Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities—Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation—can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.DOI: http://dx.doi.org/10.7554/eLife.27949.001

The effect of UVR on human basal cell carcinoma (BCC) epidemiology is complex-the incidence rises until approximately 30,000 hours of lifetime sunlight exposure and then plateaus. We hypothesize that UVR has opposing effects on BCC carcinogenesis-stimulatory via mutagenesis and inhibitory via production of hedgehog-inhibiting vitamin D (D). We find that UVR exposure of ionizing radiation-treated Ptch1 mice accelerates BCC carcinogenesis in male mice, in which UVR does not produce D. By contrast, in female mice, in which UVR does produce D, UVR fails to accelerate BCC carcinogenesis, thus mirroring the plateauing in humans. However, if D production is attenuated in female mice by deletion of keratinocyte lathosterol 5-desaturase, then UVR accelerates ionizing radiation-induced BCC carcinogenesis. Congruently, chronic topical application of D inhibits ionizing radiation-induced BCC tumorigenesis. These findings confirm that UVR-induced production of D in keratinocytes significantly restrains murine BCC tumorigenesis and demonstrate the counterintuitive conclusion that UVR has anti-BCC carcinogenic effects that can explain, at least in part, the complex relationship between exposure to UVR and BCC incidence.

Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation-can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.

In 1941 Apperly hypothesized that differential sunlight exposure is the cause of the latitudinal gradient in breast cancer mortality, and subsequently the effect was postulated to be due to the tumor inhibitory properties of skin produced vitamin D3. We first tested this latitudinal hypotheses using the C3(1)/Tag transgenic murine mammary carcinoma model and found that ultraviolet radiation (UVR) significantly prolongs tumor-free and overall survival but that dietary D3 has no such effect. The mechanism of this anti-mammary carcinogenesis effect of UVR is unclear but there is evidence that D3 is a potent inhibitor of the hedgehog (HH) - signaling pathway. To test this mechanistic hypothesis we compared the effects of the small molecule Smo-acting HH pathway inhibitor (HHI) BMS-833923/XL139 with those of UVR on breast cancer development in C3(1)/Tag mice. We have found that oral administration of the pharmacologic HHI delays Tag mammary carcinogenesis with an efficacy similar to that of UVR treatment (350 mJ/cm2, 3x/week), thus, time to first palpable tumor was delayed by 9 days by UVR treatment (p=0.016) and by 14 days by HHI treatment (p=0.009), compared control mice. Overall survival was delayed by 9 days by UVR treatment (p=0.19) and by 15 days by HHI treatment (p=0.011). And, we have found that HHI, like UVR, acts at an early stage of carcinogenesis (i.e. reduction of histologic changes at age 12 weeks), and initial studies of UVR + HHI treatments suggest that HHI and UVR have overlapping effects. Taken together our results are consistent with (albeit by no means proof of) our hypothesis that inhibition of mammary carcinogenesis by UVR-produced vitamin D3 underlies the latitudinal gradient and is mediated via HH inhibition. Moreover, our results suggest more broadly that HHI treatment before tumors develop may have more of an anti-cancer effect than does HHI treatment after tumors already have become clinically apparent. Note: This abstract was not presented at the conference. Citation Format: Anastasia M. Makarova, Subheksha KC, John A. Dolorito, Yefim I. Khaimskiy, Ervin H. Epstein, Jr. Hedgehog inhibition delays cancer growth in C3(1)/Tag transgenic murine mammary carcinoma model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A14.