Identification of mRNA binding proteins that regulate the stability of LDL receptor mRNA through AU-rich elements.
The 3'untranslated region (UTR) of human LDL receptor (LDLR) mRNA contains three AU-rich elements (AREs) responsible for rapid mRNA turnover and mediates the stabilization induced by berberine (BBR). However, the identities of the specific RNA binding proteins involved in the regulation of LDLR mRNA stability at the steady state level or upon BBR treatment are unknown. By conducting small interfering RNA library screenings, biotinylated RNA pull-down, mass spectrometry analysis, and functional assays, we now identify heterogeneous nuclear ribonucleoprotein D (hnRNP D), hnRNP I, and KH-type splicing regulatory protein (KSRP) as key modulators of LDLR mRNA stability in liver cells. We show that hnRNP D, I, and KSRP interact with AREs of the LDLR 3'UTR with sequence specificity. Silencing the expression of these proteins increased LDLR mRNA and protein levels. We further demonstrate that BBR-induced mRNA stabilization involves hnRNP I and KSRP, as their cellular depletions abolished the BBR effect and BBR treatment reduced the binding of hnRNP I and KSRP to the LDLR mRNA 3'UTR. These new findings demonstrate that LDLR mRNA stability is controlled by a group of ARE binding proteins, including hnRNP D, hnRNP I, and KSRP. Our results suggest that interference with the ability of destabilizing ARE binding proteins to interact with LDLR-ARE motifs is likely a mechanism for regulating LDLR expression by compounds such as BBR and perhaps others.
Li
Hai
H
Department of Veterans Affairs, Palo Alto Health Care System, Palo Alto, CA 94304, USA.
Chen
Wei
W
Zhou
Yue
Y
Abidi
Parveen
P
Sharpe
Orr
O
Robinson
William H
WH
Kraemer
Fredric B
FB
Liu
Jingwen
J
D020413:3' Untranslated Regions; D001483:Base Sequence; D001599:Berberine / Q000378:metabolism; D002460:Cell Line; D017930:Genes, Reporter; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C105789:KHSRP protein, human; C450006:PTBP1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011973:Receptors, LDL; D015534:Trans-Activators; D001599:Berberine; D038941:Polypyrimidine Tract-Binding Protein