RNA splicing and debranching viewed through analysis of RNA lariats
- PMID:22065066
- DOI: 10.1007/s00438-011-0635-y
RNA splicing and debranching viewed through analysis of RNA lariats
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 yeast Saccharomyces 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.
Similar articles
- Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing.Suzuki H, Zuo Y, Wang J, Zhang MQ, Malhotra A, Mayeda A.Suzuki H, et al.Nucleic Acids Res. 2006 May 8;34(8):e63. doi: 10.1093/nar/gkl151.Nucleic Acids Res. 2006.PMID:16682442Free PMC article.
- Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA.Ooi SL, Samarsky DA, Fournier MJ, Boeke JD.Ooi SL, et al.RNA. 1998 Sep;4(9):1096-110. doi: 10.1017/s1355838298980785.RNA. 1998.PMID:9740128Free PMC article.
- Yeast lariat debranching enzyme. Substrate and sequence specificity.Nam K, Hudson RH, Chapman KB, Ganeshan K, Damha MJ, Boeke JD.Nam K, et al.J Biol Chem. 1994 Aug 12;269(32):20613-21.J Biol Chem. 1994.PMID:7519612
- RNA Lariat Debranching Enzyme as a Retroviral and Long-Terminal-Repeat Retrotransposon Host Factor.Menees TM.Menees TM.Annu Rev Virol. 2020 Sep 29;7(1):189-202. doi: 10.1146/annurev-virology-012720-094902.Annu Rev Virol. 2020.PMID:32991267Review.
- A view of pre-mRNA splicing from RNase R resistant RNAs.Suzuki H, Tsukahara T.Suzuki H, et al.Int J Mol Sci. 2014 May 26;15(6):9331-42. doi: 10.3390/ijms15069331.Int J Mol Sci. 2014.PMID:24865493Free PMC article.Review.
Cited by
- Metal content and kinetic properties of yeast RNA lariat debranching enzyme Dbr1.Clark NE, Katolik A, Taggart AJ, Buerer L, Holloway SP, Miller N, Phillips JD, Farrell CP, Damha MJ, Fairbrother WG.Clark NE, et al.RNA. 2022 Jul;28(7):927-936. doi: 10.1261/rna.079159.122. Epub 2022 Apr 22.RNA. 2022.PMID:35459748Free PMC article.
- A functional link between lariat debranching enzyme and the intron-binding complex is defective in non-photosensitive trichothiodystrophy.Townley BA, Buerer L, Tsao N, Bacolla A, Mansoori F, Rusanov T, Clark N, Goodarzi N, Schmidt N, Srivatsan SN, Sun H, Sample RA, Brickner JR, McDonald D, Tsai MS, Walter MJ, Wozniak DF, Holehouse AS, Pena V, Tainer JA, Fairbrother WG, Mosammaparast N.Townley BA, et al.Mol Cell. 2023 Jul 6;83(13):2258-2275.e11. doi: 10.1016/j.molcel.2023.06.011. Epub 2023 Jun 26.Mol Cell. 2023.PMID:37369199Free PMC article.
- Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1.Clark NE, Katolik A, Roberts KM, Taylor AB, Holloway SP, Schuermann JP, Montemayor EJ, Stevens SW, Fitzpatrick PF, Damha MJ, Hart PJ.Clark NE, et al.Proc Natl Acad Sci U S A. 2016 Dec 20;113(51):14727-14732. doi: 10.1073/pnas.1612729114. Epub 2016 Dec 6.Proc Natl Acad Sci U S A. 2016.PMID:27930312Free PMC article.
- LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq.Bitton DA, Rallis C, Jeffares DC, Smith GC, Chen YY, Codlin S, Marguerat S, Bähler J.Bitton DA, et al.Genome Res. 2014 Jul;24(7):1169-79. doi: 10.1101/gr.166819.113. Epub 2014 Apr 7.Genome Res. 2014.PMID:24709818Free PMC article.
- Saccharomyces cerevisiae RNA lariat debranching enzyme, Dbr1p, is required for completion of reverse transcription by the retrovirus-like element Ty1 and cleaves branched Ty1 RNAs.Menees TM.Menees TM.Mol Genet Genomics. 2021 Mar;296(2):409-422. doi: 10.1007/s00438-020-01753-y. Epub 2021 Jan 19.Mol Genet Genomics. 2021.PMID:33464395
References
Publication types
MeSH terms
Substances
Related information
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases