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Small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), PIWI-interacting 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.

PIWI-clade proteins harness piRNAs of 24–33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwishifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR 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 Miwiknock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility.

PIWI proteins and PIWI-interacting RNAs (piRNAs) are specifically expressed in animal germlines and play essential roles during gametogenesis in animals. The primary function of PIWI/piRNAs is known to silence transposable elements for protecting genome integrity in animal germlines, while their roles beyond silencing transposons are also documented by us and others. In particular, we show that mouse PIWIL1 (MIWI)/piRNAs play a dual role in regulating protein-coding genes in mouse spermatids through interacting with different protein factors in a developmental stage-dependent manner, including translationally activating a subset of AU-rich element-containing mRNAs in round spermatids and inducing massive mRNA degradation in late spermatids. We further show that MIWI is eliminated through the ubiquitin-26S proteasome pathway during late spermiogenesis. By exploring the biological function of MIWI ubiquitination by APC/C, we identified ubiquitination-deficient mutations in human PIWIL1of infertile men and further established their causative role in male infertility in mouse model, supporting PIWIL1as a human male infertility-relevant gene. Additionally, we reported that PIWIL1, aberrantly induced in human tumors, functions as an oncoprotein in a piRNA-independent manner in cancer cells. In the current review, we summarize our latest findings regarding the roles and mechanisms of PIWIL1 and piRNAs in mouse spermatids and human diseases, and discuss the related works in the field.Beyond the primary function of silencing transposons, PIWI/piRNAs also play diverse roles in animal germ cells and human diseases.

MicroRNA (miRNA) biogenesis is known to be modulated by a variety of RNA-binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small subset of target miRNAs. Here, we report that the RNA-binding NONO–PSF heterodimer binds a large number of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha–DGCR8 Microprocessor. NONO and PSF are key components of paraspeckles organized by the long noncoding RNA (lncRNA) NEAT1. We further demonstrate that NEAT1 also has a profound effect on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with the NONO–PSF heterodimer as well as many other RBPs and that multiple RNA segments in NEAT1, including a 'pseudo pri-miRNA' near its 3′ end, help attract the Microprocessor. These findings suggest a 'bird nest' model in which an lncRNA orchestrates efficient processing of potentially an entire class of small noncoding RNAs in the nucleus.

Small noncoding RNAs (ncRNAs) are a novel class of gene regulators that modulate gene expression at transcriptional, post‐transcriptional, and epigenetic levels, and they play crucial roles in almost all cellular processes in eukaryotes. Recent studies have indicated that several types of small noncoding RNAs, including microRNAs (miRNAs), endo‐small interference RNAs (endo‐siRNAs), and Piwi‐interacting RNAs (piRNAs), are expressed in the male germline and are required for spermatogenesis in animals. In this review, we summarize the recent knowledge of these small noncoding RNAs in male germ cells and their biological functions and mechanisms of action in animal spermatogenesis. WIREs RNA2014, 5:733–745. doi: 10.1002/wrna.1252