Exploring noncoding variants in genetic diseases:from detection to functional insights

Previous studies on genetic diseases predominantly focused on protein-coding variations, overlooking the vast noncoding regions in the human genome. The development of high-throughput sequencing technologies and functional genomics tools has enabled the systematic identification of functional noncoding variants. These variants can impact gene expression, regulation, and chromatin conformation, thereby contributing to disease pathogenesis. Understanding the mechanisms that underlie the impact of noncoding variants on genetic diseases is indispensable for the development of precisely targeted therapies and the implementation of personalized medicine strategies. The intricacies of noncoding regions introduce a multitude of challenges and research opportunities. In this review, we introduce a spectrum of noncoding variants involved in genetic diseases, along with research strategies and advanced technologies for their precise identification and in-depth understanding of the complexity of the noncoding genome. We will delve into the research challenges and propose potential solutions for unraveling the genetic basis of rare and complex diseases.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility

PIWI-clade proteins harness pi RNAs of 24–33 nt in length.Of great puzzles are how PIWI-clade proteins incorporate pi RNAs of different sizes and whether the size matters to PIWI/pi RNA function.Here we report that a PIWI-Ins module unique in PIWIclade proteins helps define the length of pi RNAs.Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter pi RNAs and causes spermiogenic failure in mice,demonstrating the functional importance of this regulatory module.Mechanistically,we show that longer pi RNAs provide additional complementarity to target m RNAs,thereby enhancing the assembly of the MIWI/e IF3f/Hu R super-complex for translational activation.Importantly,we identify a c.1108C>T(p.R370W)mutation of HIWI(human PIWIL1)in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer pi RNAs.These findings reveal a critical role of PIWI-Ins-ensured longer pi RNAs in fine-tuning MIWI/pi RNA targeting capacity,proven essential for spermatid development and male fertility.

A dual role of the PIWI/pi RNA machinery in regulating mRNAs during mouse spermiogenesis

The post-meiotic phase of male germ cell development in mammals undergoes a series of dramatic morphological changes,including acrosome and flagellum formation,nuclear condensation,and cytoplasmic exclusion,which is driven by a highly regulated sequence of biochemical reactions(Meistrich and Hess,2013).Such differentiation process called spermiogenesis consists of at least 16 sequential transition steps in mice,including steps I to 8 for round spermatids,steps 9 to 11 for elongating spermatids,steps 12 to 14 for elongated or condensed spermatids,and final steps 15 to 16 for spermatozoa(Meistrich and Hess,2013).

Knockout of glutathione peroxidase 5 down-regulates the piRNAs in the caput epididymidis of aged mice

The mammalian epididymis not only plays a fun dame ntal role in the maturati on of spermatozoa,but also provides protecti on agai nst various stressors.The foremost among these is the threat posed by oxidative stress,which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids,proteins,and nucleic acids.In mice,the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5(GPX5)as a major luminal scavenger in the proximal caput epididymidal segment.Accordingly,the loss of GPX5^-/-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx57 mice.To explore the underlying mechanism,we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged(13 months old)Gpx5^-/-m mice.This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts,in eluding the downregulation of a subgroup of piRNA pathway gen es,in aged Gpx5^-/-mice.In agreeme nt with these fin dings,we also observed the loss of piRNAs,which potentially bind to the P-element-induced wimpy testis(PlWI)-like proteins PIWIL1 and PIWIL2.The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5^-/-mice.Importantly,the oxidative stress response genes tend to have more targeting piRNAs,and many of them were among the top increased genes upon the loss of GPX5^-/-.Taken together,our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible invoIvement in the aging and oxidative stress-mediated responses.

Small RNAs:An expanding world with therapeutic promises

Small non-coding RNAs(sncRNAs),such as microRNAs(miRNAs),small interfering RNAs(siRNAs),PIWIinteracting RNAs(piRNAs),and transfer RNA(tRNA)-derived small RNAs(tsRNAs),play essential roles in regulating various cellular and developmental processes.Over the past three decades,researchers have identified novel sncRNA species from various organisms.These molecules demonstrate dynamic expression and diverse functions,and they are subject to intricate regulation through RNA modifications in both healthy and diseased states.Notably,certain sncRNAs in gametes,particularly sperm,respond to environmental stimuli and facilitate epigenetic inheritance.Collectively,the in-depth understanding of sncRNA functions and mechanisms has accelerated the development of small RNA-based therapeutics.In this review,we present the recent advances in the field,including new sncRNA species and the regulatory influences of RNA modifications.We also discuss the current limitations and challenges associated with using small RNAs as either biomarkers or therapeutic drugs.