Transcription fidelity: New paradigms in epigenetic inheritance, genome instability and disease

Abstract

RNA transcription errors are transient, yet frequent, events that do have consequences for the cell. However, until recently we lacked the tools to empirically measure and study these errors. Advances in RNA library preparation and next generation sequencing (NGS) have allowed the spectrum of transcription errors to be empirically measured over the entire transcriptome and in nascent transcripts. Combining these powerful methods with forward and reverse genetic strategies has refined our understanding of transcription factors known to enhance RNA accuracy and will enable the discovery of new candidates. Furthermore, these approaches will shed additional light on the complex interplay between transcription fidelity and other DNA transactions, such as replication and repair, and explore a role for transcription errors in cellular evolution and disease.

Keywords: Backtracked RNA polymerase; DNA break repair; DNA resection; DksA; Epimutation; Genetic noise; Genomic integrity; GreA; GreB; Non-genetic phenotypic heterogeneity; TFIIS; Transcription error; Transcription factor; Transcription fidelity.

Fig. 1

Accurate RNA Consensus sequencing (ARC-seq). (1) Barcoded RNA adaptors are ligated to a pool of fragmented RNA molecules to uniquely identify individual RNA fragments. Ligated RNAs are then circularized and subjected to rolling-circle reverse transcription generating a cDNA containing tandem multiple copies of each RNA molecule (each copy is denoted by green, red and blue lines in the cDNA). (2) AscI primers are annealed to the cDNA multimer that is restricted into monomers that are copies of the original RNA fragment. (3) Each monomer is tagged with a unique index to identify the different monomer copies and then amplified and Illumina sequenced. Red circles denote the epimutation event; black and green circles are monomer-specific reverse transcription errors; blue circles are amplification/NGS errors. (4) By aligning the cDNA tags and RNA barcodes a consensus sequence is generated from the cDNA copies and the original epimutation is revealed [8]. Whole-transcriptome data is obtained using a primer against the RNA adaptor during rolling-circle reverse transcription; specific locus data is obtained using a transcript-specific primer at this step. Rep-seq and CirSeq have been recently reviewed [33].

Fig. 2

Positive feedback and all-or-none phenomena. (A) Left panel: The lac operon comprises an autocatalytic positive feedback loop (the presence of LacY permease will produce more permease) allowing a heritable epigenetic switch at a maintenance concentration of inducer. LacY is presented in green because the lacA gene in our system is replaced by gfp, and the lac operon is now lacZYA::gfp, so when permease is made GFP will also be made and the cell will fluoresce green. Stochastic events that lead to a transient depletion of repressor (in red) within a cell will result in a burst of LacY permease expression and the presence of permease will activate the positive feedback loop, so that the new induced state will be heritably maintained, mimicking mutation [29,33]. Right panel: E. coli microcolony growing in a microfluidics chamber. Lac operon Off cells were seeded and grown in minimal media plus maintenance concentration of inducer. A stochastic phenotypic switch from Lac OFF to ON occurred in a single cell and the phenotype was heritably maintained forming an ON lineage of cells. The ON lineage was torn apart due to the growth dynamics of the microcolony. The brightfield image viewed in the blue channel was merged with the GFP fluorescence image viewed in the yellow channel. (B) Left panel: A bistable RB-EF2 switch underlies the restriction point, the critical event when a mammalian cell commits to proliferation and becomes independent of growth stimulation [92]. In OFF, quiescent, cells EF2 (in green) is repressed by RB (in red). With sufficient growth stimulation by Myc, Rb repression is removed via CycD/Cdk_4,6 activation; Myc also induces EF2 which activates CycE/Cdk₂ to further block Rb. Since EF2 activates itself, two positive feedback loops are involved to shut OFF Rb and turn ON EF2 resulting in a pathway that converts graded serum inputs into an all-or-none-response. Right panel: Cellular heterogeneity in a tissue. The variation in blue represents underlying sub-threshold expression levels of a gene network in individual cells; full-blown yellow represents the activation of a network and subsequent heritable epigenetic phenotypic switch in a cell lineage. The cellular pattern is based on an M.C. Escher Alhambra tessellation sketch.

Fig. 3

Transient and permanent phenotypic consequences of an RNA transcription error. Green circle is RNAP; black lines are DNA; blue line is nascent RNA transcript; red explosion is an RNA transcription error.