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RNA splicing and debranching viewed through analysis of RNA lariats

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Abstract

Intron lariat RNAs, created by pre-mRNA splicing, are sources of information on gene expression and structure. Although produced equivalently to their corresponding mRNAs, the vast majority of intron lariat RNAs are rapidly degraded. However, their levels are enhanced in cells deficient for RNA debranching enzyme, which catalyzes linearization of these RNAs, the rate-limiting step in their degradation. Furthermore, RNA lariats are resistant to degradation by the 3′ exonuclease polynucleotide phosphorylase (PNPase), providing a means to enrich for lariat RNAs. Working with the yeastSaccharomyces cerevisiae as a model organism, our goal was to develop novel combinations of methods to enhance the use of intron lariat RNAs as objects of study. Using RT-PCR assays developed for detecting and quantifying specific lariat RNAs, we demonstrate the resistance of RNA lariats to degradation by PNPase and their sensitivity to cleavage by RNA debranching enzyme. We also employ sequential treatments with these two enzymes to produce characteristic effects on linear and lariat RNAs. We establish the utility of the methods for analyzing RNA debranching enzyme variants and in vitro debranching reactions and discuss several possible applications, including measuring relative rates of transcription and combining these methods with non-gene-specific RNA sequencing as a novel approach for genome annotation. In summary, enzymatic treatments that produce characteristic effects on linear and lariat RNAs, combined with RT-PCR or RNA sequencing, can be powerful tools to advance studies on gene expression, alternative splicing, and any process that depends on the RNA debranching enzyme.

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Acknowledgments

We thank Beate Schwer for the gift of pET16b-DBR1 and Haoping Liu for yeast sigma strain 10560-23C. Support was provided by National Science Foundation, the University of Missouri Research Board, and the University of Missouri-Kansas City School of Biological Sciences.

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  1. Zhi Cheng

    Present address: The Pediatric Surgery Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

Authors and Affiliations

  1. School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO, 64110, USA

    Zhi Cheng & Thomas M. Menees

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  1. Zhi Cheng

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  2. Thomas M. Menees

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Correspondence toThomas M. Menees.

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Communicated by A. Aguilera.

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