A Monoclonal Antibody Against 2,2,7-trimethylguanosine That Reacts With Intact, Class U, Small Nuclear Ribonucleoproteins as Well as With 7-methylguanosine-capped RNAs

Eur J Biochem. 1987 Oct 15;168(2):461-7. doi: 10.1111/j.1432-1033.1987.tb13439.x.

Abstract

A hybridoma secreting a monoclonal antibody (H-20) that recognizes the 2,2,7-trimethylguanosine(m3G)-containing cap structure of U snRNAs was derived from a mouse which was immunized with a m3G-containing human serum albumin conjugate. The antibody specifically reacts with intact small nuclear ribonucleoprotein particles, U snRNPs, and allows the snRNPs U1 to U6 to be isolated in one step from nuclear extracts of eucaryotic cells by affinity chromatography on a preparative scale. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess of the cross-reactive nucleoside 7-methylguanosine (m7G), which guarantees maintenance of their native structure. The 20 affinity column also allows the snRNPs U1, U2 and U5 to be separated from U4/U6 RNPs by sequential elution of the particles with m7G under differential salt concentrations. As determined by competitive radioimmunoassay and protein-A--Sepharose immunoprecipitation, mAb H-20 crossreacts with intact m7G cap structures. In particular we could show that non-denatured m7G-capped SP6/beta-globin RNA was precipitated efficiently by the antibody while GpppG-capped or non-capped RNAs did not react. Thus the monoclonal antibody H-20 should have a wide application, not only for studying the molecular biology and immunology of the U snRNPs from diverse organisms, but also for the characterization and isolation of m7G-capped transcripts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • Binding, Competitive
  • Chromatography, Affinity
  • Guanosine / analogs & derivatives*
  • Guanosine / immunology
  • Guanosine / metabolism
  • HeLa Cells
  • Humans
  • Immunoassay
  • Mice
  • Mice, Inbred BALB C
  • RNA Caps / genetics
  • RNA Caps / immunology*
  • Rabbits
  • Radioimmunoassay
  • Ribonucleoproteins / immunology*
  • Ribonucleoproteins, Small Nuclear
  • Transcription, Genetic

Substances

  • Antibodies, Monoclonal
  • RNA Caps
  • Ribonucleoproteins
  • Ribonucleoproteins, Small Nuclear
  • Guanosine
  • N(2),N(2),7-trimethylguanosine