Biology subjects

Chu, Y.

Publications and source records attributed to Chu, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Comparing alpha-synuclein-interactomes between multiple systems atrophy and Parkinsons disease reveals unique and shared pathological features.

IntroductionPrimary synucleinopathies, such as Parkinsons disease (PD), Dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are neurodegenerative disorders with some shared clinical and pathological features. Aggregates of alpha-synuclein (syn) phosphorylated at serine 129 (PSER129) are the hallmark of synucleinopathies, which for PD/DLB are found predominantly in neurons (Neuronal cytoplasmic inclusions "NCIs"), but for MSA, aggregates are primarily found in oligodendroglia (Glial cytoplasmic inclusions "GCIs"). It remains unclear if the distinct pathological presentation of PD/DLB and MSA are manifestations of distinct or shared pathological processes. We hypothesize that the distinct synucleinopathies MSA and PD/DLB share common molecular features. MethodsUsing the in-situ proximity labeling technique biotinylation by antibody recognition (BAR), we compare aggregated syn-interactomes (BAR-PSER129) and total syn-interactomes (BAR-MJFR1) between MSA (n=5) and PD/DLB (n=10) in forebrain and midbrain structures. ResultsFor BAR-PSER129 and BAR-MJFR1 captures, syn was the most significantly enriched protein in PD/DLB and MSA. In PD/DLB, BAR-PSER129 identified 194 syn-aggregate-interacting proteins, while BAR-MJFR1 identified 245 syn interacting proteins. In contrast, in the MSA brain, only 38 and 175 proteins were identified for each capture, respectively. When comparing MSA and PD/DLB, a high overlap (59.5%) was observed between BAR-MJFR1 captured proteins, whereas less overlap (14.4%) was observed for BAR-PSER129. Direct comparison between MSA and PD/DLB revealed 79 PD/DLB-associated proteins and only three MSA-associated proteins (CBR1, CRYAB, and GFAP). Pathway enrichment analysis revealed PD/DLB interactions were dominated by vesicle/SNARE-associated pathways, in contrast to MSA, which strongly enriched for metabolic/catabolic, iron, and cellular oxidant detoxification pathways. A subnetwork of cytosolic antioxidant enzymes called peroxiredoxins drove cellular detoxification pathways in MSA. A common network of 26 proteins, including neuronal-specific proteins (e.g., SNYGR3) with HSPA8 at the core, was shared between MSA and DLB/PD. Extracellular exosome pathways were universally enriched regardless of disease or BAR target protein. ConclusionSynucleinopathies have divergent and convergent syn-aggregate interactions, indicating unique and shared pathogenic mechanisms. MSA uniquely involves oxidant detoxification processes in glial cells, while vesicular processes in neurons dominate PD/DLB. Shared interactions, specifically SNYGR3 (i.e., a neuronal protein), between MSA and PD/DLB suggest neuronal axons origin for both diseases. In conclusion, we provide syn aggregates protein interaction maps for two distinct synucleinopathies.

neuroscience↗

bioRxiv2024

Enhancing Variant Calling in Whole Exome Sequencing (WES) Data Using Population-Matched Reference Genomes

Whole exon sequencing (WES) data are frequently used for cancer diagnosis and genome-wide association studies (GWAS), hinging upon high-coverage read mapping, informative variant calling, and high-quality reference genomes. The center position of the currently used genome assembly, GRCh38, is now challenged by two newly publicized telomere-to-telomere or T2T genomes, T2T-CHM13 and T2T-YAO, and it becomes urgent to have a comparative study to test population specificity using the three reference genomes based on real case WES data. We here report our analysis along this line for 19 tumor samples collected from Chinese patients. The primary comparison of the exon regions among the three references reveals that the sequences in up to [~]1% target regions in YAO are widely diversified from GRCh38 and may lead to off-target in sequence capture. However, YAO still outperforms GRCh38 genomes by obtaining 7.41% more mapped reads. Due to more reliable read-mapping and closer phylogenetic relationship with the samples than GRCh38, YAO reduces half of variant calls of clinical significance which are mostly benign while keeping sensitivity in identifying pathogenic variants. YAO also outperforms CHM13 in reducing calls of Chinese-specific variants. Our findings highlight the critical need for employing population-specific reference genomes in genomic analysis to ensure accurate variant analysis and the significant benefits of tailoring these approaches to the unique genetic backgrounds of each ethnic group.

