Blocked and Methylated 5′‐Terminal Cap Structures of Rat Brain Messenger Ribonucleic Acids

The presence and identity of 5′‐terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [ 3 H]sodium borohydride reduction or by β ‐elimination of 5′‐terminal nucleoside and incorporation of 32 P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures. The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7‐methylguanosine (m 7 G) as the 5′‐terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5′‐penultimate position was 6‐methyl‐2′‐ O ‐methyiadenosine [m 6 A(m)] followed by 2′‐ O ‐methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2′‐ O ‐Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2′‐ O ‐methyl‐cytidine [C(m)] and 2′‐ O ‐methyluridine [U(m)].