Identification of Gemin5 as a Novel 7-Methylguanosine Cap-Binding Protein

A unique attribute of RNA molecules synthesized by RNA polymerase II is the presence of a 7-methylguanosine (m(7)G) cap structure added co-transcriptionally to the 5' end. Through its association with trans-acting effector proteins, the m(7)G cap participates in multiple aspects of RNA metabolism including localization, translation and decay. However, at present relatively few eukaryotic proteins have been identified as factors capable of direct association with m(7)G.Employing an unbiased proteomic approach, we identified gemin5, a component of the survival of motor neuron (SMN) complex, as a factor capable of direct and specific interaction with the m(7)G cap. Gemin5 was readily purified by cap-affinity chromatography in contrast to other SMN complex proteins. Investigating the underlying basis for this observation, we found that purified gemin5 associates with m(7)G-linked sepharose in the absence of detectable eIF4E, and specifically crosslinks to radiolabeled cap structure after UV irradiation. Deletion analysis revealed that an intact set of WD repeat domains located in the N-terminal half of gemin5 are required for cap-binding. Moreover, using structural modeling and site-directed mutagenesis, we identified two proximal aromatic residues located within the WD repeat region that significantly impact m(7)G association.This study rigorously identifies gemin5 as a novel cap-binding protein and describes an unprecedented role for WD repeat domains in m(7)G recognition. The findings presented here will facilitate understanding of gemin5's role in the metabolism of non-coding snRNAs and perhaps other RNA pol II transcripts

Adenovirus-mediated TA-p73β gene transfer increases chemosensitivity of human malignant melanomas

Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (ΔTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. ΔTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73β) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73β significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73β displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models ( p < 0.05). In summary, our data show that Ad-mediated TA-p73β gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo , suggesting a new chemosensitization strategy for malignant melanomas.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44369/1/10495_2006_Article_3407.pd