genomics↗

bioRxiv2024

A host enzyme reduces non-alcoholic fatty liver disease by inactivating intestinal lipopolysaccharide

The incidence of Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) has been increasing world-wide. Since gut-derived bacterial lipopolysaccharides (LPS) can travel via the portal vein to the liver and play an important role in producing hepatic pathology, it seemed possible that (1) LPS stimulates hepatic cells to accumulate lipid, and (2) inactivating LPS can be preventive. Acyloxyacyl hydrolase (AOAH), the eukaryotic lipase that inactivates LPS and oxidized phospholipids, is produced in the intestine, liver, and other organs. We fed mice either normal chow or a high-fat diet for 28 weeks and found that Aoah-/- mice accumulated more hepatic lipid than did Aoah+/+ mice. In young mice, before increased hepatic fat accumulation was observed, Aoah-/-mouse livers increased their abundance of Sterol Regulatory Element-Binding Protein 1 (SREBP1) and the expression of its target genes that promote fatty acid synthesis. Aoah-/- mice also increased hepatic expression of CD36 and Fabp3, which mediate fatty acid uptake, and decreased expression of fatty acid oxidation-related genes Acot2 and Ppar-. Our results provide evidence that increasing AOAH abundance in the gut, bloodstream and/or liver may be an effective strategy for preventing or treating MASLD.

immunology↗

bioRxiv2024

Biochemical characterization of extended-spectrum β-lactamases from Akkermansia genus

Akkermansia muciniphila, a member of the Verrucomicrobiota phylum, is recognized as a key gut microbe and has emerged as a potential next-generation probiotic. Assessment of antibiotic resistance in probiotics is a prerequisite for their therapeutic application, while very few is known in Akkermansia species. Firstly, we screened eight representative class A {beta}-lactamases (36.90%-41.30% identity with known {beta}-lactamases) from the Akkermansia species, which could increase the minimum inhibitory concentration (MIC) of Escherichia coli to{beta}-lactams. Secondly, four{beta}-lactamases were purified and identified as extended-spectrum {beta}-lactamase because they exhibited hydrolase activity against 19 {beta}-lactam antibiotics from penicillin, cephalosporins, and monobactam classes. Based on sequence alignment, three-dimensional structure, and binding pocket information, we hypothesized and validated that serine at 51 position was catalytic amino acid. Thirdly, the genomic context analysis revealed the absence of mobile genetic elements or other antibiotic resistance genes surrounding {beta}-lactamase genes, suggesting that the {beta}-lactamases from Akkermansia species may not be transferable. The finding and biochemical characterization of {beta}-lactamase from Akkermansia species provide a foundational basis for the safety evaluation of Akkermansia species as probiotics.

microbiology↗

bioRxiv2024

SwinCell: a transformer-based framework for dense 3D cellular segmentation

Segmentation of three-dimensional (3D) cellular images is fundamental for studying and understanding cell structure and function. However, 3D cellular segmentation is challenging, particularly for dense cells and tissues. This challenge arises mainly from the complex contextual information within 3D images, anisotropic properties, and the sensitivity to internal cellular structures, which often lead to miss-segmentation. In this work, we introduce SwinCell, a 3D transformer-based framework that leverages Swin-transformer for flow prediction and effectively distinguishes individual cell instances in 3D. We demonstrate the broad utility of the SwinCell in the segmentation of nuclei, colon tissue cells, and dense cultured cells. SwinCell strikes a balance between maintaining detailed local feature recognition and understanding broader contextual information. Tested extensively with both public and in-house 3D cell imaging datasets, SwinCell shows superior performance in segmenting dense cells in 3D, making it a powerful 3D segmentation tool for cellular analysis that could expedite research in cell biology and tissue engineering.

cell biology↗

bioRxiv2024

Gut microbes of the cecum versus the colon drive more severe lethality and multi-organ damage

