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This new edition of the highly successful RNA Worlds series focuses on emerging tools for analysis of RNA biology and how these tools are driving our understanding of RNA structure and function. The contributors review new approaches for imaging RNA molecules in cells, techniques for analyzing the behavior of single RNA molecules in vitro and in vivo, transcriptome-wide analyses, and novel methods for determining the structure of RNA and RNP (ribonucleoprotein) complexes. They discuss the insights these provide into the transcription and splicing machinery, ribosomes, reverse transcriptases, and subcellular structures such as stress granules.