Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame.
The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by S1 nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse L1 repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition of a repetitive element downstream of a cryptic polyadenylylation signal.
Harendza
C J
CJ
Department of Biochemistry and Molecular Genetics, Ohio State University, Columbus 43210.
Johnson
L F
LF
D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D003062:Codon; D004251:DNA Transposable Elements; D005796:Genes; D005809:Genes, Regulator; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis; D011544:Pseudogenes; D012333:RNA, Messenger / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013940:Thymidylate Synthase / Q000235:genetics*; D014158:Transcription, Genetic
D003062:Codon; D004251:DNA Transposable Elements; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; D013940:Thymidylate Synthase; D015719:Single-Strand Specific DNA and RNA Endonucleases