The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

Cell death overarches carcinogenesis and is a center of cancer researches, especially therapy studies. There have been many nomenclatures on cell death, but only three cell death modes are genuine, i.e. apoptosis, necrosis and stress-induced cell death (SICD). Like apoptosis, SICD is programmed. Like necrosis, SICD is a pathological event and may trigger regeneration and scar formation. Therefore, SICD has subtypes of stress-induced apoptosis-like cell death (SIaLCD) and stress-induced necrosis-like cell death (SInLCD). Whereas apoptosis removes redundant but healthy cells, SICD removes useful but ill or damaged cells. Many studies on cell death involve cancer tissues that resemble parasites in the host patients, which is a complicated system as it involves immune clearance of the alien cancer cells by the host. Cancer resembles an evolutionarily lower-level organism having a weaker apoptosis potential and poorer DNA repair mechanisms. Hence, targeting apoptosis for cancer therapy, i.e. killing via SIaLCD, will be less efficacious and more toxic. On the other hand, necrosis of cancer cells releases cellular debris and components to stimulate immune function, thus counteracting therapy-caused immune suppression and making necrosis better than SIaLCD for chemo drug development.

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE), is a technique commonly used to detect specific proteins. SDS-PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography - mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non-malignant HEK293 and cancerous MDA-MB231 (MB231) cells separated using SDS-PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS-PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer-therapy studies.