Non-templated addition and template switching by Moloney murine leukemia virus (MMLV)-based reverse transcriptases co-occur and compete with each other

J Biol Chem. 2019 Nov 29;294(48):18220-18231. doi: 10.1074/jbc.RA119.010676. Epub 2019 Oct 22.

Abstract

Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3'-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3'-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5'-end (capped versus noncapped) in these biases. Our findings confirmed that the presence of a 5'-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5'-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.

Keywords: DNA polymerase; RNA; deep sequencing; enzyme mechanism; molecular biology; non-templated addition; reverse transcriptase; reverse transcription; single-cell RNA sequencing; template switching.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Complementary / biosynthesis
  • DNA, Complementary / chemistry*
  • DNA, Viral / biosynthesis
  • DNA, Viral / chemistry*
  • Moloney murine leukemia virus / enzymology*
  • Nucleotide Motifs
  • RNA, Viral / chemistry*
  • RNA, Viral / metabolism
  • RNA-Directed DNA Polymerase / chemistry*
  • RNA-Directed DNA Polymerase / metabolism
  • Reverse Transcription*

Substances

  • DNA, Complementary
  • DNA, Viral
  • RNA, Viral
  • RNA-Directed DNA Polymerase