Flexible scaffold-based cheminformatics approach for polypharmacological drug design.

Effective treatments for complex central nervous system (CNS) disorders require drugs with polypharmacology and multifunctionality, yet designing such drugs remains a challenge. Here, we present a flexible scaffold-based cheminformatics approach (FSCA) for the rational design of polypharmacological drugs. FSCA involves fitting a flexible scaffold to different receptors using different binding poses, as exemplified by IHCH-7179, which adopted a "bending-down" binding pose at 5-HT2AR to act as an antagonist and a "stretching-up" binding pose at 5-HT1AR to function as an agonist. IHCH-7179 demonstrated promising results in alleviating cognitive deficits and psychoactive symptoms in mice by blocking 5-HT2AR for psychoactive symptoms and activating 5-HT1AR to alleviate cognitive deficits. By analyzing aminergic receptor structures, we identified two featured motifs, the "agonist filter" and "conformation shaper," which determine ligand binding pose and predict activity at aminergic receptors. With these motifs, FSCA can be applied to the design of polypharmacological ligands at other receptors.

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo.

Short hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that, in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3' counting rules. These promiscuous noncanonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a "loop-counting rule," we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

3' Uridylation Confers miRNAs with Non-canonical Target Repertoires.

Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3' uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets.

Alcohol consumption may be a risk factor for cerebrovascular stenosis in acute ischemic stroke and transient ischemic attack.

BACKGROUND: Atherosclerosis are well established risk factors for ischemic stroke, however the association between alcohol consumption and atherosclerosis is controversial. This study aims to explore the potential correlation between alcohol consumption and cerebral stenosis in patients with acute ischemic stroke and transient ischemic attack (TIA). METHODS: Nine hundreds and eighty-eight patients with first acute ischemic stroke attack or TIA were recruited retrospectively. Alcohol consumption was classified into five consumption categories (non-drinkers, occasional drinkers, < 140 g per week [mild drinkers], 140-279 g per week [moderate drinkers], ≥ 280 g per week [heavy drinkers]). Computed tomography angiography (CTA) and digital subtraction angiography (DSA) were utilized to assess the carotid and cerebral artery in all patients. Five-step scale for degree of stenosis was applied: normal (0, 0 points), mild (< 50%, 1 point), moderate (50-69%, 2 points), severe (70-99%, 3 points), and occlusion (100%, 4 points). RESULTS: The carotid and cerebral artery stenosis scores were positively correlated with moderate alcohol consumption (B = 1.695, P < 0.001). Compared with nondrinkers, moderate alcohol consumption had significant increasing risk of moderate carotid and cerebral artery stenosis (OR = 4.28, 95% CI: 1.47-12.49, P = 0.008) and severe stenosis (OR = 4.24, 95% CI: 1.55-11.64, P = 0.005) and occlusion (OR = 3.87, 95% CI: 1.65-9.06, P = 0.002). Compared with nondrinkers, heavy alcohol consumption patients had significant higher risk of carotid and cerebral artery occlusion (OR = 2.71, 95% CI: 1.36-5.41, P = 0.005). CONCLUSIONS: Higher alcohol consumption may associate with higher risk and more severity of carotid and cerebrovascular stenosis.

Specifications of the ACMG/AMP Variant Classification Guidelines for Germline DICER1 Variant Curation.

Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification is essential for reliable diagnosis of DICER1-related tumor predisposition and identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP), to create DICER1- specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known Pathogenic/Likely Pathogenic (P/LP) variants, 12 known Benign/Likely Benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

Visible-light-initiated external photocatalyst-free synthesis of α,α-difluoro-ß-ketoamides from 4-aminocoumarins.

