Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.
The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.
Düvel
Katrin
K
Institute of Microbiology and Genetics, Georg-August-University, Göttingen, Germany.
Valerius
Oliver
O
Mangus
David A
DA
Jacobson
Allan
A
Braus
Gerhard H
GH
D020413:3' Untranslated Regions / Q000235:genetics* / Q000378:metabolism; D021581:Active Transport, Cell Nucleus; D000877:Anthranilate Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002455:Cell Division / Q000502:physiology; D015870:Gene Expression / Q000235:genetics; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000378:metabolism
D020413:3' Untranslated Regions; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D011061:Poly A; D014364:Tryptophan; D000877:Anthranilate Phosphoribosyltransferase