A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.

Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.

An inappropriate decline in ribosome levels drives a diverse set of neurodevelopmental disorders.

Many neurodevelopmental defects are linked to perturbations in genes involved in housekeeping functions, such as those encoding ribosome biogenesis factors. However, how reductions in ribosome biogenesis can result in tissue and developmental specific defects remains a mystery. Here we describe new allelic variants in the ribosome biogenesis factor AIRIM primarily associated with neurodevelopmental disorders. Using human cerebral organoids in combination with proteomic analysis, single-cell transcriptome analysis across multiple developmental stages, and single organoid translatome analysis, we identify a previously unappreciated mechanism linking changes in ribosome levels and the timing of cell fate specification during early brain development. We find ribosome levels decrease during neuroepithelial differentiation, making differentiating cells particularly vulnerable to perturbations in ribosome biogenesis during this time. Reduced ribosome availability more profoundly impacts the translation of specific transcripts, disrupting both survival and cell fate commitment of transitioning neuroepithelia. Enhancing mTOR activity by both genetic and pharmacologic approaches ameliorates the growth and developmental defects associated with intellectual disability linked variants, identifying potential treatment options for specific brain ribosomopathies. This work reveals the cellular and molecular origins of protein synthesis defect-related disorders of human brain development. Highlights: AIRIM variants reduce ribosome levels specifically in neural progenitor cells. Inappropriately low ribosome levels cause a transient delay in radial glia fate commitment.Reduced ribosome levels impair translation of a selected subset of mRNAs.Genetic and pharmacologic activation of mTORC1 suppresses AIRIM-linked phenotypes.

The Role of the Ascorbic Acid-Glutathione Cycle in Young Wheat Ears' Response to Spring Freezing Stress.

Freezing stress in spring often causes the death and abnormal development of young ears of wheat, leading to a significant reduction in grain production. However, the mechanisms of young wheat ears responding to freezing are largely unclear. In this study, the role of the ascorbic acid-glutathione cycle (AsA-GSH cycle) in alleviating freezing-caused oxidative damage in young wheat ears at the anther connective tissue formation phase (ACFP) was investigated. The results showed that the release rate of reactive oxygen species (ROS) and the relative electrolyte conductivity in young ears of Jimai22 (JM22, freezing-tolerant) were significantly lower than those in young ears of Xumai33 (XM33, freezing-sensitive) under freezing. The level of the GSH pool (231.8~392.3 µg/g FW) was strikingly higher than that of the AsA pool (98.86~123.4 µg/g FW) in young wheat ears at the ACFP. Freezing significantly increased the level of the AsA pool and the activities of ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) in the young ears of both varieties. The level of the GSH pool increased in the young ears of XM33 under freezing but decreased in the young ears of JM22. The young ears of JM22 showed higher activities of glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione peroxidase (GPX) than the young ears of XM33 under freezing. Collectively, these results suggest that the AsA-GSH cycle plays a positive role in alleviating freezing-induced oxidative damage in young wheat ears. Furthermore, the ability of utilizing GSH as a substrate to scavenge ROS is an important factor affecting the freezing tolerance of young wheat ears. In addition, abscisic acid (ABA), salicylic acid (SA), 3-indolebutyric acid (IBA) and cis-zeatin (cZ) may be involved in regulating the AsA-GSH cycle metabolism in young wheat ears under freezing.

Ribosome biogenesis and function in development and disease.

Although differential transcription drives the development of multicellular organisms, the ultimate readout of a protein-coding gene is ribosome-dependent mRNA translation. Ribosomes were once thought of as uniform molecular machines, but emerging evidence indicates that the complexity and diversity of ribosome biogenesis and function should be given a fresh look in the context of development. This Review begins with a discussion of different developmental disorders that have been linked with perturbations in ribosome production and function. We then highlight recent studies that reveal how different cells and tissues exhibit variable levels of ribosome production and protein synthesis, and how changes in protein synthesis capacity can influence specific cell fate decisions. We finish by touching upon ribosome heterogeneity in stress responses and development. These discussions highlight the importance of considering both ribosome levels and functional specialization in the context of development and disease.

Genome-Scale CRISPR Screening Reveals Host Factors Required for Ribosome Formation and Viral Replication.