A concise and efficient ring-opening difluorination strategy was developed for the synthesis of highly functionalized hydroxy-containing α,α-difluoro-ß-ketoamides from the one-pot multicomponent reaction of 4-aminocoumarins, NFSI, and water in dimethyl carbonate (DMC) as a green solvent. The reactions were smoothly achieved under visible light irradiation in air at room temperature without the addition of any other external photocatalysts. With this protocol, various α,α-difluoro-ß-ketoamides were successfully synthesized under mild conditions (25 examples, 73-91% yields). This transition-metal-free synthetic procedure shows good functional group compatibility and attractive practical potential for large-scale synthesis.

Mining electronic health records using artificial intelligence: Bibliometric and content analyses for current research status and product conversion.

BACKGROUND: The use of Electronic Health Records is the most important milestone in the digitization and intelligence of the entire medical industry. AI can effectively mine the immense medical information contained in EHRs, potentially assist doctors in reducing many medical errors. OBJECTIVE: This article aims to summarize the research status and trends in using AI to mine medical information from EHRs for the past thirteen years and investigate its information application. METHODS: A systematic search was carried out in 5 databases, including Web of Science Core Collection and PubMed, to identify research using AI to mine medical information from EHRs for the past thirteen years. Furthermore, bibliometric and content analysis were used to explore the research hotspots and trends, and systematically analyze the conversion rate of research resources in this field. RESULTS: A total of 631 articles were included and analyzed. The number of published articles has increased rapidly after 2017, with an average annual growth rate of 55.73%. The US (41.68%) and China (19.65%) publish the most articles, but there is a lack of international cooperation. The extraction of disease lesions is a hot topic at present, and the research topic is gradually shifting from disease risk grading to disease risk prediction. Classification (66%), and regress (15%) are the main implemented AI tasks. For AI algorithms, deep learning (31.70%), decision tree algorithms family (26.47%), and regression algorithms family (17.43%) are used most frequently. The funding rate for publications is 69.26%, and the input-output conversion rate is 21.05%. CONCLUSIONS: Over the past decade, the use of AI to mine medical information from EHRs has been developing rapidly. However, it is necessary to strengthen international cooperation, improve EHRs data availability, focus on interpretable AI algorithms, and improve the resource conversion rate in future research.

Serum lactate dehydrogenase level predicts the prognosis in bladder cancer patients.

BACKGROUND: Recently, several studies investigated the association between lactate dehydrogenase (LDH) level and the prognosis of urothelial carcinoma. However, no studies explored the role of serum LDH level in the survival of overall bladder cancer (BC). In this study, we intended to address the association of LDH level with the prognosis of BC. METHODS: 206 patients with BC were included in this study. The clinical data and blood samples of patients were collected. The overall survival and progression-free survival were used. Kaplan-Meier method and Log rank test were used to evaluate the effects of LDH level on the survival of BC. Univariate and multivariate Cox regression analyses were utilized to identify prognosis predictors of BC. RESULTS: Data indicated that serum LDH level in the BC patients was significantly higher than those in controls. In addition, this study suggested that serum LDH level was associated with T stage, N stage, tumor size, M stage, pathological type, and lymphovascular invasion. The Kaplan-Meier analysis found significant differences in the OS and PFS rate between lower and higher serum LDH level groups (LDH ≥ 225 U/L and < 225 U/L). Multivariate Cox regression indicated that pathological type, T2-3, and higher level of LDH were independently associated with adverse prognosis in BC patients. CONCLUSION: The higher serum LDH level (≥ 225 U/L) is associated with poor prognosis in patients with BC. Serum LDH level could be used as a novel predictive biomarker for BC patients.

Histone methyltransferase enzyme enhancer of zeste homolog 2 counteracts ischemic brain injury via H3K27me3-mediated regulation of PI3K/AKT/mTOR signaling pathway.

