9.3 Photocontrol of RNA Processing Steven G. Chaulk, Oliver A. Kent, and Andrew M. MacMillan 9.3.1 Introduction In addition to being a carrier of genetic information, RNA is involved in the regulation of both transcription and translation, plays a key role in the splicing of pre-mRNA in eukaryotes, and is responsible for catalysis of amide bond for- mation at the heart of the ribosome [1, 2]. The study of many RNA systems is complicated by the dynamic nature of RNA secondary and tertiary structures, the transient nature of RNA–RNA or RNA–protein complexes, and in some cases the chemical reactivity of the RNA itself. One useful approach to the study of discrete RNA (and DNA) structures has been to limit available conformations through site-specific intra-strand crosslinking [3–7]. A complementary approach which allows the isolation of specific RNA structures or complexes involves the transient blocking or “caging” of RNA functional groups involved in the transi- tion between two different states. The caging of cofactors or reactive substrates with photolabile groups has proven useful in a wide variety of investigations ranging from mechanistic enzymology to cell biology [8–10]. Caging of specific functionalities within proteins has been used to control reactivity in these sys- tems, e.g. Noren et al. caged a specific serine residue in Thermococcus litoralis Vent polymerase for studies on the mechanism of protein splicing [11]. In the context of an RNA molecule, a caging approach might be used to block either chemical reactivity of the RNA or formation of secondary or tertiary struc- ture. The caged RNA system can be studied both before and after photolysis, thus permitting characterization of the two states and the transition between them. The first application of the caging approach to studies of RNA structure and function has involved blocking the chemical reactivity associated with an RNA functionality, the 2'-hydroxyl group, since specific RNA 2'-hydroxyls act as nucleophiles in a number of biologically important transesterifications [12–17]. Caging of a single 2'-hydroxyl in a short synthetic oligonucleotide blocks the cleavage reaction catalyzed by the hammerhead ribozyme; cleavage is initiated by photolysis of the ribozyme–substrate complex [18]. This approach has also been extended to the caging of the branch adenosine in a full-length pre-mRNA for studies of RNA processing by the mammalian spliceosome [19]. Caging ef- fectively isolates spliceosome assembly from catalysis of the splicing transesteri- fications permitting a closer examination of the mechanisms of each. 9.3 Photocontrol of RNA Processing 495