Bian, T. | Search bioRxiv

bioRxiv2022

Structural Insights into Branch Site Proofreading by Human Spliceosome

Selection of the pre-mRNA branch site (BS) by U2 snRNP is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection; but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem loop (BSL) of U2 snRNA is shielded by the splicing factor TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2/BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA; the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection/proofreading by PRP5.

biochemistry↗

bioRxiv2021

NNAL, a major metabolite of tobacco-specific carcinogen NNK, promotes lung cancer progression through deactivating LKB1 in an isomer-dependent manner

Smoking is associated with worse clinical outcomes for lung cancer patients. Cell-based studies suggest that NNK (a tobacco specific carcinogen) promotes lung cancer progression. Given its short half-life, the physiological relevance of these in vitro results remains elusive. NNAL, a major metabolite of NNK with a similar structure, a chiral center, and a longer half-life, has never been evaluated in cancer cells. In this study, we characterized the effect of NNAL and its enantiomers on cancer progression among a panel of NSCLC cell lines and explored the associated mechanisms. We found that (R)-NNAL promotes cell proliferation, enhances migration, and induces drug resistance while (S)-NNAL has much weaker effects. Mechanistically, (R)-NNAL phosphorylates and deactivates LKB1 via the {beta}-AR signaling in the LKB1 wild type NSCLC cell lines, contributing to the enhanced proliferation, migration, and drug resistance. Of note, NNK exposure also increases the phosphorylation of LKB1 in A/J mice. More importantly, human lung cancer tissues appear to have elevated LKB1 phosphorylation. Our results reveal, for the first time, that NNAL may promote lung cancer progression through LKB1 deactivation in an isomer-dependent manner.

cancer biology↗