Photochemical Cross-Linking of Cap Binding Proteins to Eucaryotic mRNAs: Effect of mRNA 5′ Secondary Structure

Abstract

We used UV light-induced cross-linking to study the interactions of cap binding proteins with the 5' cap structure of eucaryotic mRNAs. Thymidine kinase gene (herpes simplex virus type 1) transcripts prepared in vitro using the SP6 RNA polymerase transcription system were capped and methylated posttranscriptionally with [alpha-32P]GTP and S-adenosyl-L-methionine to yield cap-labeled transcripts. Irradiation of capped transcripts with crude rabbit reticulocyte initiation factors in the presence of ATP-Mg2+ resulted in the cap-specific cross-linking of two polypeptides with molecular masses of 24 and 80 kilodaltons (kDa). The cross-linking characteristics of these polypeptides resemble those of the cap-binding proteins previously detected by a chemical cross-linking assay (N. Sonenberg, D. Guertin, D. Cleveland, and H. Trachsel, Cell 27:563-572, 1981). However, the relative efficiency of the cross-linking of these two polypeptides to the cap structure was different from that in previous studies, and there was no detectable cross-linking of the previously described 50-kDa polypeptide. In addition, we present data indicating that the insertion of secondary structure into the 5' noncoding region of tk mRNA, 6 nucleotides from the cap structure, decreases the cap-specific cross-linking of the 80-kDa but not the 24-kDa polypeptide. In contrast, the insertion of secondary structure 37 nucleotides from the cap structure had no significant effect on the cross-linking of either the 24- or the 80-kDa cap-specific polypeptide. These results demonstrate that the position of mRNA 5'-proximal secondary structure relative to the cap structure can influence the cap-specific interaction between the mRNA and a translation initiation factor.

Inhibition of eukaryotic translation by nucleoside 5'-monophosphate analogs of mRNA 5'-cap: changes in N7 substituent affect analog activity

“…One possibility for the difference in activity of bn7GMP vs m7GMP was that the former analogue affected individual cap binding proteins differently. Since several initiation factors have been shown to interact with a portion of the mRNA at or near the cap (Grifo et al, 1982;Pelletier & Sonenberg, 1985), it is possible that the affected protein(s) for the observed inhibition by bn7GMP is (are) not the same as that for m7GMP. In order to help answer this question, we examined the ability of m7GMP, e7GMP, and bn7GMP to inhibit UV cross-linking of radiolabeled mRNA to cap binding proteins.…”

“…Transcripts were transcriptionally capped with m7G, e7G, and bn7G by addition of the corresponding NpppG dinucleotide in the transcription mixture; uncapped mRNA was synthesized by omitting the cap dinucleotide. The samples (50000 cpm) were digested with T2 RNase for 15 min at 37 °C, and the resulting products were analyzed by two-dimensional chromatography locyte salt wash, followed by UV irradiation to cross-link RNA-protein complexes (Pelletier & Sonenberg, 1985). After…”

“…UV Photo-Cross-Linking of mRNA to Protein. Photocross-linking of radiolabeled mRNA to proteins was performed as described by Pelletier and Sonenberg (1985) with yeast rp51AA2 mRNA (Ruskin et al, 1986). Uncapped 3H-labeled mRNA was synthesized by transcription of rp51AA2 cDNA with SP6 RNA polymerase.…”

“…0006-2960/89/0428-4771S01.50/0 © 1989 American Chemical Society Tahara et al, 1981), and eIF-4F or CBP II (Tahara et al, 1981;Grifo et al, 1982Grifo et al, , 1983Edery et al, 1983). Of these, only eIF-4F and eIF-4E have been shown to possess true cap binding activity; eIF-4A probably does not bind the cap directly but instead binds at a site very near to the cap structure, as does eIF-4B (Pelletier & Sonenberg, 1985). The chemical features of the cap which are recognized include (1) the N7 substituent of the capping guanine nucleotide (Adams et al, 1978;Furuichi et al, 1979), (2) the purine C2 amino and C6 keto substituents (Adams et al, 1978), and (3) the a-phosphate of the capping nucleotide (Darzynkiewicz et al, 1981(Darzynkiewicz et al, , 1987.…”

Fluorescence study of the binding of m7GpppG and rabbit globin mRNA to protein synthesis initiation factors 4A, 4E, and 4F

“…The interaction of eIF-4E and eIF-4A with mRNA has been extensively studied. eIF-4E, either separately or as part of eIF-4F, binds to the cap in the absence of ATP (Sonenberg, 1981;Hellmann et al, 1982) and independent of the degree of mRNA secondary structure (Pelletier and Sonenberg, 1985; Lawson et al, 1986). eIF-4A binds to single-stranded regions of mRNA with concomitant hydrolysis of ATP and may unwind mRNA secondary structure (Grifo et al, 1982;Ray et al, 1985;Goss et al, 1987).…”