Location of oligo(uridylic acid) sequences within messenger ribonucleic acid molecules of HeLa cells

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Abstract
A significant fraction of the polyadenylated mRNa of HeLA cells contain an oligo(uridylic acid) [oligo(U)] sequence of 15-30 nucleotides. Several different experimental approaches were used to determine if these oligo(U) occupied similar sites within all mRNA. In 1 approach, poly(adenylic acid)-containing mRNA [poly (A+)mRNA] averaging 2800 nucleotides in length were reduced to an average size of 500 nucleotide by controlled alkaline hydrolysis. Over 20% of the oligo(U)-containing fragments isolated from the hydrolysate retained a poly(A) sequence, showing that oligo(U) were not exclusively located near 5'' ends of mRNA although 20% were apparently close to 3'' ends. To confirm these observations, oligo(U)-containing mRNA [oligo(U+) mRNA] was exposed to the 3''-exonucleolytic activity of polynucleotide phosphorylase to produce fragments containing the 5'' regions of mRNA. Each of a set of fragments of decreasing length generated by increased times of exposure of the mRNA to the enzyme was found to have about the same oligo(U) content, including the shortest that averaged 550 nucleotides. These data not only eliminated an exclusive location for oligo(U) in either 3'' or 5'' ends of mRNA but also suggested that oligo(U)''s might be close to the 5'' end of some mRNA. To verify this last observation, periodate-oxidized poly(A+) mRNA was labeled at the 5'' caps and at 3''-adenosine residues by sodium [3H]borohydride reduction before it was nicked 3-5 times with alkali to produce 5'' and 3'' end-labeled piecies that could be separated with oligo(thymidylic acid)-cellulose. Quantitation of radioactivity in the oligo(U+) fragments (averaging 450 nucleotides in length) isolated from each population showed that oligo(U) was associated with the 3''-polyadenylated fragments 3 times more often than with 5''-capped fragments of the same average length. However, at least half of the oligo(U) were not associated with these end-labeled fragments, confirming the experiments cited above that suggested that most oligo(U) sequences are not close to the ends of mRNA molecules. Thus, oligo(U) sequences occupy different sites in different mRNA. Their distribution is not random, however, since 3'' regions are favored over 5'' regions. The localization of oligo(U) sequences in untranslated regions of mRNA is discussed in relation to the sequence data currently available for eukaryotic mRNA.

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