New Ideas for in Vivo Detection of RNA

The parallel discovery of the catalytic potential of RNA by Tom Cech and Sydney Altman at the beginning of the 1980s completely changed our view of the capabilities of RNA molecules and won them the Nobel prize in 1989 (Kruger et al., 1982; Guerrier-Takada et al., 1983). Since then, many new roles have been discovered and assigned to RNA, including the ability to form complex multitasking, supramolecular machines, such as ribosomes or spliceosomes, in which RNAs play leading roles. While only about 1.5 % of the human genome codes directly for protein sequences, a large fraction of it is nonetheless transcribed to produce many noncoding RNAs (ncRNA) that carry out important cellular functions (International Human Genome Sequencing Consortium, 2004). The structures and functions of some ncRNA have been extensively characterized, for example, ribosomal RNAs (rRNA), transfer RNAs (tRNA), small nuclear and nucleolar RNAs (snRNA and snoRNA). Moreover, applications of recent technological advances are revealing new classes of ncRNA molecules with novel or yet unknown functions, ranging in size from very small (20-30 nts) to thousands of nucleotides long. Small ncRNA (~20-30nts in length) include microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (ta-siRNA), natural antisense transcript siRNA (nat-siRNA) and small scan RNA (scnRNA) all of which help regulate various stages of gene expression (Choudhuri, 2009). The functions of “ long noncoding RNA” are still not understood, but they seem to be involved in transcriptional and epigenetic gene regulation (Ponting et al., 2009). Many ncRNAs form distinct yet very diverse three-dimensional structures to carry out their functions (Noller, 2005; Staple & Butcher, 2005; Montange & Batey, 2008). These functions include fundamental roles at all stages of gene expression including chromosome remodeling, transcriptional and translation regulation, mRNA processing, transport, and localization, protein translocation and posttranslational modification. RNA molecules use various mechanisms to participate in these processes. For example, miRNA, siRNA and piRNA inhibit gene expression by hybridizing to complementary sequences of target mRNAs in association with RISC ribonucleoprotein complexes and either temporarily block translation or target the mRNA for destruction (Carthew, 2006; Bushati & Cohen, 2007; Boyd, 2008). Riboswitches, usually located in the 5' untranslated region (5’-UTR) of mRNA, also inhibit translation, but do so by forming specific three-dimensional structures, that respond to small molecule metabolites that are products of the pathway to which the gene belongs. Upon binding cognate metabolites, riboswitches undergo conformational changes in their secondary and tertiary structures which usually result in suppression of