The presence of modified nucleotides in cellular RNAs has been known for decades and over 100 distinct RNA modifications have been characterized to date. While the exact role of many of these modifications is still unclear, many are highly conserved across evolution and most contribute to the overall fitness of the organism. In recent years, new methods and bioinformatics approaches have been developed for the dissection of modification pathways and functions. These methods intersect a number of related fields, ranging from RNA processing to comparative genomics and systems biology. In addition, many of the techniques described in this volume have broad applicability, particularly in regards to the isolation, characterization, and reconstitution of ribonucleoprotein complexes, expanding the experimental repertoire available to all RNA researchers.
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Chapter 1: Identifying modifications in RNA by mass spectrometry.
Chapter 2: Identification of modified residues in RNAs by reverse transcription-based methods.
SECTION II. tRNA Modifications
Chapter 3: Detection of enzymatic activity of transfer RNA modification enzymes using radiolabelled tRNA substrates.
Chapter 4: In vitro detection of the enzymatic activity of folate-dependent tRNA(U54, C5)-methyltransferase.
Chapter 5: Probing the intermediacy of covalent RNA.enzyme complexes in RNA modification enzymes.
Chapter 6: Identification and characterization of modification enzymes by biochemical analysis of the proteome.
Chapter 7: Identification of genes encoding tRNA modification enzymes via comparative genomics.
Chapter 8: Identification and characterization of archaeal and fungal tRNA methyltransferases.
Chapter 9: Mass spectrometric identification and characterization of RNA-modifying enzymes.
Chapter 10: Chaplet column chromatography: isolation of a large set of individual RNAs in a single step.
SECTION III. SNO-mediated Modifications
Chapter 11: Biochemical Purification of box H/ACA RNPs involved in pseudouridylation.
Chapter 12: In Vitro Reconstitution and Affinity Purification of Catalytically Active Archaeal Box C/D sRNP Complexes.
Chapter 13: Identifying effects of snoRNA-guided modifications on the synthesis and function of the yeast ribosome.
Chapter 14: The U1 snRNA hairpin II as a RNA affinity tag for selecting snoRNP complexes.
Chapter 15: A Dedicated Computational Approach for the Identification of Archaeal H/ACA sRNAs.
Chapter 16: Reconstitution of archaeal H/ACA sRNPs and test of their activity