Abstract
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.
Highlights
Before reaching cytoplasm, most vertebrate primary transcripts are processed at their 5Ј end by capping [1] and at their 3Ј end by polyadenylation [2], and they are spliced to remove introns [3, 4]
To study the effect of the length of the 3Ј-untranslated region (UTR) on ␣-globin mRNA levels, transfections were performed in nonerythroid JEG-3 cells to eliminate possible effects of putative tissue-specific elements
We have clearly demonstrated that increasing the length of the 3Ј-UTR by insertion of DNA segments had a strong negative effect on ␣-globin mRNA level
Summary
Most vertebrate primary transcripts are processed at their 5Ј end by capping [1] and at their 3Ј end by polyadenylation [2], and they are spliced to remove introns [3, 4]. The insertion of DNA fragments of various unrelated sequences into the 3-UTR of the human ␣-globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG-3 cells. Co-transfection of the resulting clones with the parental plasmid into a choriocarcinoma cell line, JEG-3, that does not express the endogenous globin genes revealed that the length of the 3Ј-UTR strongly influences the level of ␣-globin mRNA.
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