In the decades leading up to the turn of the century, as analytical sensitivity improved, many less abundant RNAs that are not involved in protein synthesis were identified. Small antisense RNAs (‘riboregulators’) were shown to control gene expression in bacteria and eukaryotes, and cis-acting RNA structures (‘riboswitches’) to control transcription and translation by allosteric sensing of metabolites and environmental signals in bacteria. Synthetic antisense oligonucleotides began to be used to artificially control gene expression. Overlapping ‘antisense’ transcription and genes within genes were observed in many species, hinting at complex genetic arrangements and regulatory complexity. Differentially transcribed long ‘untranslated’ RNAs were described in the regulatory regions of bithorax and other homeotic loci and heat shock induced genes in Drosophila, and in immunoglobulin class-switching, cancer-associated and parentally imprinted loci in mammals, among others. Xist was identified as a long non-coding RNA that mediates female X-chromosome inactivation in mammals, and analogous RNAs mediating male X-chromosome activation identified in Drosophila, different approaches using a common RNA-based mechanism for sex chromosome dosage compensation. Small RNAs antisense to 3′UTRs were found to control developmental timing in C. elegans. Later 3′UTR sequences were found to be expressed and to transmit genetic information independently of their normally associated protein-coding sequences. Although some speculated that these RNAs may be the first examples of a more extensive RNA regulatory landscape in cell and developmental biology, they were generally regarded as oddities.