Recent advancements in mRNA technology, utilized in vaccines, immunotherapies, protein replacement therapies, and genome editing, have emerged as promising and increasingly viable treatments. The rapid, potent, and transient properties of mRNA-encoded proteins make them attractive tools for the effective treatment of a variety of conditions, ranging from infectious diseases to cancer and single-gene disorders. The capability for rapid and large-scale production of mRNA therapeutics fueled the global response to the COVID-19 pandemic. For effective clinical implementation, it is crucial to deeply characterize and control important mRNA attributes such as purity/integrity, identity, structural quality features, and functionality. This implies the use of powerful and advanced analytical techniques for quality control and characterization of mRNA. Improvements in analytical techniques such as electrophoresis, chromatography, mass spectrometry, sequencing, and functionality assessments have significantly enhanced the quality and detail of information available for product and process characterization, as well as for routine stability and release testing. Here, we review the latest advancements in analytical techniques for the characterization of mRNA-based therapeutics, typically employed by the biopharmaceutical industry for eventual market release.

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.

A gene encoding the transcription factor RTF1 has been associated with an increased risk of ulcerative colitis (UC). In this study, we investigated its function in modulating T cells expressing interleukin-17A (Th17 cells), a cardinal cell type promoting intestinal inflammation. Our results indicate that Rtf1 deficiency disrupts the differentiation of Th17 cells, while leaving regulatory T cells (Treg) unaffected. Mechanistically, RTF1 facilitates histone H2B monoubiquitination (H2Bub1), which requires its histone modification domain (HMD), for supporting Th17 cell function. Impaired Th17 differentiation was also observed in cells lacking the H2Bub1 E3 ligase subunit RNF40, an enzyme known to physically interact with RTF1. Thus, our study underscores the essential role of RTF1 in H2Bub1-mediated epigenetic regulation of Th17 cell differentiation. Understanding this process will likely provide valuable insights into addressing Th17-associated inflammatory disorders. (Images were created with BioRender).

A gene encoding the transcription factor RTF1 has been associated with an increased risk of ulcerative colitis (UC). In this study, we investigated its function in modulating T cells expressing interleukin-17A (Th17 cells), a cardinal cell type promoting intestinal inflammation. Our results indicate that Rtf1 deficiency disrupts the differentiation of Th17 cells, while leaving regulatory T cells (Treg) unaffected. Mechanistically, RTF1 facilitates histone H2B monoubiquitination (H2Bub1), which requires its histone modification domain (HMD), for supporting Th17 cell function. Impaired Th17 differentiation was also observed in cells lacking the H2Bub1 E3 ligase subunit RNF40, an enzyme known to physically interact with RTF1. Thus, our study underscores the essential role of RTF1 in H2Bub1-mediated epigenetic regulation of Th17 cell differentiation. Understanding this process will likely provide valuable insights into addressing Th17-associated inflammatory disorders. Key Points: RTF1 is crucial for Th17 cell differentiation via histone H2B monoubiquitination RTF1 deficiency impairs Th17 differentiation, similar to H2Bub1 ligase RNF40 deficiency RTF1 and H2Bub1 colocalize on genes regulating Th17 cells, indicating an epigenetic role