Viruses are known to co-opt host machinery for translation initiation, but less is known about which host factors are required for the formation of ribosomes used to synthesize viral proteins. Using a loss-of-function CRISPR screen, we show that synthesis of a flavivirus-encoded fluorescent reporter depends on multiple host factors, including several 60S ribosome biogenesis proteins. Viral phenotyping revealed that two of these factors, SBDS, a known ribosome biogenesis factor, and the relatively uncharacterized protein SPATA5, were broadly required for replication of flaviviruses, coronaviruses, alphaviruses, paramyxoviruses, an enterovirus, and a poxvirus. Mechanistic studies revealed that loss of SPATA5 caused defects in rRNA processing and ribosome assembly, suggesting that this human protein may be a functional ortholog of yeast Drg1. These studies implicate specific ribosome biogenesis proteins as viral host dependency factors that are required for synthesis of virally encoded protein and accordingly, optimal viral replication. IMPORTANCE Viruses are well known for their ability to co-opt host ribosomes to synthesize viral proteins. The specific factors involved in translation of viral RNAs are not fully described. In this study, we implemented a unique genome-scale CRISPR screen to identify previously uncharacterized host factors that are important for the synthesis of virally encoded protein. We found that multiple genes involved in 60S ribosome biogenesis were required for viral RNA translation. Loss of these factors severely impaired viral replication. Mechanistic studies on the AAA ATPase SPATA5 indicate that this host factor is required for a late step in ribosome formation. These findings reveal insight into the identity and function of specific ribosome biogenesis proteins that are critical for viral infections.

The homeostatic regulation of ribosome biogenesis.

The continued integrity of biological systems depends on a balance between interdependent elements at the molecular, cellular, and organismal levels. This is particularly true for the generation of ribosomes, which influence almost every aspect of cell and organismal biology. Ribosome biogenesis (RiBi) is an energetically demanding process that involves all three RNA polymerases, numerous RNA processing factors, chaperones, and the coordinated expression of 79-80 ribosomal proteins (r-proteins). Work over the last several decades has revealed that the dynamic regulation of ribosome production represents a major mechanism by which cells maintain homeostasis in response to changing environmental conditions and acute stress. More recent studies suggest that cells and tissues within multicellular organisms exhibit dramatically different levels of ribosome production and protein synthesis, marked by the differential expression of RiBi factors. Thus, distinct bottlenecks in the RiBi process, downstream of rRNA transcription, may exist within different cell populations of multicellular organisms during development and in adulthood. This review will focus on our current understanding of the mechanisms that link the complex molecular process of ribosome biogenesis with cellular and organismal physiology. We will discuss diverse topics including how different steps in the RiBi process are coordinated with one another, how MYC and mTOR impact RiBi, and how RiBi levels change between stem cells and their differentiated progeny. In turn, we will also review how regulated changes in ribosome production itself can feedback to influence cell fate and function.

Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly.

Although features of ribosome assembly are shared between species, our understanding of the diversity, complexity, dynamics, and regulation of ribosome production in multicellular organisms remains incomplete. To gain insights into ribosome biogenesis in human cells, we perform a genome-wide loss-of-function screen combined with differential labeling of pre-existing and newly assembled ribosomes. These efforts identify two functionally uncharacterized genes, C1orf109 and SPATA5. We provide evidence that these factors, together with CINP and SPATA5L1, control a late step of human pre-60S maturation in the cytoplasm. Loss of either C1orf109 or SPATA5 impairs global protein synthesis. These results link ribosome assembly with neurodevelopmental disorders associated with recessive SPATA5 mutations. Based on these findings, we propose that the expanded repertoire of ribosome biogenesis factors likely enables multicellular organisms to coordinate multiple steps of ribosome production in response to different developmental and environmental stimuli.

The Dynamic Regulation of mRNA Translation and Ribosome Biogenesis During Germ Cell Development and Reproductive Aging.

The regulation of mRNA translation, both globally and at the level of individual transcripts, plays a central role in the development and function of germ cells across species. Genetic studies using flies, worms, zebrafish and mice have highlighted the importance of specific RNA binding proteins in driving various aspects of germ cell formation and function. Many of these mRNA binding proteins, including Pumilio, Nanos, Vasa and Dazl have been conserved through evolution, specifically mark germ cells, and carry out similar functions across species. These proteins typically influence mRNA translation by binding to specific elements within the 3' untranslated region (UTR) of target messages. Emerging evidence indicates that the global regulation of mRNA translation also plays an important role in germ cell development. For example, ribosome biogenesis is often regulated in a stage specific manner during gametogenesis. Moreover, oocytes need to produce and store a sufficient number of ribosomes to support the development of the early embryo until the initiation of zygotic transcription. Accumulating evidence indicates that disruption of mRNA translation regulatory mechanisms likely contributes to infertility and reproductive aging in humans. These findings highlight the importance of gaining further insights into the mechanisms that control mRNA translation within germ cells. Future work in this area will likely have important impacts beyond germ cell biology.

Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus.

Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.