Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases—N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11—as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central “hub” protein that regulates the activity of at least seven methyltransferases.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain–Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood–brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 10⁴ plaque-forming units (p.f.u.) ml⁻¹ in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1.

Melanoma-associated antigen A (MAGEA) subfamily proteins are normally expressed in testis and/or placenta. However, aberrant expression is detected in the tumour cells of multiple types of human cancer. MAGEA expression is mainly observed in cancers that have acquired malignant phenotypes, invasiveness and metastasis, and the expression of MAGEA family proteins has been linked to poor prognosis in cancer patients. All MAGE proteins share the common MAGE homology domain (MHD) which encompasses up to 70% of the protein; however, the areas flanking the MHD region vary between family members and are poorly conserved. To investigate the molecular basis of MAGEA10 expression and anomalous mobility in gel, deletion and point-mutation, analyses of the MAGEA10 protein were performed. Our data show that the intrinsically disordered N-terminal domain and, specifically, the first seven amino acids containing a unique linear motif, PRAPKR, are responsible for its expression, aberrant migration in SDS-PAGE and nuclear localisation. The aberrant migration in gel and nuclear localisation are not related to each other. Hiding the N-terminus with an epitope tag strongly affected its mobility in gel and expression in cells. Our results suggest that the intrinsically disordered domains flanking the MHD determine the unique properties of individual MAGEA proteins.

Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of “specific” non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research.