Sequence content of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

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Abstract
Oligo(uridylic acid)-containing [oligo(U+)] RNA was isolated from poly(A+)mRNA from HeLa cells by using either formaldehyde pretreatment or poly(A) removal, both of which resulted in increased accessibility of oligo(U)-rich sequences to a poly(A)-agarose affinity column. The sequence content of oligo(U+) RNA was compared with that of molecules lacking oligo(U) [oligo(U-) RNA] by their relative hybridization to c[complementary]DNA reverse-transcribed from poly(A+) mRNA and by comparison of their in vitro translation products synthesized in a rabbit reticulocyte lysate. Formaldehyde-modified poly(A+) RNA, treated to remove the formol adjuncts, was inactive as a template for in vitro protein synthesis; consequently, only depolyadenylated RNA, which retains its translatability, could be used in the translation studies. The hybridization kinetic experiments revealed that oligo(U+) RNA contained most of the sequence information present in oligo(U-) RNA but at a reduced level (ca. [circa] 25%), the majority of the oligo(U+) RNA sequences being poorly represented in the cDNA. This result was supported by 1- and 2-dimensional gel analysis of their in vitro translation products which showed that oligo(U+) RNA, although less effective as a template for translation than oligo(U-) RNA, coded for proteins, the most abundant of which were encoded by rare messages not highly represented in oligo(U-) RNA or the total poly(A+) RNA. Although some minor products were synthesized by both oligo(U+) and oligo(U-) RNA, at least 33 proteins were unique to or highly enriched in the pattern of products directed by oligo(U+) RNA. Of these, only 2 spp., which have the mobility characteristics of .beta.-actin and its unacetylated derivative, were also abundant oligo(U-) RNA products.