{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T18:30:26Z","timestamp":1770748226358,"version":"3.49.0"},"reference-count":0,"publisher":"Medtext Publishing Services Pvt Ltd","issue":"1","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["AIMM"],"abstract":"<jats:p>Molecular Biology, a branch of science established to examine the flow of information from \u201cletters\u201d encrypted intoDNA structure to functional proteins, was initially defined by a concept of DNA-to-RNA-to-Protein information movement, a notion termed the Central Dogma of Molecular Biology. RNA-dependent mRNA  amplification,  a  novel  mode  of  eukaryotic  protein-encoding  RNA-to-RNA-to-Protein  genomic  information  transfer,  constitutes  the  extension  of  the  Central Dogma in the context of mammalian cells. It was shown to occur in cellular circumstances requiring exceptionally high levels of production of specific polypeptides,  e.g.  globin  chains  during  erythroid  differentiation  or  defined  secreted  proteins  in  the  context  of  extracellular  matrix  deposition.  Its  potency  is  reflected in the observed cellular levels of the resulting amplified mRNA product: At the peak of the erythroid differentiation, for example, the amount of globin mRNA  produced  in  the  amplification  pathway  is  about  1500-fold  higher  than  the  amount  of  its  conventionally  generated  counterpart  in  the  same  cells.  The  cellular enzymatic machinery at the core of this process, RNA-dependent RNA polymerase activity (RdRp), albeit in a non-conventional form, was shown to be constitutively and ubiquitously present, and RNA-dependent RNA synthesis (RdRs) appeared to regularly occur, in mammalian cells. Under most circumstances, the mammalian RdRp activity produces only short antisense RNA transcripts. Generation of complete antisense RNA transcripts and amplification of mRNA molecules require the activation of inducible components of the mammalian RdRp complex. The mechanism of such activation is not clear. The present article suggests that it is triggered by a variety of cellular stresses and occurs in the context of stress responses in general and within the framework of the integrated stress  response  (ISR)  in  particular.  In  this  process,  various  cellular  stresses  activate,  in  a  stress  type-specific  manner,  defined  members  of  the  mammalian  translation  initiation  factor  2\u03b1,  eIF2\u03b1,  kinase  family:  PKR,  GCN2,  PERK  and  HRI.  Any  of  these  kinases,  in  an  activated  form,  phosphorylates  eIF2\u03b1.  This  results in suppression of global cellular protein synthesis but also in activation of expression of select group of transcription factors including ATF4, ATF5 and CHOP.  These  transcription  factors  either  function  as  inducible  components  of  the  RdRp  complex  or  enable  their  expression.  The  assembly  of  the  competent  RdRp complex activates mammalian RNA-dependent mRNA amplification, which appears to be a two-tier process. Tier One is a \u201cchimeric\u201d pathway, named so because it results in an amplified chimeric mRNA molecule containing a fragment of the antisense RNA strand at its 5\u2019 terminus. Tier Two further amplifies one of the two RNA end products of the chimeric pathway and constitutes the physiologically occurring intracellular polymerase chain reaction, iPCR. Depending on the structure of the initial mRNA amplification progenitor, the chimeric pathway, Tier One, may result in multiple outcomes including chimeric mRNA that produces  either  a  polypeptide  identical  to  the  original,  conventional  mRNA  progenitor-encoded  protein  or  only  its  C-terminal  fragment,  CTF.  The  chimeric  RNA end product of Tier One may also produce a polypeptide that is non-contiguously encoded in the genome, activate translation from an open reading frame, which is \u201csilent\u201d in a conventionally transcribed mRNA, or initiate an abortive translation. In sharp contrast, regardless of the outcome of Tier One, the mRNA end product of Tier Two of mammalian mRNA amplification, the iPCR pathway, always produces a polypeptide identical to a conventional mRNA progenitor-encoded protein. This discordance is referred to as the Two-Tier Paradox and discussed in detail in the present article. On the other hand, both Tiers are similar in  that  they  result  in  heavily  modified  mRNA  molecules  resistant  to  reverse  transcription,   undetectable   by   reverse   transcription-based   methods   of   sequencing  and  therefore  constituting  a  proverbial  \u201cDark  Matter\u201d  mRNA,  despite  being  highly  ubiquitous.  