Functional alpha-fragment of beta-galactosidase can be expressed from the mobile group I intron PpLSU3 embedded in yeast pre-ribosomal RNA derived from the chromosomal rDNA locus.
PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.
Lin
J
J
Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.
Vogt
V M
VM
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D007438:Introns / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D016839:Physarum polycephalum / Q000201:enzymology / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D004274:DNA, Recombinant; D004275:DNA, Ribosomal; D010446:Peptide Fragments; D012322:RNA Precursors; D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D012318:RNA Polymerase I; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease; D001616:beta-Galactosidase