Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

Volloch, Vladimir; Schweitzer, Bruce; Rits, Sophia

doi:10.1083/jcb.105.1.137

Cited by 23 publications

(11 citation statements)

References 34 publications

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“…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.…”

Section: Discussionsupporting

confidence: 89%

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

Volloch

Rits

Olsen

2018

Preprint

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The aim of the present study was to test for the occurrence of key elements predicted by the previously postulated mammalian RNA-dependent mRNA amplification model in a tissue producing massive amounts of extracellular matrix proteins. At the core of RNA-dependent mRNA amplification, until now only described in one mammalian system, is the self-priming of an antisense strand and extension of its 3' terminus into a sense-oriented RNA containing the protein-coding information of a conventional mRNA. The resulting product constitutes a new type of biomolecule. It is chimeric in that it contains covalently connected antisense and sense sequences in a hairpin configuration. Cleavage of this chimeric intermediate in the loop region of a hairpin structure releases mRNA which contains an antisense segment in its 5'UTR; depending on the position of self-priming, the chimeric end product may encode the entire protein or its C-terminal fragment. The occurrence of such composite chimeric molecules is unique for this type of mRNA amplification and represents a conclusive "identifier" of this process. We report here the detection, by next generation sequencing, of such chimeric junction sequences for mRNAs molecules encoding a1, b1, and g1 chains of laminin in cells of the extracellular matrix-generating Engelbreth-Holm-Swarm (EHS) mouse tumor, best known for producing extraordinarily large amounts of "Matrigel".

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Section: Discussionsupporting

confidence: 89%

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

Volloch

Rits

Olsen

2018

Preprint

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“…They were enucleated by centrifugation through a Ficoll density gradient in the presence of cytochalasin B (Volloch et al, 1987). They were enucleated by centrifugation through a Ficoll density gradient in the presence of cytochalasin B (Volloch et al, 1987).…”

Section: Nuclear Transfermentioning

confidence: 99%

“…Nuclei from the four tumor cell lines were isolated as karyoplasts, and cells from the two normal fibroblast cell lines were enucleated by Ficoll gradient centrifugation (Volloch et al, 1987). Tumor nuclei were then introduced into the enucleated fibroblasts by chemical fusion (Kishi et al, 2003) or electroporation (Peura and Vajta, 2003).…”

Section: Nuclear Transfer-induced Correction Of Aberrant Igf2 Imprintmentioning

confidence: 99%

“…Cells in which IGF2 imprinting was maintained (HBF1 and MBW2) were used as the acceptor cells. They were enucleated by centrifugation through a Ficoll density gradient in the presence of cytochalasin B (Volloch et al, 1987). After centrifugation, the enucleated HBF1 and MBW2 cells (cytoplasts) were collected between the 15 and 18% Ficoll interfaces.…”

Section: Nuclear Transfermentioning

confidence: 99%

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Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming

Chen

Qiu

et al. 2006

EMBO J

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Loss of genomic imprinting of insulin-like growth factor II (IGF2) is a hallmark of many human neoplasms. We attempted to correct this aberrant epigenotype by transferring nuclei from human tumor cells that showed loss of IGF2 imprinting into enucleated mouse and human fibroblasts that had maintained normal IGF2 imprinting. After nuclear transfer, the abnormal biallelic expression of IGF2 in tumor nuclei transiently converted to normal monoallelic imprinted expression in the reconstructed diploid cells. In tetraploid hybrid cells, however, normal IGF2 imprinting was permanently restored in the tumor genome. Inhibition of the synthesis of putative trans imprinting factors with cycloheximide led to loss of IGF2 imprinting in normal cultured fibroblasts, suggesting that normal cells produce proteins that act in trans to induce or maintain genomic imprinting. These data demonstrate that an abnormal tumor epigenotype can be corrected by in vitro reprogramming, and suggest that loss of imprinting is associated with the loss of activity of non-CTCF trans imprinting factor(s) that are either inactivated or mutated in tumors.

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“…The process of RdRp-facilitated, antisense RNA-mediated amplification of mammalian mRNA has been studied in quite some detail in cells and tissues undergoing erythroid differentiation [1][2][3][4][5][6]38]. If this phenomenon is not limited to specialized circumstances of terminal differentiation, it is conceivable that its principles, deduced in investigations with the erythroid model, [5,6,38] would apply in other physiological situations as well.…”

Section: Introductionmentioning

confidence: 99%

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

Volloch

Rits

Olsen

2019

AIMM

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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...

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Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

Cited by 23 publications

References 34 publications

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

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

Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming

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

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