Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases.
Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.
D000818:Animals; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D005914:Globins; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012333:RNA, Messenger; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis; D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases
D012333:RNA, Messenger; D005914:Globins; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases