.…”

“…Previously, we demonstrated the occurrence of RNA-dependent amplification of mRNA encoding all three chains of laminin in mouse tissue producing very large quantities of this protein [50]. It was suggested [48–50] that the initial ER stress resulting from increased transcription and subsequent translation of conventional mRNA encoding a secreted protein could be one of potentially multiple cellular events which may trigger mRNA amplification.…”