A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.

J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified ß-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the ß-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP ß-hairpin as a highly specific target for cancer therapeutics.

Microprocessor depends on hemin to recognize the apical loop of primary microRNA.

Microprocessor, which consists of a ribonuclease III DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) maturation by cleaving primary miRNA transcripts (pri-miRNAs). We recently demonstrated that the DGCR8 dimer recognizes the apical elements of pri-miRNAs, including the UGU motif, to accurately locate and orient Microprocessor on pri-miRNAs. However, the mechanism underlying the selective RNA binding remains unknown. In this study, we find that hemin, a ferric ion-containing porphyrin, enhances the specific interaction between the apical UGU motif and the DGCR8 dimer, allowing Microprocessor to achieve high efficiency and fidelity of pri-miRNA processing in vitro. Furthermore, by generating a DGCR8 mutant cell line and carrying out rescue experiments, we discover that hemin preferentially stimulates the expression of miRNAs possessing the UGU motif, thereby conferring differential regulation of miRNA maturation. Our findings reveal the molecular action mechanism of hemin in pri-miRNA processing and establish a novel function of hemin in inducing specific RNA-protein interaction.

Select amino acids in DGCR8 are essential for the UGU-pri-miRNA interaction and processing.

Microprocessor, composed of DROSHA and DGCR8, processes primary microRNAs (pri-miRNAs) in miRNA biogenesis. Its cleavage efficiency and accuracy are enhanced because DGCR8 interacts with the apical UGU motif of pri-miRNAs. However, the mechanism and influence of DGCR8-UGU interaction on cellular miRNA expression are still elusive. In this study, we demonstrated that Rhed (i.e., the RNA-binding heme domain, amino acids 285-478) of DGCR8 interacts with UGU. In addition, we identified three amino acids 461-463 in Rhed, which are critical for the UGU interaction and essential for Microprocessor to accurately and efficiently process UGU-pri-miRNAs in vitro and UGU-miRNA expression in human cells. Furthermore, we found that within the DGCR8 dimer, the amino acids 461-463 from one monomer are capable of discriminating between UGU- and noUGU-pri-miRNAs. Our findings improve the current understanding of the substrate-recognizing mechanism of DGCR8 and implicate the roles of this recognition in differentiating miRNA expression in human cells.