Ribosomal protein L24 mediates mammalian microRNA processing in an evolutionarily conserved manner

To investigate the mechanism(s) underlying the expression of primate-specific microRNAs (miRs), we sought DNA regulatory elements and proteins mediating expression of the primate-specific hsa-miR-608 (miR-608), which is located in the SEMA4G gene and facilitates the cholinergic blockade of inflammation by targeting acetylcholinesterase mRNA. 'Humanized' mice carrying pre-miR-608 flanked by 250 bases of endogenous sequences inserted into the murine Sema4g gene successfully expressed miR-608. Moreover, by flanking miR-608 by shortened fragments of its human genome region we identified an active independent promoter within the 150 nucleotides 5' to pre-miR-608, which elevated mature miR-608 levels by 100-fold in transfected mouse- and human-originated cells. This highlighted a regulatory role of the 5' flank as enabling miR-608 expression. Moreover, pull-down of the 150-base 5' sequence revealed its interaction with ribosomal protein L24 (RPL24), implicating an additional mechanism controlling miR-608 levels. Furthermore, RPL24 knockdown altered the expression of multiple miRs, and RPL24 immunoprecipitation indicated that up- or down-regulation of the mature miRs depended on whether their precursors bind RPL24 directly. Finally, further tests showed that RPL24 interacts directly with DDX5, a component of the large microprocessor complex, to inhibit miR processing. Our findings reveal that RPL24, which has previously been shown to play a role in miR processing in Arabidopsis thaliana, has a similar evolutionarily conserved function in miR biogenesis in mammals. We thus characterize a novel extra-ribosomal role of RPL24 in primate miR regulation

Sex-specific declines in cholinergic-targeting tRNA fragments in the nucleus accumbens in Alzheimer\u27s disease

INTRODUCTION: Females with Alzheimer\u27s disease (AD) suffer accelerated dementia and loss of cholinergic neurons compared to males, but the underlying mechanisms are unknown. Seeking causal contributors to both these phenomena, we pursued changes in transfer RNS (tRNA) fragments (tRFs) targeting cholinergic transcripts (CholinotRFs). METHODS: We analyzed small RNA-sequencing (RNA-Seq) data from the nucleus accumbens (NAc) brain region which is enriched in cholinergic neurons, compared to hypothalamic or cortical tissues from AD brains; and explored small RNA expression in neuronal cell lines undergoing cholinergic differentiation. RESULTS: NAc CholinotRFs of mitochondrial genome origin showed reduced levels that correlated with elevations in their predicted cholinergic-associated mRNA targets. Single-cell RNA seq from AD temporal cortices showed altered sex-specific levels of cholinergic transcripts in diverse cell types; inversely, human-originated neuroblastoma cells under cholinergic differentiation presented sex-specific CholinotRF elevations. DISCUSSION: Our findings support CholinotRFs contributions to cholinergic regulation, predicting their involvement in AD sex-specific cholinergic loss and dementia

Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via β-Klotho and perilipin 2

Objective: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5′tRF transfer RNA fragments and microRNA miR-194-5p. Methods: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5′tRF and miR-194-5p. Results: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5′tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5′tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5′tRF mimic elevated mRNA levels of the metabolic regulator β-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5′tRF levels. Conclusion: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5′tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis

Knockout of the longevity gene Klotho perturbs aging and Alzheimer\u27s disease-linked brain microRNAs and tRNA fragments.

Overexpression of the longevity gene Klotho prolongs lifespan, while its knockout shortens lifespan and impairs cognition via perturbation of myelination and synapse formation. However, comprehensive analysis of Klotho knockout effects on mammalian brain transcriptomics is lacking. Here, we report that Klotho knockout alters the levels of aging- and cognition related mRNAs, long non-coding RNAs, microRNAs and tRNA fragments. These include altered neuronal and glial regulators in murine models of aging and Alzheimer\u27s disease and in human Alzheimer\u27s disease post-mortem brains. We further demonstrate interaction of the knockout-elevated tRNA fragments with the spliceosome, possibly affecting RNA processing. Last, we present cell type-specific short RNA-seq datasets from FACS-sorted neurons and microglia of live human brain tissue demonstrating in-depth cell-type association of Klotho knockout-perturbed microRNAs. Together, our findings reveal multiple RNA transcripts in both neurons and glia from murine and human brain that are perturbed in Klotho deficiency and are aging- and neurodegeneration-related

Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a “changing of the guards” between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level

Transfer RNA fragments replace microRNA regulators of the cholinergic post-stroke immune blockade

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced two days after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by RT-qPCR validated the top 6 upregulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes upregulated the top 6 stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using an ssRNA mimic induced downregulation of immune regulator Z-DNA binding protein 1 (Zbp1). In summary, we identified a “changing of the guards” between RNA types that may systemically affect homeostasis in post-stroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein- and RNA-level. Significance Statement Ischemic stroke triggers peripheral immunosuppression, increasing the susceptibility to post-stroke pneumonia that is linked with poor survival. The post-stroke brain initiates intensive communication with the immune system, and acetylcholine contributes to these messages; but the responsible molecules are yet unknown. We discovered a “changing of the guards,” where microRNA levels decreased but small transfer RNA fragments (tRFs) increased in post-stroke blood. This molecular switch may re-balance acetylcholine signaling in CD14+ monocytes by regulating their gene expression and modulating post-stroke immunity. Our observations point out to tRFs as molecular regulators of post-stroke immune responses that may be potential therapeutic targets