In Vitro Splicing of Pre-mRNA in HeLa Extracts

  • Johannes Schenkel
  • Frank Jung
  • Apostolia Guialis
  • Angela Krämer
Part of the Springer Lab Manual book series (SLM)

Abstract

The protein-coding sequences in most messenger RNA precursors (pre-mRNAs) of higher eukaryotes are interrupted by intervening sequences (or introns), which are removed from the primary transcript in a process termed splicing (for review see Moore et al. 1993; Krämer 1996). The reaction occurs in the nucleus either co- or post-transcriptionally. The coding sequences (or exons) are joined and the mature mRNA is exported to the cytoplasm where it is translated into protein. Intron removal is an essential process because introns usually have no or only little protein-coding potential and the failure to remove them from the mRNA can introduce in-frame stop codons resulting in premature termination of translation and thus nonfunctional proteins. Many pre-mRNAs contain more than one intron, which provides a means for the regulation of gene expression by alternative splicing events. The selection of alternative 5’ and 3’ splice sites or the inclusion/exclusion of entire exons can result in an on/off switch of a particular gene or in the translation of different protein isoforms (often with specific functions) from one and the same pre-mRNA, thus augmenting the coding repertoire of the genome.

Keywords

Glycerol Phenol Urea EDTA Chloroform 

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References

  1. Beggs JD (1995) Yeast splicing factors and genetic strategies for their analysis. In: Lamond AI (ed) Pre-mRNA processing. RG Landes, Austin, pp 79 - 95Google Scholar
  2. Chabot B (1994) Synthesis and purification of RNA substrates. In: Higgins SJ, Hames BD (eds) RNA processing: a practical approach, vol I. IRL, Oxford, pp 1 - 29Google Scholar
  3. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11: 1475 - 1489PubMedCrossRefGoogle Scholar
  4. Dignam JD (1990) Preparation of extracts from higher eukaryotes. Methods Enzymol 182: 194 - 203PubMedCrossRefGoogle Scholar
  5. Eperon IC, Krainer AR (1994) Splicing of mRNA precursors in mammalian cells. In: Higgins SJ, Hames BD (eds) RNA processing: a practical approach, vol I. IRL, Oxford, pp 57 - 101Google Scholar
  6. Frendewey D, Krämer A, Keller W (1987) Different small nuclear ribonucleoprotein particles are involved in different steps of splicing complex formation. CSH Symp. Quant. Biol. 52, 287-298Google Scholar
  7. Grabowski PJ (1994) Characterization of RNA. In: Higgins SJ, Hames BD (eds) RNA processing: a practical approach, vol I. IRL, Oxford, pp 31 - 55Google Scholar
  8. Green MR, Maniatis T, Melton DA (1983) Human 13-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32: 681 - 694PubMedCrossRefGoogle Scholar
  9. Konarska MM (1989) Analysis of splicing complexes and small nuclear ribonucleoprotein particles by native gel electrophoresis. Methods Enzymol 180: 442 - 453PubMedCrossRefGoogle Scholar
  10. Konarska MM, Sharp PA (1987) Interactions between small nuclear ribonucleoprotein particles in the formation of spliceosomes. Cell 49: 763 - 774PubMedCrossRefGoogle Scholar
  11. Krainer AR, Conway GC, Kozak D (1990) Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev 4: 1158 - 1171PubMedCrossRefGoogle Scholar
  12. Krämer A (1996) The structure and function of proteins involved in nuclear pre-mRNA splicing. Annu Rev Biochem 65: 367 - 409PubMedCrossRefGoogle Scholar
  13. Krämer A, Keller W (1990) Preparation and fractionation of mammalian extracts active in pre-mRNA splicing. Methods Enzymol 181: 3 - 19PubMedCrossRefGoogle Scholar
  14. Lamond AI, Konarska MM, Sharp PA (1987) A mutational analysis of spliceosome assembly: evidence for splice site collaboration during spliceosome formation. Genes Dev 1: 532 - 543PubMedCrossRefGoogle Scholar
  15. Lee KA, Green MR (1990) Small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. Methods Enzymol 181: 20 - 30PubMedCrossRefGoogle Scholar
  16. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12: 7035 - 7056PubMedCrossRefGoogle Scholar
  17. Moore MJ, Query CC, Sharp PA (1993) Splicing of precursors to mRNA by the spliceosome. In: Gesteland RF, Atkins JF (eds) The RNA world. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 303 - 357Google Scholar
  18. Newman A (1994) Analysis of pre-mRNA splicing in yeast. In: Higgins SJ, Hames BD (eds) RNA processing: a practical approach, vol I. IRL, Oxford, pp 57 - 101Google Scholar
  19. Ruskin B, Green MR (1985) An RNA processing activity that debranches RNA lariats. Science 229: 135 - 140PubMedCrossRefGoogle Scholar
  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  21. Yisraeli JK, Melton DA (1989) Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases. Methods Enzymol 180: 42 - 50PubMedCrossRefGoogle Scholar
  22. Zahler AM, Lane WS, Stolk JA, Roth MB (1992) SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev 6: 837 - 847PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Johannes Schenkel
    • 1
  • Frank Jung
    • 2
  • Apostolia Guialis
    • 3
  • Angela Krämer
    • 4
  1. 1.II. Institute of PhysiologyUniversity of HeidelbergHeidelbergGermany
  2. 2.Institute of Molecular GeneticsUniversity of HeidelbergHeidelbergGermany
  3. 3.Institute of Biological Research and BiotechnologyThe National Hellenic Research FoundationAthensGreece
  4. 4.Department of Cell BiologyUniversity of GenevaGeneva 4Switzerland

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