Length increase of the human alpha -globin 3'-untranslated region disrupts stability of the pre-mRNA but not that of the mature mRNA.
Polyadenylation increases the stability of mRNA molecules. By studying the effect of the length of 3'-untranslated region (UTR) on mRNA levels, we have found that alpha-globin pre-mRNA is stabilized by a mechanism that does not modulate the half-life of mature mRNA. The insertion of DNA fragments of various unrelated sequences into the 3'-UTR of the human alpha-globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG-3 cells. We found an inverse relationship between mRNA levels and the length of the introduced fragments. In fact, mRNA levels as low as 1% were observed after inserting a 477-nucleotide (nt) fragment, whereas inserting a fragment of 86 nt at the same position had no effect on mRNA accumulation. DNA insertion induced no change in transcription rate or in half-life of mature mRNA. Semi-quantitative reverse transcription-polymerase chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR resulted in decreased levels of nuclear pre-mRNA in proportion to that observed for mature mRNA. In contrast, the insertion of the 477-nt exogenous DNA in the last intron had no effect on mRNA levels despite the presence of intronic sequences in the pre-mRNA. This shows that the reduction of pre-mRNA level was not due to the insertion of putative ribonuclease cleavage sites or the insertion of a segment DNA that reduces the elongation efficiency. Taken together, our results strongly support the existence of a pre-mRNA stabilizing mechanism that can be disrupted by increasing the length of the 3'-UTR. The fact that the half-life of mature mRNA is not affected by DNA insertion is compatible with a pre-mRNA-specific stabilizing mechanism that acts specifically before polyadenylation.
Provost
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PR
Laboratory of Ontogeny and Reproduction, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Canada.
Tremblay
Y
Y
D020413:3' Untranslated Regions; D005914:Globins / Q000235:genetics*; D006801:Humans; D011061:Poly A / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D015138:RNA, Nuclear / Q000378:metabolism
D020413:3' Untranslated Regions; D012322:RNA Precursors; D012333:RNA, Messenger; D015138:RNA, Nuclear; D011061:Poly A; D005914:Globins