PCIF1 is partly cytoplasmic, dynamically localizes to stress granules and binds mRNA coding regions upon oxidative stress.

PCIF1 (Phosphorylated CTD-Interacting Factor 1) is the mRNA (2'-O-methyladenosine-N(6)-)-methyltransferase that catalyzes the formation of cap-adjacent N6,2'-O-dimethyladenosine (m6Am) by methylating adenosines at the first transcribed position of capped mRNAs. While previous studies assumed that PCIF1 was nuclear, cell fractionation and immunofluorescence both show that a population of PCIF1 is localized to the cytoplasm. Further, PCIF1 redistributes to stress granules upon oxidative stress. Immunoprecipitation studies with stressed cells show that PCIF1 also physically interacts with G3BP and other stress granule components. In addition, PCIF1 behaves as a stress granule component as it disassociates from stress granules upon recovery from stress. Overexpressing full-length PCIF1 also inhibits stress granule formation, while knocking out PCIF1 slows stress granule disassembly. Next, our enhanced crosslinking and immunoprecipitation (eCLIP) data show that PCIF1 binds mRNAs in their coding sequences rather than cap-proximal regions. Further PCIF1's association with mRNAs increased upon NaAsO2 stress. In contrast to eCLIP data, ChIP-Seq experiments show that PCIF1 is predominantly associated with transcription start sites rather than gene bodies, indicating that PCIF1's association with mature mRNA is not co-transcriptional. Collectively, our data suggest that PCIF1 has cytoplasmic RNA surveillance role(s) independent of transcription-associated cap-adjacent mRNA modification, particularly during the stress response.

RNA Therapeutics: the Next Generation of Drugs for Cardiovascular Diseases.

PURPOSE OF REVIEW: RNA therapeutics are a new and rapidly expanding class of drugs to prevent or treat a wide spectrum of diseases. We discuss the defining characteristics of the diverse family of molecules under the RNA therapeutics umbrella. RECENT FINDINGS: RNA therapeutics are designed to regulate gene expression in a transient manner. For example, depending upon the strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. RNA therapies include antisense nucleotides, microRNAs and small interfering RNAs, RNA aptamers, and messenger RNAs. Further, we discuss the mechanism(s) by which different RNA therapies either reduce or increase the expression of their targets. We review the RNA therapeutics approved (and those in trials) to treat cardiovascular indications. RNA-based therapeutics are a new, rapidly growing class of drugs that will offer new alternatives for an increasing array of cardiovascular conditions.

Cyclin E/Cdk2-dependent phosphorylation of Mcl-1 determines its stability and cellular sensitivity to BH3 mimetics.

Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E-/- mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors.