ABSTRACT
It has been shown that CA repeats in the 3'-untranslated region (UTR) of bcl-2 mRNA contribute the constitutive decay of bcl-2 mRNA and that hnRNP L (heterogenous nuclear ribonucleoprotein L) interacts with CA repeats in the 3'-UTR of bcl-2 mRNA, both in vitro and in vivo. The aim of this study was to determine whether the alteration of hnRNP L affects the stability of bcl-2 mRNA in vivo. Human breast carcinoma MCF-7 cells were transfected with hnRNP L-specific shRNA or hnRNP L-expressing vector to decrease or increase hnRNP L levels, respectively, followed by an actinomycin D chase. An RT-PCR analysis showed that the rate of degradation of endogenous bcl-2 mRNA was not affected by the decrease or increase in the hnRNP L levels. Furthermore, during apoptosis or autophagy, in which bcl-2 expression has been reported to decrease, no difference in the degradation of bcl-2 mRNA was observed between control and hnRNP L-knock down MCF-7 Cells. On the other hand, the levels of AUF-1 and nucleolin, transacting factors for ARE in the 3'UTR of bcl-2 mRNA, were not significantly affected by the decrease in hnRNP L, suggesting that a disturbance in the quantitative balance between these transacting factors is not likely to interfere with the effect of hnRNP L. Collectively, the findings indicate that the decay of bcl-2 mRNA does not appear to be directly controlled by hnRNP L in vivo.
Subject(s)
Humans , 3' Untranslated Regions , Apoptosis , Autophagy , Breast , Dactinomycin , Hand , Heterogeneous-Nuclear Ribonucleoprotein L , Heterogeneous-Nuclear Ribonucleoproteins , MCF-7 Cells , Phosphoproteins , Ribonucleoproteins , RNA, Messenger , RNA, Small Interfering , RNA-Binding ProteinsABSTRACT
Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is one of the principal pre-mRNA-binding proteins found in human cells. The hnRNP L protein is fairly abundant. However,it is not restricted to the nucleus, and instead shuttles between the nucleus and the cytoplasm. It is composed of 558 amino acid residues and harbours four loosely conserved RNP-consensus RNA-binding domains.In an attempt to characterize the interaction occurring between cellular proteins and hnRNP L, yeast two-hybrid screening was conducted using a HeLa cDNA library. Some of the cDNA clones were found to harbour a partial human hnRNP D/AUF1 cDNA (GeneBank accession number NM_031369). In this study,we determined that hnRNP L interacts specifically with the hnRNP D/AUF1 in the yeast two-hybrid system. This interaction was verified via an in vitro pull-down assay.