Small-scale nuclear extracts for functional assays of gene-expression machineries

Journal of Visualized Experiments

Volumn , Issue 64, 2012, Pages 1-6

  Folco, Eric G ^a   Lei, Haixin ^a   Hsu, Jeanne L ^a   Reed, Robin ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

Author keywords

Cellular biology; Coupled transcription splicing; Genetics; Hela nuclear extract; In vitro gene expression assays; Issue 64; Pre mRNA splicing; RNA polymerase ii transcription; RNAi; Small scale extract

Indexed keywords

EID: 84863799471     PISSN: 1940087X     EISSN: None     Source Type: Journal    
DOI: 10.3791/4140     Document Type: Article

References (9)

1. Krainer, A.R., Maniatis, T., Ruskin, B., & Green, M.R: Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 36, 993-1005 (1984).
2. Ghosh, S. & Garcia-Blanco, M.A: Coupled in vitro synthesis and splicing of RNA polymerase II transcripts. Rna. 6, 1325-1334 (2000).
3. Das, R., et al. Functional coupling of RNAP II transcription to spliceosome assembly. Genes Dev. 20, 1100-1109 (2006).
4. Hicks, M.J., Yang, C.R., Kotlajich, M.V., & Hertel, K.J: Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns. PLoS Biol. 4, e147 (2006).
5. Yu, Y., Das, R., Folco, E.G., & Reed, R: A model in vitro system for co-transcriptional splicing. Nucleic Acids Res. 38, 7570-7578, doi:10.1093/nar/gkq620 [pii] gkq620 (2010).
6. Kotake, Y., et al. Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat. Chem Biol. 3, 570-575, doi:10.1038/nchembio.2007.16 [pii] nchembio.2007.16 (2007).
7. Folco, E.G., Coil, K.E., & Reed, R: The anti-tumor drug E7107 reveals an essential role for SF3b in remodeling U2 snRNP to expose the branch point-binding region. Genes & development. 25, 440-444, doi:10.1101/gad.2009411 (2011).
8. Dignam, J.D., Lebovitz, R.M., & Roeder, R.G: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475-1489 (1983).
9. Lee, K.A., Bindereif, A., & Green, M.R: A small-scale procedure for preparation of nuclear extracts that support efficient transcription and premRNA splicing. Gene Anal. Tech. 5, 22-31 (1988).