An intestinal perforation or puncture leads to a high risk of sepsis-associated morbidity. A perforation initiates the transfer of the intestinal contents (ICs) to the peritoneal cavity, leading to abdominal infections and varying with different prognoses. However, the mechanisms associated with different perforations in the cecum and colon remain unknown. We sought to examine how different gut flora contribute to prognoses in different intestinal perforation sites. We compared the microbiome of the ICs in the cecum and colon in a fecal-induced peritonitis mouse model. The results showed that cecum ICs developed more severe sepsis than colon ICs, including a shorter median survival time, increased biochemical indicators, more pathological changes in multiple organs and overwhelmed systematic inflammation. Moreover, our results demonstrated that cecum ICs hold more bacterial burden in unit weight than colon ICs, and the microbial communities differed between the ICs from the cecum and colon. A more detailed comparison of the two microbiome groups showed that the abundance of potentially pathogenic bacteria increased in the cecum ICs. Our data suggest that the sepsis severity developed by perforation was associated with bacterial burden and increased abundance of potentially pathogenic bacteria in the cecum. Our findings first compared the differences in the lethality associated with the ICs of the cecum and colon, which pointed out that the site of perforation could help providers predict the severity of sepsis.

immunology↗

bioRxiv2024

Exploring the conformational space of protein-protein complex with transformer-based generative model

Protein-protein interactions are the basis of many protein functions, and understanding the contact and conformational changes of protein-protein interactions is crucial for linking protein structure to biological function. Although difficult to detect experimentally, molecular dynamics (MD) simulations are widely used to study the conformational ensembles and dynamics of protein-protein complexes, but there are significant limitations in sampling efficiency and computational costs. In this study, a generative neural network was trained on protein-protein complex conformations obtained from molecular simulations to directly generate novel conformations with physical realism. We demonstrated the use of a deep learning model based on the transformer architecture to explore the conformational ensembles of protein-protein complexes through MD simulations. The results showed that the learned latent space can be used to generate unsampled conformations of protein-protein complexes for obtaining new conformations complementing pre-existing ones, which can be used as an exploratory tool for the analysis and enhancement of molecular simulations of protein-protein complexes.

bioinformatics↗

bioRxiv2024

Characterization of RNA editing profiles in rice endosperm development

Rice (Oryza sativa L.) endosperm provides nutrients for seed germination and determines grain yield. RNA editing, a post-transcriptional modification essential for plant development, unfortunately, is not fully characterized during rice endosperm development. Here, we conduct genome re-sequencing and RNA sequencing for rice endosperms across five successive developmental stages and perform systematic analyses to characterize RNA editing profiles during rice endosperm development. We find that the majority of their editing sites are C-to-U CDS-recoding in mitochondria, leading to increased hydrophobic amino acids, and affecting structures and functions of mitochondrial proteins. Comparative analysis of RNA editing profiles across the five developmental stages reveals that CDS-recoding sites present higher editing frequencies with lower variabilities, and recoded amino acids, particularly caused by these sites with higher editing frequencies, tend to exhibit stronger evolutionary conservation across many land plants. Based on these results, we further classify mitochondrial genes into three groups that present distinct patterns in terms of editing frequency and variability of CDS-recoding sites. Besides, we identify a series of P- and PLS-class pentatricopeptide repeat (PPR) proteins with editing potential and construct PPR-RNA binding profiles, yielding candidate PPR editing factors related to rice endosperm development. Taken together, our findings provide valuable insights for deciphering fundamental mechanisms of rice endosperm development underlying RNA editing machinery. Author summaryRice endosperm development, a critical process determining quality and yield of our mankinds essential food, is regulated by RNA editing that provokes RNA base alterations by protein factors. However, our understanding of this regulation is incomplete. Hence, we systematically characterize RNA editing profiles during rice endosperm development. We find that editing sites resulting in amino acid changes, called "CDS-recoding", predominate in mitochondria, leading to increased hydrophobic amino acids and affecting structures and functions of proteins. Comparative analysis of RNA editing profiles during rice endosperm development reveals that CDS-recoding sites present higher editing frequencies with lower variabilities. Furthermore, evolutionary conservation of recoded amino acids caused by these CDS-recoding sites is positively correlated with editing frequency across many land plants. We classify mitochondrial genes into three groups that present distinct patterns in terms of editing frequency and variability of CDS-recoding sites, indicating different effects of these genes on rice endosperm development. In addition, we identify candidate protein factors associated closely with RNA editing regulation. To sum up, our findings provide valuable insights for fully understanding the role of RNA editing during rice endosperm development.

