Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs

  1. Joshua E. Babiarz1,3,
  2. J. Graham Ruby2,3,
  3. Yangming Wang1,
  4. David P. Bartel2,5, and
  5. Robert Blelloch1,4
  1. 1 Institute for Regeneration Medicine, Center for Reproductive Sciences, and Department of Urology, University of California at San Francisco, San Francisco, California 94143, USA;
  2. 2 Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA
  1. 3 These authors contributed equally to this work.

Abstract

Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Δ/Δ mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Δ/Δ mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Δ/Δ, and dicer1Δ/Δ mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.

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