It  appears  that  in  addition  to  their  other  functions,  the  modifications  of  the  amplified  mRNA  render  it  compatible,  unlike the bulk of cellular mRNA, with phosphorylated eIF2\u03b1 in translation, implying that in addition to being extraordinarily abundant due to the method of  its  generation,  amplified  mRNA  is  also  preferentially  translated  under  the  ISR  conditions,  thus  augmenting  the  efficiency  of  the  amplification  process.  The  vital  importance  of  powerful  mechanisms  of  amplification  of  protein-encoding  genomic  information  in  normal  physiology  is  self-evident.  Their  malfunctions or misuse appear to be associated with two types of abnormalities, the  deficiency  of  a  protein  normally  produced  by  these  mechanisms  and  the  mRNA  amplification-mediated  overproduction  of  a  protein  normally  not  generated by such a process. Certain classes of beta-thalassemia exemplify the first type, whereas the second type is represented by overproduction of beta-amyloid in Alzheimer\u2019s disease. Both examples are discussed in detail in the present article, which summarizes and systematizes our current understanding of the field and describes two categories of reporter constructs, one for the chimeric Tier of mRNA amplification, another for the iPCR pathway; both reporter types are essential for elucidating underlying molecular mechanisms. It also suggests, in light of the recently demonstrated feasibility of RNA-based vaccines, that the targeted  intracellular  amplification  of  exogenously  introduced  amplification-eligible  antigen-encoding  mRNAs  via  the  induced  or  naturally  occurring  RNA-dependent mRNA amplification pathway could be of substantial benefit in triggering a fast and potent immune response and instrumental in the development of future vaccines. Similar approaches can also be effective in achieving efficient and sustained expression of exogenous mRNA in mRNA therapeutics.<\/jats:p>","DOI":"10.33597\/aimm.02-1009","type":"journal-article","created":{"date-parts":[[2021,2,25]],"date-time":"2021-02-25T05:01:56Z","timestamp":1614229316000},"page":"0131-0173","source":"Crossref","is-referenced-by-count":9,"title":["News from Mars: Two-Tier Paradox, Intracellular PCR, Chimeric Junction Shift, Dark Matter mRNA and Other Remarkable Features of Mammalian RNA-Dependent mRNA Amplification. Implications for Alzheimer\u2019s Disease, RNA-Based Vaccines and mRNA Therapeutics"],"prefix":"10.33597","volume":"2","author":[{"given":"Vladimir","family":"Volloch","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sophia","family":"Rits-Volloch","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"19244","published-online":{"date-parts":[[2019,3,1]]},"container-title":["Annals of Integrative Molecular Medicine"],"original-title":[],"deposited":{"date-parts":[[2021,2,25]],"date-time":"2021-02-25T05:01:57Z","timestamp":1614229317000},"score":1,"resource":{"primary":{"URL":"http:\/\/www.medtextpublications.com\/open-access\/news-from-mars-two-tier-paradox-intracellular-pcr-chimeric-junction-shift-712.pdf?__cf_chl_jschl_tk__=fb9517f7a2c20e0f819b554f7ecbce9f3284c49f-1614228255-0-AYqiEhEaqhrx0xlbIr1VRBN1HYs7zWwGdWdrhjTT5huf5zRpExCEAr9s0s5jq__qRfb4hAOLIwtqucrc14cp9Oe-fi4FjGyixEhDl1H6s-qMQVKhmjLoYpf6hViIrAygkYiFoJ-QlpZxa6Xs3AXj03SkKHhdjMCPUJQrHivSvZdPLReY3dLl9tkx_NaYr0Ku_l7NHxcQjbjU9YpyShybqJoUZ1sVdc4WoNFvlTsMJdnOAOH0J2igYYIWa_YeDF7r1verK5cIesuwII8aQTwjVkVWG4L1B7-XBakj3hkwB5_VU-fLVtkbrkfWDFJQL4t9v3cSFtLnDaji-xPL1RSnQk29XNy5uMXy5Nwx0Kxz1jvw1ocLnl_2KzQgboKssu2xlreyWjwAHwQXCK82ep40_tARiso-Qy0qFdNLu0uPMc91ieO0QmDmR9xlNJSd9T5LBg"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,3,1]]},"references-count":0,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2019,3,1]]}},"URL":"https:\/\/doi.org\/10.33597\/aimm.02-1009","relation":{},"ISSN":["2690-0319"],"issn-type":[{"value":"2690-0319","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,3,1]]}}}