High-resolution mapping of human RNA polymerase III reveals transcription termination as a rate-limiting step
Mikolajczyk, J.; Kollaran, A.; Weidenhausen, J.; Muller, C. W.; Turowski, T. W.
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Transfer RNA (tRNA) molecules play a central role in the flow of genetic information, translating nucleic acid sequences into the functional protein portfolio of an organism. Despite the high abundance of tRNAs in cells, studying their biology remains challenging due to the repetitive nature of genomic sequences and the numerous modifications of mature molecules. tRNA genes (tDNAs) are transcribed exclusively by RNA polymerase III (RNAPIII), which has been recently linked to rare genetic disorders but also longevity. Here, we present the first mapping of actively transcribing RNAPIII in the human, using K562 cell line and UV-crosslinking followed by stringent purification. Our data reveals high variability in expression across tDNAs, common occurrences of transcriptional read-through, and unique transcription dynamics across transcription units, with the lowest kinetics associated with transcription termination. Further analysis revealed that release of the nascent transcript is a critical step in tRNA transcription. Unexpectedly, shorter terminators promote more efficient termination, which becomes the rate-limiting step for human RNAPIII transcription of highly expressed tDNAs. Our dataset provides insights into actively transcribing RNAPIII dynamics in human cells, allowing for a comprehensive analysis of genomic loci transcribed by RNAPIII. Our high resolution data and resulting kinetic information reveal that human RNAPIII contradicts the paradigm that initial steps of transcription are the main determinants of its output. This is significant as RNAPIII is commonly used to transcribe synthetic RNA constructs, such as short hairpin RNAs or guide RNAs used in gene editing technologies. With our study we offer a functional framework for the analysis of RNAPIII activity, addressing the need for comprehensive understanding of RNAPIII transcription fueled by a growing number of described genetic disorders caused by mutations in this enzyme.
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