bioinformatics↗

bioRxiv2024

Characterization of Cancer Stem Cells in Laryngeal Squamous Cell Carcinoma by Single-Cell RNA Sequencing

Cancer stem cells (CSCs) constitute a pivotal element within the tumor microenvironment (TME), driving the initiation and progression of cancer. However, the identification of CSCs and their underlying molecular mechanisms in laryngeal squamous cell carcinoma (LSCC) remains a formidable challenge. We employed single-cell RNA sequencing of matched primary tumor tissues, paracancerous tissues, and local lymph nodes from three LSCC patients. Two distinct clusters of stem cells originating from epithelial populations were delineated and verified as CSCs and normal stem cells (NSCs) respectively. CSCs were abundant in the paracancerous tissues compared to the tumor tissues. CSCs showed high expression of stem cell marker genes such as PROM1, ALDH1A1, and SOX4, and increased activity of tumor-related hypoxia, Wnt/{beta}-catenin, and notch signaling pathways. We then explored the intricate crosstalk between CSCs and the TME cells and identified targets within the TME that related with CSCs. We also find eight marker genes of CSCs that correlated significantly with the prognosis of LSCC patients. Furthermore, bioinformatics analyses showed that drugs such as erlotinib, OSI-027, and ibrutinib selectively targeted the CSC-specifically expressed genes. In conclusion, our results represent the first comprehensive characterization of CSCs properties in LSCC at the single-cell level.

bioinformatics↗

bioRxiv2023

A 5' UTR Language Model for Decoding Untranslated Regions of mRNA and Function Predictions

The 5 UTR, a regulatory region at the beginning of an mRNA molecule, plays a crucial role in regulating the translation process and impacts the protein expression level. Language models have showcased their effectiveness in decoding the functions of protein and genome sequences. Here, we introduced a language model for 5 UTR, which we refer to as the UTR-LM. The UTR-LM is pre-trained on endogenous 5 UTRs from multiple species and is further augmented with supervised information including secondary structure and minimum free energy. We fine-tuned the UTR-LM in a variety of downstream tasks. The model outperformed the best-known benchmark by up to 42% for predicting the Mean Ribosome Loading, and by up to 60% for predicting the Translation Efficiency and the mRNA Expression Level. The model also applies to identifying unannotated Internal Ribosome Entry Sites within the untranslated region and improves the AUPR from 0.37 to 0.52 compared to the best baseline. Further, we designed a library of 211 novel 5 UTRs with high predicted values of translation efficiency and evaluated them via a wet-lab assay. Experiment results confirmed that our top designs achieved a 32.5% increase in protein production level relative to well-established 5 UTR optimized for therapeutics.

bioinformatics↗

bioRxiv2023

Conditioned pain modulation of pressure pain is associated with reduced activation in the parietal operculum and posterior insula

Conditioned pain modulation (CPM) is the phenomenon of pain inhibiting further pain. CPM is impaired in patients suffering from various chronic pain conditions and may resemble a pro-nociceptive profile. A possible neural mechanism for CPM is descending control from cortical regions via brainstem that modulates neural activity at the level of the dorsal horn of the spinal cord where noxious input first arrives. We studied the involvement of the descending pain modulatory pathway by simultaneous functional imaging of the brain, brainstem, rostral medulla, and cervical spinal cord during CPM with pressure cuff algometry in 42 healthy human participants. Subjective CPM as measured by decreased pain ratings developed over the experiment with application of repeated short test stimuli for long conditioning pain compared with non-painful conditioning. Test stimuli paired with painful conditioning were associated with decreased BOLD fMRI activity in the parietal operculum (secondary somatosensory cortex) and posterior insula, as well as increased responses in bilateral primary somatosensory cortices. Subjective CPM reflected in pain ratings was associated with decreased test stimulus responses in somatosensory and pain-responsive brain regions such as the anterior and posterior insula and the parietal operculum. Conditioning pain and test pain were associated with increased neural activity in stimulus-ipsilateral spinal cord dorsal horn at segment C6 and C4, respectively. No evidence for neural correlates of CPM or associations with subjective CPM was observed in the spinal cord. Functional connectivity between the brain and spinal cord was altered during both conditioning and test pain but did not depend on conditioning intensity. Taken together, we observed neural activity associated with both negative (CPM) and positive modulation of pain in different somatosensory and pain-associated brain regions, and an association of these and other neural responses with subjective CPM. While we found that painful stimuli altered the connectivity along the descending pain modulatory pathway, we did not find evidence for a modulation by conditioning intensity nor neural correlates of CPM in the spinal cord.