BACKGROUND: Epigenetic histone methylation plays a crucial role in cerebral ischemic injury, particularly in the context of ischemic stroke. However, the complete understanding of regulators involved in histone methylation, such as Enhancer of Zeste Homolog 2 (EZH2), along with their functional effects and underlying mechanisms, remains incomplete. METHODS: Here, we employed a rat model of MCAO (Middle cerebral artery occlusion) and an OGD (Oxygen-Glucose Deprivation) model of primary cortical neurons to study the role of EZH2 and H3K27me3 in cerebral ischemia-reperfusion injury. The infarct volume was measured through TTC staining, while cell apoptosis was detected using TUNEL staining. The mRNA expression levels were quantified through quantitative real-time polymerase chain reaction (qPCR), whereas protein expressions were evaluated via western blotting and immunofluorescence experiments. RESULTS: The expression levels of EZH2 and H3K27me3 were upregulated in OGD; these expression levels were further enhanced by GSK-J4 but reduced by EPZ-6438 and AKT inhibitor (LY294002) under OGD conditions. Similar trends were observed for mTOR, AKT, and PI3K while contrasting results were noted for UTX and JMJD3. The phosphorylation levels of mTOR, AKT, and PI3K were activated by OGD, further stimulated by GSK-J4, but inhibited by EPZ-6438 and AKT inhibitor. Inhibition of EZH2 or AKT effectively counteracted OGD-/MCAO-induced cell apoptosis. Additionally, inhibition of EZH2 or AKT mitigated MCAO-induced infarct size and neurological deficit in vivo. CONCLUSIONS: Collectively, our results demonstrate that EZH2 inhibition exerts a protective effect against ischemic brain injury by modulating the H3K27me3/PI3K/AKT/mTOR signaling pathway. The results provide novel insights into potential therapeutic mechanisms for stroke treatment.

Tumor IsomiR Encyclopedia (TIE): a pan-cancer database of miRNA isoforms.

SUMMARY: MicroRNAs (miRNAs) are master regulators of gene expression in cancers. Their sequence variants or isoforms (isomiRs) are highly abundant and possess unique functions. Given their short sequence length and high heterogeneity, mapping isomiRs can be challenging; without adequate depth and data aggregation, low frequency events are often disregarded. To address these challenges, we present the Tumor IsomiR Encyclopedia (TIE): a dynamic database of isomiRs from over 10 000 adult and pediatric tumor samples in The Cancer Genome Atlas (TCGA) and The Therapeutically Applicable Research to Generate Effective Treatments (TARGET) projects. A key novelty of TIE is its ability to annotate heterogeneous isomiR sequences and aggregate the variants obtained across all datasets. Results can be browsed online or downloaded as spreadsheets. Here, we show analysis of isomiRs of miR-21 and miR-30a to demonstrate the utility of TIE. AVAILABILITY AND IMPLEMENTATION: TIE search engine and data are freely available to use at https://isomir.ccr.cancer.gov/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Flexible pri-miRNA structures enable tunable production of 5' isomiRs.

The Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5' isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that in addition to previously known features, the overall structural flexibility of pri-miRNA impact Drosha cleavage fidelity. Internal loops and nearby G · U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5' isomiR production. By analysing patient data deposited in the Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5' isomiR biogenesis in tumours.[Figure: see text].

Develop a stepwise integrated method to screen biomarkers of Baihe-Dihuang Tang on the treatment of depression in rats applying with composition screened, untargeted, and targeted metabolomics analysis.

Baihe-Dihuang Tang is a commonly prescribed remedy for depression. In this study, component screening with untargeted and targeted metabolomics was used to identify potential biomarkers for depression in chronic unpredictable mildly stressed rats. Using this novel identification method, the screening of organic acids, lily saponins, iridoids, and other ingredients formed the basis for subsequent metabolomics research. Baihe-Dihuang Tang supplementation in chronic unpredictable mild-stress-induced depression models, increased their body weight, sucrose preference, brain-derived neurotrophic factor deposition, and spatial exploring. Untargeted metabolomics revealed that Baihe-Dihuang Tang exerts its antidepressant effects by regulating the levels of lipids, organic acids, and its derivatives, and benzenoids in the brain, plasma, and urine of the depressed rats. Moreover, it also modulates the d-glutamine and d-glutamate metabolism and purine metabolism. Targeted metabolomics demonstrated significant reduction in l-glutamate levels in the brains of depressed rats. This could be a potential biomarker for depression. Baihe-Dihuang Tang alleviated depression by regulating the levels of l-glutamate, xanthine, and adenine in the brains of depressed rats. Together, these findings conclusively established the promising therapeutic effect of Baihe-Dihuang Tang on depression and also unraveled the underlying molecular mechanism of its potential antidepressant function.

Adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin signaling participates in the protective effect of chronic intermittent hypobaric hypoxia on vascular endothelium of metabolic syndrome rats.

Our previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) protects vascular endothelium function through ameliorating autophagy in mesenteric arteries of metabolic syndrome (MS) rats. This study aimed to investigate the role of adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling in CIHH effect. Six-week-old male Sprague-Dawley rats were divided into control (CON), MS model, CIHH treatment (CIHH), and MS + CIHH groups. Serum pro-inflammatory cytokines were measured. The endothelium dependent relaxation (EDR), endothelial ultrastructure and autophagosomes were observed in mesenteric arteries. The expression of phosphor (p)-AMPKα, p-mTOR, autophagy-related and endoplasmic reticulum stress-related proteins, p-endothelial nitric oxide synthase, and cathepsin D were assayed. In MS rats, pro-inflammatory cytokines were increased, EDR was attenuated, and endothelial integrity was impaired. In addition, the expression level of p-AMPKα and cathepsin D was down-regulated, but the level of p-mTOR was up-regulated. While in MS + CIHH rats, all aforementioned abnormalities were ameliorated, and the beneficial effect of CIHH was cancelled by AMPKα inhibitor. In conclusion, AMPK-mTOR signaling pathway participates in the protection of CIHH on vascular endothelium of MS rats.

Ligand Strain Energy in Large Library Docking.

While small molecule internal strain is crucial to molecular docking, using it in evaluating ligand scores has remained elusive. Here, we investigate a technique that calculates strain using relative torsional populations in the Cambridge Structural Database, enabling fast precalculation of these energies. In retrospective studies of large docking screens of the dopamine D4 receptor and of AmpC ß-lactamase, where close to 600 docking hits were tested experimentally, including such strain energies improved hit rates by preferentially reducing the ranks of strained high-scoring decoy molecules. In a 40-target subset of the DUD-E benchmark, we found two thresholds that usefully distinguished between ligands and decoys: one based on the total strain energy of the small molecules and another based on the maximum strain allowed for any given torsion within them. Using these criteria, about 75% of the benchmark targets had improved enrichment after strain filtering. Relying on precalculated population distributions, this approach is rapid, taking less than 0.04 s to evaluate a conformation on a standard core, making it pragmatic for precalculating strain in even ultralarge libraries. Since it is scoring function agnostic, it may be useful to multiple docking approaches; it is openly available at http://tldr.docking.org.

A novel sensitive ACNTs-MoO2 SERS substrate boosted by synergistic enhancement effect.

Integrating chemical enhancement (CM) and electromagnetic enhancement (EM) into one substrate is of great significance, but as far as we know, little research has been done on this project. In this paper, the novel bead chain like acidified carbon nanotubes-MoO2 (ACNTs-M) were designed by a simple two-step hydrothermal synthesis method. Benefitting from a good adsorption capacity, chemical enhancement and surface electromagnetic field enhancement effect, ACNTs-M exhibits a stunning SERS performance. The maximum enhancement factor (EF) of 5.13 × 107 is obtained with R6G molecules on ACNTs-M. The limit of detection (LOD) of R6G is 10-10 M. In addition, ACNTs-M also exhibits SERS sensitivity of other organic dyes (CV, RhB and MB). The results of Raman signal enhancement mechanism research verified that the synergy of CM and EM is the reason for the high SERS sensitivity of ACNTs-M. We believe that our work may bring cutting edge of development of stable and highly sensitive nonmetal SERS substrates.

