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Related Concept Videos

LTR Retrotransposons03:08

LTR Retrotransposons

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LTR retrotransposons are class I transposable elements with long terminal repeats flanking an internal coding region. These elements are less abundant in mammals compared to other class I transposable elements. About 8 percent of human genomic DNA comprises LTR retrotransposons. Some of the common examples of LTR retrotransposons are Ty elements in yeast and Copia elements in Drosophila.
The internal coding region of LTR retrotransposons and their mechanism of transposition closely resembles a...
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Non-LTR Retrotransposons03:18

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As the name suggests, non-LTR retrotransposons lack the long terminal repeats characteristic of the LTR retrotransposons. Additionally, both LTR and non-LTR retrotransposons use distinct mechanisms of mobilization. Non-LTR retrotransposons are further divided into two classes - Long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), both of which occur abundantly in most mammals, including humans. Some of the active non-LTR retrotransposons in humans are L1...
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Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the...
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse

Heather E Upton1,2, Lucas Ferguson1,3, Morayma M Temoche-Diaz4

  • 1Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

Proceedings of the National Academy of Sciences of the United States of America
|October 15, 2021
PubMed
Summary

Researchers harnessed reverse transcriptases (RTs) from retroelements and introns to develop Ordered Two-Template Relay (OTTR). This novel method enables efficient, low-bias RNA sequencing, outperforming commercial kits for complex samples.

Keywords:
RNA sequencingmiRNAnon-LTR retroelement reverse transcriptasenoncoding RNAtRNA

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genomics

Background:

  • Non-long terminal repeat (non-LTR) retroelements and group II introns possess reverse transcriptases (RTs) capable of initiating DNA synthesis without extensive primer-template pairing.
  • Recombinant expression challenges have limited the biochemical characterization and biotechnological application of these unique RTs.

Purpose of the Study:

  • To investigate the enzymatic activities of representative non-LTR and group II intron RTs.
  • To develop a streamlined protocol for next-generation sequencing library preparation using these RTs.

Main Methods:

  • Characterization of modified non-LTR RT from *Bombyx mori* and group II intron RT from *Eubacterium rectale*.
  • Development of the Ordered Two-Template Relay (OTTR) protocol by combining template jumping and terminal deoxynucleotidyl transferase activities.
  • Benchmarking OTTR against commercial kits and home-brew protocols using microRNA reference pools.

Main Results:

  • The non-LTR RT exhibited robust template jumping and terminal deoxynucleotidyl transferase activity.
  • OTTR enabled streamlined, single-reaction fusion of sequencing adaptors to cDNA.
  • OTTR demonstrated superior performance in library production efficiency and low bias compared to commercial kits.
  • Application of OTTR to extracellular vesicle RNA revealed diverse noncoding RNAs (ncRNAs) and ncRNA fragments.

Conclusions:

  • The characterized RTs possess unique enzymatic activities exploitable for molecular biology applications.
  • OTTR is an efficient, low-bias method for end-to-end RNA sequencing, suitable for complex ncRNA samples.
  • OTTR facilitates automation-friendly RNA inventorying from various biological sources.