Detection and structural definition of a minor splicing product from the hα-globin gene. (A) MEL/tTA cells transfected with the hα-globin gene express a predominant full-length hα-globin mRNA and a minor species with distinct polysome profiles. MEL/tTA cells⁷  were transfected with a plasmid p(Tet-αWT) expressing the full-length hα-globin gene.⁷  RPA was performed across the sucrose gradient. A diagram of the RPA is shown below the gel image; the entire 3-exon α-globin gene is shown along with its 2 introns. The upper band (132 bp) in the gel (hα-globin mRNA) represents protection of the ³²P internally labeled 244-bp probe by the hα-globin transcript spliced at the cognate exon 1/intron 1 splice donor site. The smaller protected fragment of ∼80 bp (minor mRNA) suggests the presence of a small population of hα-globin RNA generated by an alternative processing pathway. The distribution of the minor RNA is shifted strongly to the left (ie, to lighter fractions) compared with the full-length hα-globin mRNA. A 25-bp size marker ladder (M) is shown on the left of the gel; 19 fractions are labeled on the top of the gel and align with the sucrose gradient tracing (OD254 nm). (B) Amplification of hα-globin complementary DNAs from a transfected hα-globin gene in MEL/tTA cells. RNA was isolated from cells 48 hours posttransfection. Three bands were generated by RT-PCR with a primer set bracketing intron 1 (horizontal arrows; top diagram); the largest band (355 bp) corresponds to the unprocessed hα-globin transcript with intron 1 (pre-mRNA), the second band (238 bp) corresponds to the hα-globin mRNA subsequent to excision of intron 1 from the cognate donor site (hα-globin mRNA), and the third band (189 bp; minor mRNA) was of unknown structure. The minor DNA fragment was excised and sequenced (bottom). M indicates 100-bp size marker ladder. (C) The hα-globin transcript undergoes low-frequency splicing from a cryptic splice donor site located within exon 1. The sequence of the minor mRNA revealed utilization of a cryptic splice donor located within exon 1 ligated to the cognate splice acceptor of exon 2. Sequences are shown at the bottom of the figure; capital letters indicate exons 1 and 2, and lower case letters indicate sequences in introns 1 and 2. The region of exon 1 that is converted to intronic sequence by the use of the cryptic splice donor within exon 1 is underlined. The splicing from this cryptic splice donor generates a shorter α-globin mRNA that would be out of frame with the WT α-globin mRNA with a new in-frame stop codon within exon 2 (TGA; red font). This shorter α-globin mRNA also contains a second potential translation initiation site within exon 2 (green font) that would be in frame with the WT hα-globin mRNA.