Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.

Abstract

Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5') GM, was oxidized with sodium periodate to conveit the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5' termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes. A "cap" structure, m7GpppX, is present at the 5' end of most eukaryotic mRNAs (1). Several lines of evidence indicate that the cap in reovirus mRNA and other viral and cellular mRNAs has an important functional role. Reovirus capped and methylated mRNA (5'-m7GpppGm) is translated more efficiently than unmethylated (5'-GpppG) or unblocked (5'-ppG) viral mRNAs (2). Removal of the m7G from methylated mRNA by chemical (3) or enzymatic treatment (4) lowers its template activity in cell-free protein-synthesizing systems, and the absence of a 5'-blocked structure results in a decrease in the stability of reovirus mRNA (5). In addition, compounds structurally related to the cap-e.g., m7GMP and m7GDP-inhibit translation in vitro (6-9). These "cap analogs" interfere with the binding of mRNA to 40S ribsomal subunits in initiation compexes, presumably by competing with the 5' end of mRNA for some essential site or component(s).Previously it was reported that wheat germ 40S ribosomal subunits bind to the 5'-terminal region of reovirus mRNAs and protect cap-containing sequences against RNase digestion (10). Because initiation complexes include protein factors capable of binding mRNA, protection of 5' sequences could be due to an interaction of one or more of these proteins with mRNA at or near the cap. Proteins that bind to m7GpppGm-C, the 5'-terminal oligonucleotide derived from reovirus mRNA by T2 RNase digestion, have been detected in high-salt washes of Artemia salina ribosomes (11). Furthermore, the binding of rabbit reticulocyte initiation factor M3 (eIF-4B) to histone mRNA is inhibited by m7GMP, suggesting that binding involves the cap (12). As part of an effort to understand how the cap influences eukaryotic mRNA function, we have assayed initiation complexes for the presence of proteins that are locatedThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore...

Interaction of a limited set of proteins with different mRNAs and protection of 5′-caps against pyrophosphatase digestion in initiation complexes

“…Similarly, a limited and reproducible group of proteins could be crosslinked to the capped end of oxidized reovirus mRNA in initiation complexes fomed in wheat germ extracts (7). In the present study we have tested several additional viral mRNAs and observed that the same proteins interact with the 5'-caps of different mRNAs in initiation complexes.…”

“…Previously we showed that oxidized reovirus mRNA bound to wheat germ or rabbit reticulocyte initiation complexes can be cross-linked by the 5'-cap m G to a limited set of proteins (7). Because reovirus mRNA consists of a mixture of large, medium and small molecules (17) lyzed by SDS-polyacrylamide gel electrophoresis and fluorography (7).…”

“…The same technique was used to identify proteins adjacent to the 5'-cap of mRNA in initiation complexes formed in rabbit reticulocyte lysate or in incubation mixtures containing mouse ascites cell ribosomes and rabbit reticulocyte factors. Several different proteins were specifically cross-linked to reovirus mRNA under these conditions (7).…”

“…We have described a chemical cross-linking procedure as an alternative approach for identifying proteins that interact with the 5'-ends of mRNAs (7). 3H-Methyl-labeled mRNA is oxidized with periodate to convert the radioactive 5'-terminal m7G to a reactive dialdehyde.…”

“…An aliquot (10%) was assayed for binding to 80S and 40S ribosomes by sedimentation in glycerol gradients (15). The remainder was treated with NaBH3CN, digested with RNase and analyzed by fluorography after polyacrylamide gel electrophoresis as described in detail previously (7). Fluorographs were traced using an Ortec 4310 densitometer.…”