Reverse transcription—a brief introduction

Reverse transcription involves a broad family of enzymes called reverse transcriptases that play a unique role in the flow of genetic information. Since their discovery, researchers have used these enzymes as fundamental tools in a wide range of molecular biology applications.

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Discovery

The original central dogma of molecular biology held that DNA was transcribed to RNA, which in turn was translated into protein. However, this concept was challenged in the 1970s when two scientific teams, one led by Howard Temin at the University of Wisconsin and the other led by David Baltimore at MIT, independently identified new enzymes associated with replication of RNA viruses called retroviruses [1,2]. These enzymes convert the viral RNA genome into a complementary DNA (cDNA) molecule, which then is capable of integrating into the host’s genome. These are RNA-dependent DNA polymerases and are called reverse transcriptase because, in contrast to the DNA-to-RNA flow of the central dogma, they transcribe RNA templates into cDNA molecules (Figure 1). In 1975, Temin and Baltimore received the Nobel Prize in Physiology or Medicine (shared with Renato Dulbecco for related work on tumor-inducing viruses) for their pioneering work in identifying reverse transcriptases [3].

Current understanding of the flow of genetic information includes reverse transcription
Figure 1. Current understanding of the flow of genetic information includes reverse transcription.

Prevalance

Reverse transcriptases have been identified in many organisms, including viruses, bacteria, animals, and plants. In these organisms, the general role of reverse transcriptase is to convert RNA sequences to cDNA sequences that are capable of inserting into different areas of the genome. In this manner, reverse transcription contributes to (Figure 2):

  • Propagation of retroviruses—e.g., human immunodeficiency virus (HIV), Moloney murine leukemia virus (M-MuLV), and avian myeloblastosis virus (AMV) [1,2]
  • Genetic diversity in eukaryotes via mobile transposable elements called retrotransposons [4]
  • Replication of chromosomal ends called telomeres [5,6]
  • Synthesis of extrachromosomal DNA/RNA chimeric elements called multicopy single-stranded DNA (msDNA) in bacteria [7,8]

Figure 2. Roles of reverse transcriptase in biological systems. (A) Viral RNA is reverse-transcribed for integration into the host genome. (B) In retrotransposition, an RNA intermediate is reverse-transcribed to insert DNA copies into other areas of the genome. (C) Telomerase reverse transcriptase (TERT) uses RNA as a template to elongate and maintain eukaryotic chromosome ends. (D) Reverse transcription is an intermediate step in the formation of multicopy single-stranded DNA (msDNA) in bacteria.

Applications

While reverse transcriptases have functional roles in biological systems, they also serve as important tools for studying RNA populations. One of the first molecular biology protocols utilizing reverse transcriptases was for the production of cDNA to build libraries that contained DNA copies of mRNA from cells and tissues [9,10]. These cDNA libraries aid in understanding actively expressed genes and their functions at a specific time point.

Although the creation of cDNA libraries was an important step forward in characterizing expressed genes, challenges remained for the study of low-abundance RNAs. These were subsequently addressed with the development of the polymerase chain reaction (PCR), a technique to amplify small amounts of genetic material. Reverse transcription combined with PCR, or reverse transcription PCR (RT-PCR), allows detection of RNA even at very low levels of gene expression and paves the way for detection of circulating RNA, RNA viruses, and cancerous gene fusions in molecular diagnostics [11-13].

In addition, cDNAs serve as templates in applications such as microarray and RNA sequencing to characterize unknown RNAs in a high-throughput manner [14-17]. (Learn more about reverse transcription applications.)

References

  1. Temin HM, Mizutani S (1970) RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226(5252):1211–1213.
  2. Baltimore D (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226(5252):1209–1211.
  3. Nobel Media AB (2014) The Nobel Prize in Physiology or Medicine 1975. Nobelprize.org 
  4. Dombroski BA, Feng Q, Mathias SL et al. (1994) An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Sacchromyces cerevisiae. Mol Cell Biol 14(7):4485–4492.
  5. Weinrich SL, Pruzan R, Ma L et al. (1997) Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 17(4): 498–502.
  6. Poole JC, Andrews LG, Tollefsbol TO (2001) Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene 269(1-2):1–12.
  7. Inouye S, Herzer PJ, Inouye M (1990) Two independent retrons with highly diverse reverse transcriptases in Myxococcus xanthus. Proc Natl Acad Sci U S A 87(3):942–945.
  8. Lampson BC, Inouye M, Inouye S (2005) Retrons, msDNA, and the bacterial genome. Cytogenet Genome Res 110(1-4):491–499.
  9. Okayama, H, Berg P (1982) High-efficiency cloning of full-length cDNA. Mol Cell Biol 2(2):161–170.
  10. Gubler, U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25(2-3):263–269.
  11. Kawasaki ES, Clark SS, Coyne MY et al. (1988) Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplified in vitro. Proc Natl Acad Sci U S A 85(15):5698–5702.
  12. Mayer G, Müller J, Lünse CE (2011) RNA diagnostics: real-time RT-PCR strategies and promising novel target RNAs. Wiley Interdiscip Rev RNA 2(1):32–41.
  13. Bridge JA (2016) Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors. Cancer Cytopathol. doi: 10.1002/cncy.21762. [Epub ahead of print]
  14. Schena M, Shalon D, Davis RW et al. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270(5235):467–470.
  15. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63.
  16. Mortazavi A, Williams BA, McCue K (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5(7):621–628.
  17. Parkhomchuk D, Borodina T, Amstislavskiy V (2009) Transcriptome analysis by strand-specific sequencing of complementary DNA. Nucleic Acids Res 37(18):e123.

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