Partially unspliced and fully spliced ELF3 mRNA, including a new Alu element in human breast cancer.
Using modified representational difference analysis, a DNA fragment (GC3) was isolated as a difference between a breast cancer and a normal cell line from the same patient. GC3 proved to be a fragment of intron 7 of the ELF3 gene, an ets family transcription factor, amplified in the breast cancer cell line. Using genomic walking technology, a new Alu (Alu(kwd)) was found downstream of GC3 in an antisense position between nt 8762 and nt 8763 within intron 8 of the ELF3 gene. This ELF3 intron fragment(GC3) was expressed in human breast cancer cell lines and four of six breast cancer tissues, but not in matched normal cell lines and tissues. Similarly, Alu(kwd) was also found in the same breast cancer cell lines and five of eight other breast cancer tissues, but not in matched normal cell lines and tissue. This was confirmed by RNase and DNase digestion analysis. Moreover, GC3 and Alu(kwd) were detected in both the nuclear and cytoplasmic RNA fractions of breast cancer cell lines. The finding of cytoplasmic intron retention was verified with northern blotting and the 5' and 3' rapid amplification cDNA ends procedure (5' and 3'RACE) to search for cDNA sequences in RNA from these cancer cell lines. Partially unspliced ELF3 mRNA and fully spliced ELF3 mRNA was found in the same breast cancer cell line. Partially unspliced ELF3 mRNA contained introns 4-7 without any nucleotide mutation at intron/exon splice junction borders. Fully spliced 1959 bp ELF3 mRNA showed a different 5'UTR from the published ELF3 mRNA, and was predicted to encode a 371 amino acid protein sharing 98% homology with the ELF3 protein sequence. This is the first report of intron retention of ELF3 as well as the pathological appearance of both spliced and unspliced cytoplasmic ELF3 mRNA in human breast cancer cells.
Department of Medicine, North Shore University Hospital, Manhasset, NY 11030, USA. mkaplan@nshs.edu
Mark H
MH
Kaplan
Xue-Ping
XP
Wang
Hao-Peng
HP
Xu
Michael H
MH
Dosik
D020087:Alu Elements / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D001940:Breast / Q000473:pathology*; D001943:Breast Neoplasms / Q000235:genetics*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C105529:ELF3 protein, human; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D014157:Transcription Factors