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(m 3 G)‐containing cap structure of U snRNAs was derived from a mouse which was immunized with a m 3 G‐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 (m 7 G), 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 m 7 G under differential salt concentrations. As determined by competitive radioimmunoassay and protein‐A‐Sepharose immunoprecipitation, mAb H‐20 crossreacts with intact m 7 G cap structures. In particular we could show that non‐denatured m 7 G‐capped SP6/β‐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 m 7 G‐capped transcripts.