Mode of transfection influences the stability of ectopically expressed mRNA.
In the study of mRNA metabolism, modified mRNAs are often analyzed after corresponding mRNA expression vectors have been transfected, either transiently or stably, into cells. Two differently transfected templates might be localized in distinct nuclear compartments: in transient transfection they remain in the nucleoplasm while in stable transfection they are integrated in the chromatin. Consequently, nascent transcripts may encounter different environments which may affect the physical state of mRNA and its fate. In this work, we addressed the question whether the two different modes of transfection affect the stability of expressed mRNA. We compared globin mRNA, which is characteristically stable, and globin-delta AU mRNA, which contains the 3' untranslated region of urokinase-type plasminogen activator mRNA and is unstable. In stably transfected cells, these mRNAs were degraded in a manner which mimicked the endogenous mRNA, whereas in transiently transfected cells, the regulated degradation of both mRNAs was impaired. However, when lower amounts of template DNA were used in transient transfection, mRNA was degraded in a manner similar to that of stably expressed mRNA, indicating that mRNA levels affect its stability. To monitor potential differences in the physical state of mRNAs in vivo, we developed a method based on a combination of chemical modification of cellular RNA and a modified RT-PCR. We found that patterns of chemical modification vary with the levels of mRNA expressed. Our results suggest that a proper interaction of mRNA with specific cellular proteins is important for regulated degradation and that overexpression of mRNA destroys such proper stoichiometry.
Nanbu
R
R
Friedrich Miescher Institute, Basel, Switzerland.
Nagamine
Y
Y
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D018374:LLC-PK1 Cells; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D013552:Swine; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D018076:DNA, Complementary; D012333:RNA, Messenger; D005914:Globins; D014568:Urokinase-Type Plasminogen Activator