Abstract
A nuclease S1 mapping procedure was used to identify sites accessible to nucleases in the 3'-noncoding region of the rabbit globin mRNAs. A complex structure was evident in the alpha-globin species, with one highly accessible single-stranded site, large portions in an accessible double-stranded configuration, and a portion not accessible to any of the nucleases. In the beta-globin mRNA, the region was more uniformly accessible to RNase T1 and to a cobra venom enzyme specific for double-stranded RNA, but it had only a single site highly accessible to a bulkier Neurospora endonuclease. The patterns of cleavage were nearly identical in the deproteinized mRNAs and in the mRNAs associated with polyribosomes in reticulocyte extracts. In both species, a zone of secondary structure occurred around the poly(A) junction. In each species, virtually all the molecules had a poly(A) sequence of at least 20-25 AMP residues. A periodicity in poly(A) size distribution was observed. These results indicate that the beginning of this sequence is well protected against degradation inside the cell and that zones of partial protection occur at measured intervals. In crude extracts, where the poly(A) is covered with proteins, this sequence was protected against nuclease digestion.
Highlights
Globin mRNA, the region was more uniformly accessible to RNase TIand to a cobra venom enzyme specific for double-stranded RNA, but it had only a single site highly accessible to a bulkier Neurospora endonuclease
Themapping with thepresent probe yielded a small population of fragments with sizes greater than 187 nucleotides but smaller than200 nucleotides, whereas fragments of tions used for deproteinized RNA, with cycloheximide (50 pg/ml) as sizes 171-187 nucleotides were virtuallynonexistent.The an additionalingredienttopreventany possibleribosomerun-off results indicate that essentially all the @-globin mRNA molduring the incubation.I t was verified that the mRNA in the samples of lysate used for fragmentation was almost entirely inpolyribosomes
I t appears from the analysis of poly(A) sizes that the first 2025 AMP residues in this sequence are well protected from the degradation process that causes poly(A) shortening in the cells
Summary
Preparation of Cell Extracts and of RNA from Reticulocytes- ments. The length of labeled probe complementary to intact Reticulocyteswere obtained from New Zealand White long-eared a-globin mRNA, up to the poly(A) junicsti1o6n3,nucleotides. rahbits made anemic with phenylhydrazine as described by Pelham The corresponding length of the [+probe is 170 nucleotides and dackson (1976). The length of labeled probe complementary to intact Reticulocyteswere obtained from New Zealand White long-eared a-globin mRNA, up to the poly(A) junicsti1o6n3,nucleotides. Themapping with thepresent probe yielded a small population of fragments with sizes greater than 187 nucleotides but smaller than200 nucleotides, whereas fragments of tions used for deproteinized RNA, with cycloheximide (50 pg/ml) as sizes 171-187 nucleotides were virtuallynonexistent.The an additionalingredienttopreventany possibleribosomerun-off results indicate that essentially all the @-globin mRNA molduring the incubation.I t was verified that the mRNA in the samples of lysate used for fragmentation was almost entirely inpolyribosomes (data not shown). The procedures for cleavingthe plasmids with EcoRI, labeling the terminiwith [n-"PIdATP, hybridization with the fragmented RNA preparations, treatment with nuclease S1 (3-10 units), and separating the fragmenwtesre as described. Distribution of Poly(A) Sizes-The S1 mapping procedure involved hybridization of fragmented RNA with probes labeled at the 3' end as indicated in Fig. 1 and removal by UI. The sizes of the resulting probe fragments correspond to the distances between the EcoRI siitne each of the
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