Evidence that the smaller species of mouse GM-CSF mRNA represents deadenylated mRNA.

BMSCs were stimulated with TNF-α (10 ng/mL) for 2 or 3 hours, and total cellular RNA was harvested as described in “Materials and Methods.” As indicated, the RNA samples were treated with 1 unit of RNase H for 40 minutes at 37°C, then loaded onto a gel and used for Northern blotting. −, no RNase H was used, and 16 μg of total cellular RNA from BMSCs treated with TNF-α for 2 hours (samples 2Htz and 2KO in Figure 3) was loaded into each lane. +, oligonucleotide poly(dT)_12-18 (1 μg) and RNase H were added to 10 μg of RNA from BMSCs treated with TNF-α for 3 hours (samples 3Htz and 3KO in Figure 3). The Northern blot was probed with a ³²P-labeled mouse GM-CSF cDNA. The positions of the 18S and 28S ribosomal RNA are indicated as 1.8 kb and 4.4 kb, respectively. The position of the fully deadenylated mouse GM-CSF mRNA is indicated as 0.8 kb. (The mouse GM-CSF mRNA without its polyA tail contains 775 bases.) The position of the polyA-containing mouse GM-CSF mRNA is indicated as 1.0 kb; this approximate size was obtained from a semi-logarithmic plot derived from the migration distances of the 18S and 28S ribosomal RNA and deadenylated mouse GM-CSF mRNA.