LTR-Retrotransposon Control by tRNA-Derived Small RNAs.

Transposon reactivation is an inherent danger in cells that lose epigenetic silencing during developmental reprogramming. In the mouse, long terminal repeat (LTR)-retrotransposons, or endogenous retroviruses (ERV), account for most novel insertions and are expressed in the absence of histone H3 lysine 9 trimethylation in preimplantation stem cells. We found abundant 18 nt tRNA-derived small RNA (tRF) in these cells and ubiquitously expressed 22 nt tRFs that include the 3' terminal CCA of mature tRNAs and target the tRNA primer binding site (PBS) essential for ERV reverse transcription. We show that the two most active ERV families, IAP and MusD/ETn, are major targets and are strongly inhibited by tRFs in retrotransposition assays. 22 nt tRFs post-transcriptionally silence coding-competent ERVs, while 18 nt tRFs specifically interfere with reverse transcription and retrotransposon mobility. The PBS offers a unique target to specifically inhibit LTR-retrotransposons, and tRF-targeting is a potentially highly conserved mechanism of small RNA-mediated transposon control.

Conserved chromosomal functions of RNA interference.

RNA interference (RNAi), a cellular process through which small RNAs target and regulate complementary RNA transcripts, has well-characterized roles in post-transcriptional gene regulation and transposon repression. Recent studies have revealed additional conserved roles for RNAi proteins, such as Argonaute and Dicer, in chromosome function. By guiding chromatin modification, RNAi components promote chromosome segregation during both mitosis and meiosis and regulate chromosomal and genomic dosage response. Small RNAs and the RNAi machinery also participate in the resolution of DNA damage. Interestingly, many of these lesser-studied functions seem to be more strongly conserved across eukaryotes than are well-characterized functions such as the processing of microRNAs. These findings have implications for the evolution of RNAi since the last eukaryotic common ancestor, and they provide a more complete view of the functions of RNAi.

A synthetic transcription platform for programmable gene expression in mammalian cells.

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner. We demonstrate the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1α promoter, in multiple cell types. We use these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-γ production. Antibody titers and interferon-γ concentrations significantly correlate with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications.