Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

Abstract

The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.

The RNA chaperon activity of the human La protein (LARP3)

“…Mapping the La binding site in cyclin D1 mRNA revealed that La binds in close proximity to the translation start site [36] . It has been shown earlier that the two RRMs of La are both required and sufficient for binding to hepatitis B virus RNA [21] and both RRMs are also sufficient to bind cyclin D1 RNA [36] .…”

“…Mapping the La binding site in cyclin D1 mRNA revealed that La binds in close proximity to the translation start site [36] . It has been shown earlier that the two RRMs of La are both required and sufficient for binding to hepatitis B virus RNA [21] and both RRMs are also sufficient to bind cyclin D1 RNA [36] . Interestingly, computer calculated (mfold, [37] ) structure of the La binding site in cyclin D1 RNA predicts a stem-loop structure in which the translation start site (AUG) is buried.…”

“…We proposed that the RNA chaperone activity of La destabilizes the helical region and thereby easing the access of the 43S subunit to the AUG. To test the first part of the assumption an RNA chaperone assay has been developed using the La binding site in cyclin D1 RNA as molecular beacon (D1-AUG-MB). This molecular beacon has a fluorescence group at the 5'-end and a quencher at the 3'-end [36] . Based on the structural prediction of the D1-AUG-MB the 5'-and 3'-end should be located next to each other closing the stem.…”

“…The RNA chaperone assay bases on the assumption that if the La protein destabilizes the stem the fluorescence molecule (5'-end) would be spatially separated from the quencher (3'-end), allowing fluorescence light emission after excitation. Using the RNA chaperone assay it has recently been shown that recombinant human La protein can destabilize the helical region of the D1-AUG-MB in an ATP-independent manner [36] . However, the question remains how La is able to assist structural changes in an ATP-independent manner.…”

“…Previous work suggested that a basic stretch of amino acids in the C-terminal domain might be involved in RNA-binding and additional work implied that the C-terminal domain is required for polio virus IRES-mediated translation [35,40] . By introducing two sets of mutations in the C-terminal domain focusing on aromatic and positively charged amino acids known to preferentially interact with RNA, it has been demonstrated recently that the mutated amino acids, referred to as RNA chaperone domain (RCD), were critical for La to assist structural changes in D1-AUG-MB [36] . This finding suggests that RRM1 and RRM2 are required for binding of the D1-AUG-MB and that the RNA chaperone domain of La [1] ; RRM = RNA Recognition motif [1] ; RCD = RNA Chaperone domain [36] ; S366 = Casein Kinase II phosphorylation site [42] ; T389 = AKT phosphorylation site [36] ; T301 = AKT phosphorylation site in murine La [43] .…”