Substrate recognition by RNA 5-methyluridine methyltransferases and pseudouridine synthases: a structural perspective.

Through all kingdoms of life RNAs are modified as, or after, they are synthesized. The types and sites of modification are often conserved, implying conservation of function. Many of these modifications are located at key functional regions of the ribosome or other RNAs. The enzymes that carry out the modifications exhibit unique or limited multisite specificity. Substrate recognition by RNA-modifying enzymes is more challenging than for DNA-modifying enzymes, because the complex tertiary folds of RNA often prevent direct read out of the target sequence. In some cases, there is no consensus target sequence for the multiple substrates of an RNA-modifying enzyme beyond the target base itself. A challenge for biologists is to determine how anRNA-modifying enzyme selects the correct segment from a large folded or partially folded RNA and then recognizes a single target base in the segment without relying exclusively on sequence. Several crystal structures of RNA-modifying enzymes with and without substrate bound have recently been determined. These structures are beginning to “decode” the basis for selectivity of enzymes for the many different states and folds of RNA. We focus on two of the classes of RNA-modifying enzymes for which mechanisms of substrate recognition are understood to some degree. These are the 5-methyluridine (m5U)2methyltransferases (MTases) and the pseudouridine synthases ( synthases). These families catalyze some of the most common modifications of the more than 100 different types of posttranscriptional modification seen in RNA. Each modification is evoked at a particular stage in RNA folding suggesting that the determinants of selectivity often involve unique three-dimensional folded structures. In some cases a fragment of the physiological substrate, for example an isolated RNA stem-loop, can act as substrate; in others only folded or partially folded ribosome or full tRNA suffices. Both of these classes of RNA-modifying enzymes use covalent addition of anucleophilic sidechain (CysorAsp) to initiate thechemicalmodificationof uridine. Syntheticminimal substrates containing 5-fluorouridine at the target site, when reacted with the proteins, yield covalent protein-substrate analog complexes, providing an effective means to crystallize enzyme-RNA complexes.