Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.

Cap-binding proteins have been routinely isolated using m7GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m7GTP. Here, we report the synthesis of new affinity resins, m7GpCH2pp- and m7GpCH2ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m7GpppA-Sepharose, bind recombinant mouse eIF4E²8⁻²¹7 specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m7GpCH2pp- and m7GpCH2ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m7GpCH2ppA-Sepharose. Our data prove the applicability of these novel resins, especially m7GpCH2ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.

The integrator complex recognizes a new double mark on the RNA polymerase II carboxyl-terminal domain.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) comprises multiple tandem repeats of the heptapeptide Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7). This unusual structure serves as a platform for the binding of factors required for expression of pol II-transcribed genes, including the small nuclear RNA (snRNA) gene-specific Integrator complex. The pol II CTD specifically mediates recruitment of Integrator to the promoter of snRNA genes to activate transcription and direct 3' end processing of the transcripts. Phosphorylation of the CTD and a serine in position 7 are necessary for Integrator recruitment. Here, we have further investigated the requirement of the serines in the CTD heptapeptide and their phosphorylation for Integrator binding. We show that both Ser(2) and Ser(7) of the CTD are required and that phosphorylation of these residues is necessary and sufficient for efficient binding. Using synthetic phosphopeptides, we have determined the pattern of the minimal Ser(2)/Ser(7) double phosphorylation mark required for Integrator to interact with the CTD. This novel double phosphorylation mark is a new addition to the functional repertoire of the CTD code and may be a specific signal for snRNA gene expression.