Profiling of T helper cell-derived small RNAs reveals unique antisense transcripts and differential association of miRNAs with argonaute proteins 1 and 2

Nucleic Acids Res. 2013 Jan;41(2):1164-77. doi: 10.1093/nar/gks1098. Epub 2012 Nov 26.


RNA interference mediated through antisense transcripts is a fundamentally important mechanism regulating gene expression that remains incompletely understood. Here, we have used next-generation sequencing to determine from mouse CD4+ T cells the functional implications of antisense transcripts binding to argonaute (AGO) proteins that mediate RNA interference and post-transcriptional gene silencing. This effort identified 90 new microRNAs (miRNAs) and six endogenous hairpin RNA-derived small interfering RNAs (siRNAs) mapping to distinct introns. Unexpectedly, 69 miRNAs were expressed as non-canonical isomiRs as the dominant AGO-binding transcript, with extensive 3' terminal nucleotide modifications. Furthermore, differential expression analysis between AGO1- and AGO2-bound miRNAs suggested preferential binding of isomiRs ending with 3' adenine residues to AGO1 and 3' uridine residues to AGO2. Analysis of the putative targets of all miRNAs suggested a striking preference for regulating transcription and transcription factors with additional evidence of a functional division of labor between AGO proteins in this regard. We further provide evidence that multiple mitochondrial genomic loci serve as the source of endogenous cis-natural antisense transcripts. These findings imply diversity in AGO protein function based on differential miRNA binding and indicate that RNA interference-based gene regulation is more complex than previously recognized.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Argonaute Proteins / metabolism*
  • Eukaryotic Initiation Factors / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • MicroRNAs / chemistry
  • MicroRNAs / metabolism*
  • RNA / genetics
  • RNA Precursors / chemistry
  • RNA Precursors / genetics
  • RNA, Antisense / genetics
  • RNA, Antisense / metabolism*
  • RNA, Mitochondrial
  • RNA, Small Interfering / chemistry
  • RNA, Small Interfering / genetics
  • T-Lymphocytes, Helper-Inducer / metabolism*


  • Ago1 protein, mouse
  • Ago2 protein, mouse
  • Argonaute Proteins
  • Eukaryotic Initiation Factors
  • MicroRNAs
  • RNA Precursors
  • RNA, Antisense
  • RNA, Mitochondrial
  • RNA, Small Interfering
  • RNA