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.

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 snR.NPs, 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/β-globin RNA was precipitated efficiently by the antibody while GpppG-capped or non-capped RN As 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. [ABSTRACT FROM AUTHOR]