Non-Coding RNA

Background/Objectives: The telomerase RNA (TR) is an indispensable part of the telomerase protein complex responsible for telomere elongation in most eukaryotic species. Although the telomere terminal repeat sequence (TTAGGC)_n in Caenorhabditis elegans has been known for years, a telomerase RNA gene was not identified in the entire phylum of Nematoda until recently. Methods: In this exploratory study, we employ a combination of different approaches to identify likely telomerase RNA candidates among putative non-coding transcripts. Results: A detailed analysis of our prime candidate shows compelling evidence that it encodes the missing RNA element of the telomerase complex, which is notably located in an intron of the coding gene nmy-2. Using nmy-2 homologs in other nematodes as anchors, we annotate the conserved TR gene in 21 Caenorhabditis species. We furthermore show that the intronic localization of the TR gene is conserved in two distinct branching groups of the Caenorhabditis phylogeny and demonstrate that this property likely emerged from a single point of origin. Conclusions: While the intronic TR represents a very interesting evolutionary adaption that seems to have been successful in the Elegans and Japonica groups, the question regarding the macroscopic nematode TR evolution remains.

Background/Objectives: Human milk is a complex biological fluid containing not only macro- and micronutrients but also diverse bioactive molecules, including extracellular RNAs. Although RNA has been detected in milk for decades, only a subset of RNA species has been characterized in detail, and abundant families such as tRNA-, yRNA-, and rRNA-derived fragments remain underexplored. This study aimed to define the composition, fragmentation patterns, stability, and exploratory functional activity of these highly abundant RNAs in human milk. Methods: We performed small RNA sequencing on skim milk samples and analyzed the resulting profiles in comparison with publicly available milk and biofluid datasets. RNA stability assays, Northern blotting, and RT-qPCR were conducted to validate RNA abundance and degradation kinetics. Extracellular vesicles (EVs) and non-vesicular fractions were analyzed to determine the subcellular distribution of RNA species. Exploratory functional assays using synthetic RNA fragments were carried out to assess their ability to modulate cellular responses in vitro. Results: Human milk was found to be highly enriched in small RNA fragments derived from tRNA, yRNA, and rRNA, dominated by a limited set of discrete sequences. These profiles were highly reproducible across independent datasets and distinct biofluids. Orthologal validation assays confirmed their abundance and stability, with RNA levels exceeding those of serum by over two orders of magnitude. Full-length transcripts were enriched in EVs, whereas shorter fragments predominated in the non-vesicular fraction. Synthetic milk-derived exRNAs showed detectable pro-survival activity under stress conditions in vitro. Conclusions: This study reveals that human milk carries a limited set of highly abundant stable sRNA molecules, primarily derived from tRNAs, yRNAs, and rRNAs. These findings provide new insights into the RNA cargo of human milk and offer preliminary evidence that selected sRNA fragments can modulate cellular stress responses in in vitro models.

This review provides a thorough survey of long noncoding RNAs that bear the RNA modification N6-methyladenosine (m6A) and current work to understand the resulting mechanistic and biological consequences. We give an overview of lncRNA and m6A biology first, describing the writers, erasers, and readers of m6A and their targeting of lncRNAs. Next, we give an in-depth review of the field of nuclear lncRNAs that regulate chromatin and their regulation via m6A. We then describe the growing appreciation of liquid–liquid phase separation properties in lncRNA and m6A biology. Finally, we cover examples of cytoplasmic lncRNAs regulated by m6A. Overall, this review aims to emphasize how epitranscriptomics influences noncoding RNA mechanisms to provide additional layers of regulation, integrated into downstream biological processes.