neuroscience↗

bioRxiv2023

Actomyosin-II Proactively Shields Axons of the Central Nervous System from Mild Mechanical Stress

SummaryPan et al found that actomyosin-II-driven radial contractility underpins the resilience of central axons to mild mechanical stress by suppressing the propagation and firing of injurious Ca2+ waves. Boosting actomyosin-II activity alleviates axon degeneration in mice with traumatic brain injury. Traumatic brain injury (TBI) remains a significant and unmet health challenge. However, our understanding of how neurons, particularly their fragile axons, withstand the abrupt mechanical impacts within the central nervous system remains largely unknown. Using a microfluidic device applying discrete levels of transverse forces to axons, we identified the stress levels that most axons could resist and explored their instant responses at nanoscale resolution. Mild stress induces rapid and reversible axon beading, driven by actomyosin-II-dependent radial contraction, which restricts the spreading and bursting of stress-induced Ca2+ waves. More severe stress causes irreversible focal swelling and Ca2+ overload, ultimately leading to focal axonal swelling and degeneration. Up-regulating actomyosin-II activity prevented the progression of initial injury in vivo, protecting commissural axons from degeneration in a mice TBI model. Our study established a scalable axon injury model and uncovered the critical roles of actomyosin-II in shielding neurons against detrimental mechanical stress.

neuroscience↗

bioRxiv2023

Correlative single-cell X-ray tomography and X-ray fluorescence imaging

X-ray tomography (XRT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XRT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XRT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.

cell biology↗

bioRxiv2023

T2T-YAO: a Telomere-to-telomere Assembled Diploid Reference Genome for Han Chinese

Since its initial release in 2001, the human reference genome has been continuously improved in both continuity and accuracy, and the recently-released telomere-to-telomere version--T2T-CHM13--reaches its top quality after 20 years of effort. However, T2T-CHM13 does not represent an authentic diploid human genome, but rather one derived from a simplified, nearly homozygous genome of a hydatidiform mole cell line. To address this limitation and provide an alternative pertinent to the Chinese population, the largest ethnic group in the world, we have assembled a complete diploid human genome of a male Han Chinese, T2T-YAO, which includes telomere-to-telomere assemblies for all the 22+X+M and 22+Y chromosomes in his two haploids inherited separately from his parents. Both haplotypes contain no artificial sequences or model nucleotides and possess a high quality comparable to CHM13, with fewer than one error per [~]14 Mb. Derived from the individual who lives in the aboriginal region of Han Chinese, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors of the Han population. Each haplotype of T2T-YAO possesses [~]340 Mb exclusive sequences and [~]3100 unique genes as compared to CHM13, and their genome sequences show greater genetic distance to CHM13 than to each other in terms of nucleotide polymorphism and structural variations. The construction of T2T-YAO would serve as a high-quality diploid reference that enables precise delineation of genomic variations in a haplotype-sensitive manner, which could advance our understandings in human evolution, hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population.

genetics↗

bioRxiv2023

Modification of social cheating facilitates the stabilization and chronic infection of polymorphic Pseudomonas aeruginosa population

