The FASTK family of proteins have been recently reported to play a key role in the post-transcriptional regulation of mitochondrial gene expression, including mRNA stability and translation. Accumulated studies have provided evidence that the expression of some FASTK genes is altered in certain types of cancer, in agreement with the central role of mitochondria in cancer development. Here, we obtained a pan-cancer overview of the genomic and transcriptomic alterations of FASTK genes. FASTK, FASTKD1, FASTKD3 and FASTKD5 showed the highest rates of genetic alterations. FASTK and FASTKD3 alterations consisted mainly of amplifications that were seen in more than 8% of ovarian and lung cancers, respectively. FASTKD1 and FASTKD5 were the most frequently mutated FASTK genes, and the mutations were identified in 5–7% of uterine cancers, as well as in 4% of melanomas. Our results also showed that the mRNA levels of all FASTK members were strongly upregulated in esophageal, stomach, liver and lung cancers. Finally, the protein-protein interaction network for FASTK proteins uncovers the interaction of FASTK, FASTKD2, FASTKD4 and FASTKD5 with cancer signaling pathways. These results serve as a starting point for future research into the potential of the FASTK family members as diagnostic and therapeutic targets for certain types of cancer.

Voltage‐dependent potassium channel Kv1.3 plays a key role on T‐cell activation; however, lack of reliable antibodies has prevented its accurate detection under endogenous circumstances. To overcome this limitation, we created a Jurkat T‐cell line with endogenous Kv1.3 channel tagged, to determine the expression, location, and changes upon activation of the native Kv1.3 channels. CRISPR‐Cas9 technique was used to insert a Flag‐Myc peptide at the C terminus of the KCNA3 gene. Basal or activated channel expression was studied using western blot analysis and imaging techniques. We identified two isoforms of Kv1.3 other than the canonical channel (54 KDa) differing on their N terminus: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms were upregulated after T‐cell activation. We focused on the functional characterization of the truncated isoform (short form, SF), because it has not been previously described and could be present in the available Kv1.3−/− mice models. Overexpression of SF in HEK cells elicited small amplitude Kv1.3‐like currents, which, contrary to canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. Although the canonical isoform (long form) localizes mainly at the plasma membrane, SF remains intracellular, accumulating perinuclearly. Accordingly, SF Jurkat cells did not show Kv1.3 currents and exhibited depolarized resting membrane potential (VM), decreased Ca2+ influx, and a reduction in the [Ca2+]i increase upon stimulation. Functional characterization of these Kv1.3 channel isoforms showed their differential contribution to signaling pathways involved in formation of the immunological synapse. We conclude that alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells that exhibit different functional roles. For some of these functions, Kv1.3 proteins do not need to form functional plasma membrane channels.

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3’ truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5’ truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.