The molecular mechanism of dsRNA processing by a bacterial Dicer.

Members of the ribonuclease (RNase) III family regulate gene expression by processing dsRNAs. It was previously shown that Escherichia coli (Ec) RNase III recognizes dsRNA with little sequence specificity and the cleavage products are mainly 11 nucleotides (nt) long. It was also shown that the mutation of a glutamate (EcE38) to an alanine promotes generation of siRNA-like products typically 22 nt long. To fully characterize substrate specificity and product size of RNase IIIs, we performed in vitro cleavage of dsRNAs by Ec and Aquifex aeolicus (Aa) enzymes and delineated their products by next-generation sequencing. Surprisingly, we found that both enzymes cleave dsRNA at preferred sites, among which a guanine nucleotide was enriched at a specific position (+3G). Based on sequence and structure analyses, we conclude that RNase IIIs recognize +3G via a conserved glutamine (EcQ165/AaQ161) side chain. Abolishing this interaction by mutating the glutamine to an alanine eliminates the observed +3G preference. Furthermore, we identified a second glutamate (EcE65/AaE64), which, when mutated to alanine, also enhances the production of siRNA-like products. Based on these findings, we created a bacterial Dicer that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.

KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182.

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.

Differential Roles of Extracellular Histidine Residues of GPR68 for Proton-Sensing and Allosteric Modulation by Divalent Metal Ions.

GPR68, an orphan G-protein coupled receptor, senses protons, couples to multiple G-proteins, and is also activated or inhibited by divalent metal ions. It has seven extracellular histidine residues, although it is not clear how these histidine residues play a role in both proton-sensing and metal ion modulation. Here we demonstrate that divalent metal ions are allosteric modulators that can activate or inhibit proton activity in a concentration- and pH-dependent manner. We then show that single histidine mutants have differential and varying degrees of effects on proton-sensing and metal ion modulation. Some histidine residues play dual roles in proton-sensing and metal ion modulation, while others are important in one or the other but not both. Two extracellular disulfide bonds are predicted to constrain histidine residues to be spatially close to each other. Combining histidine mutations leads to reduced proton activity and resistance to metal ion modulation, while breaking the less conserved disulfide bond results in a more severe reduction in proton-sensing over metal modulation. The small-molecule positive allosteric modulators (PAMs) ogerin and lorazepam are not affected by these mutations and remain active at mutants with severely reduced proton activity or are resistant to metal ion modulation. These results suggest GPR68 possesses two independent allosteric modulation systems, one through interaction with divalent metal ions at the extracellular surface and another through small-molecule PAMs in the transmembrane domains. A new GPR68 model is developed to accommodate the findings which could serve as a template for further studies and ligand discovery by virtual ligand docking.

QuagmiR: a cloud-based application for isomiR big data analytics.

SUMMARY: MicroRNAs (miRNAs) function as master regulators of gene expression. Recent studies demonstrate that miRNA isoforms (isomiRs) play a unique role in cancer development. Here, we present QuagmiR, the first cloud-based tool to analyze isomiRs from next generation sequencing data. Using a novel and flexible searching algorithm designed for the detection and annotation of heterogeneous isomiRs, it permits extensive customization of the query process and reference databases to meet the user 's diverse research needs. AVAILABILITY AND IMPLEMENTATION: QuagmiR is written in Python and can be obtained freely from GitHub (https://github.com/Gu-Lab-RBL-NCI/QuagmiR). QuagmiR can be run from the command line on local machines, as well as on high-performance servers. A web-accessible version of the tool has also been made available for use by academic researchers through the National Cancer Institute-funded Seven Bridges Cancer Genomics Cloud (https://cancergenomicscloud.org). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.