Chronic infection of the common bacterial pathogen Pseudomonas aeruginosa frequently leads to the coexistence of heterogeneous individuals to engage in several group behaviors. However, further evolution of the polymorphic P. aeruginosa population, including the dynamic change of social cooperation and its impact on host immune system, still remain elusive. We show that the evolution of P. aeruginosa in the patients with chronic obstructive pulmonary disease frequently selects the isolates deficient in producing the costly and sharable extracellular products for nutrient acquisition. The evolution of polymorphic P. aeruginosa population is mainly concentrated on modifying the adaptability of lasR mutants, which are typical cheaters in the competition of quorum-sensing-controlled extracellular proteases. Importantly, lasR mutants with varying degrees of evolution interact with wild-type P. aeruginosa in a framework termed cascaded public goods game to compete for extracellular proteases and siderophores, and thus perpetuate social cooperation under different conditions. Finally, we find that a polymorphic population comprised of lasR-intact P. aeruginosa and evolved lasR-mutant can minimize the host immune fluctuation for persistent colonization. This study demonstrates the multistage evolution and complex interaction of P. aeruginosa in adaptation to host lungs, and provides an explanation for the success of cooperation in public goods game.

microbiology↗

bioRxiv2023

Ventral premotor cortex influences spinal cord activation during force generation

Force generation is a crucial element of dexterity and a highly relevant skill of the human motor system. How cerebral and spinal components interact and how spinal activation is influenced by cerebral primary motor and premotor areas is poorly understood. Here we conducted combined cortico-spinal functional MRI during a simple visually guided isometric force generation task in a group of 20 healthy young subjects. Activation was localized in the ipsilateral cervical spinal cord and contralateral primary motor and premotor areas. The main finding is that spinal activation was influenced by ventral premotor cortex activation. Spinal activation was furthermore significantly correlated with primary motor cortex activation while increasing target forces led to an increase in the amount of activation. These data indicate that human premotor areas such as the ventral premotor cortex might be functionally connected to the lower cervical spinal cord contributing to distal upper limb functions, a finding which extends our understanding about human motor function beyond the animal literature.

neuroscience↗

bioRxiv2023

Impact of Peripheral Auditory Structure on the Development of Auditory-Language Network in Children with Profound Sensorineural Hearing Loss

Profound congenital sensorineural hearing loss (SNHL) prevents children from developing spoken language. Cochlear implantation and auditory brainstem implantation can provide hearing sensation, but language development outcomes can vary, particularly for patients with inner ear malformations and/or cochlear nerve deficiency (IEM&CND). Currently, the peripheral auditory structure is evaluated through visual inspection of clinical imaging, but this method is insufficient for surgical planning and prognosis. The central auditory pathway is also challenging to examine in vivo due to its delicate subcortical structures. Previous attempts to locate subcortical auditory nuclei using fMRI responses to sounds are not applicable to deaf patients. In this study, we developed a new pipeline for mapping the auditory pathway using structural and diffusional MRI. We used a fixel-based approach to investigate the structural development of the auditory-language network for profound SNHL children with normal peripheral structure and those with IEM&CND under six years old. Our findings indicate that the language pathway is more sensitive to peripheral auditory condition than the central auditory pathway, highlighting the importance of early intervention for profound SNHL children to provide timely speech inputs. We also propose a comprehensive pre-surgical evaluation extending from the cochlea to the auditory-language network, which has promising clinical potential.

neuroscience↗

bioRxiv2022

Prioritizing Annotated miRNAs: Only a Small Percentage are Candidates for Biological Regulation

The potential for miRNAs to regulate gene expression remains controversial. DROSHA initiates the biogenesis of miRNAs while Argonaute (AGO) and TNRC6 proteins form complexes with miRNAs that recognize RNA. Here we investigate the fate of miRNAs in the absence of critical RNAi protein factors. Knockout of DROSHA expression reduced levels of some miRNAs, but not others. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of miRNAs. Quantitative analysis indicates compensation to maintain the overall pool of AGO after knockout of AGO variants. Evaluation of miRNA binding to AGO proteins revealed that association between AGO and miRNAs was similar for AGO1 - 4. Contrary to the assumptions underlying many peer-reviewed reports, not all annotated miRNAs have equal potential as biological regulators. Cellular abundance, DROSHA dependence, and physical association with AGO must be considered when forming hypotheses related to their function. Our data prioritize sixty miRNAs - under two percent of the overall annotated miRNA repertoire - as being most likely to function as robust gene regulators. Our approach will facilitate identifying biologically active miRNAs.

biochemistry↗