hnRNP: Methods for Preparation

Abstract

During transcription in higher eukaryotes, the elongating pre‐messenger RNA is bound by a unique set of abundant nuclear proteins to form a repeating array of 20–22‐nm particles termed 30–40S heterogeneous nuclear ribonucleoprotein (hnRNP) particles. A variety of methods exist for the isolation and purification of hnRNP complexes and for the in vitro assembly of hnRNP assembly intermediates.

Keywords: ribonucleoprotein; hnRNA; hnRNP; pre‐mRNA; RNA‐binding protein

Figure 1.

This coomassie‐stained SDS‐PAGE (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis) gel shows the proteins present in each successive 0.5‐mL fraction of a 15–30% glycerol gradient after centrifugation of a nuclear sonicate containing 40S heterogeneous nuclear ribonucleoprotein (hnRNP) monoparticles. The top of the gradient is to the left. The protein bands labelled near the middle of the gradient are termed the ‘core particle’ proteins. This stoichiometric grouping of major proteins is dissociated from the packaged pre‐messenger RNA (pre‐mRNA) fragment (approximately 700 nucleotides in length) by the SDS present in the electrophoresis sample buffer. The fast‐sedimenting proteins labelled with the asterisk overlap the position of the core particle but are not exclusively enriched in hnRNP complexes.

Figure 2.

As in Figure , panels (a) and (b) show the distribution of the core particle proteins in 15–30% glycerol gradients; however, in these experiments the spin time was reduced to emphasize the presence of large polyparticle complexes in preparations where nuclease activity is inhibited (a). The electron micrograph insets in (a) reveal 20‐nm monoparticles near the top of the gradient, dimers and trimers near the midpoint of the gradient, and polyparticle complexes in fast‐sedimenting fractions near the bottom. The solid tracings reveal the distribution of RNA (OD260 material) in the gradients.

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References

Beyer AL, Christensen ME, Walker BW and LeStourgeon WM (1977) Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell 11: 127–138.

Choi YD and Dreyfuss G (1984) Isolation of the heterogeneous nuclear RNA‐ribonucleoprotein complex (hnRNP): a unique supramolecular assembly. Proceedings of the National Academy of Sciences of the USA 81: 7471–7475.

Huang M, Rech JE, Northington SJ et al. (1994) The C‐protein tetramer binds 230–240 nucleotides of pre‐mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles. Molecular and Cellular Biology 14: 518–533.

LeStourgeon WM, Barnett SF and Northington SJ (1990) Tetramers of the core proteins of 40S nuclear ribonucleoprotein particles assemble to package nascent transcripts into a repeating array of regular particles. In: Strauss PR and Wilson SH (eds) The Eukaryotic Nucleus: Molecular Biochemistry and Macromolecular Assemblies, vol. 2, pp. 477–502. Caldwell, NJ: Telford Press.

McAfee JG, Iyengar S and LeStourgeon WM (1998) Isolation of 40S monomer hnRNP core particles, polyparticle complexes, and the assembly of hnRNP particles in vitro. In: Cells JE (ed.) Cell Biology: a Laboratory Handbook, 2nd edn, vol. 2, pp. 165–173. New York: Academic Press.

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Tan, Jia‐huai, and LeStourgeon, Wallace M(Apr 2001) hnRNP: Methods for Preparation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002603]