Biology subjects

Chakrabarti, A.

Publications and source records attributed to Chakrabarti, A..

16 recordsLinked to original sources

bioRxiv2025

Antifungal susceptibility profile of clinically relevant species of the genus Sporothrix: establishment of Epidemiological Cutoff Values (ECVs) according to CLSI broth microdilution methodology

Sporotrichosis is a globally distributed subcutaneous mycosis caused mainly by Sporothrix brasiliensis, S. schenckii, and S. globosa. Cat-transmitted sporotrichosis, primarily caused by S. brasiliensis in South America and to a lesser extent by S. schenckii in Southeast Asia, is emerging as a significant public health concern, due its outbreak potential. Itraconazole is the first-choice drug for treatment of human and cats, but reduced susceptibility has been reported based on previously proposed epidemiological cut-off values (ECVs). To support resistance surveillance, we aimed to establish CLSI-endorsed ECVs for these clinically relevant Sporothrix species. A total of 3,588 minimum inhibitory concentration (MIC) values for seven antifungal agents (amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, olorofim, and terbinafine) were obtained from 19 international laboratories. Four of seven antifungals met the CLSI M57 guidelines criteria to determine the ECV. Established ECVs for amphotericin B were found to be high with 8 {micro}g/mL for S. brasiliensis and S. globosa, and 4 {micro}g/mL for S. schenckii. Itraconazole ECVs were 4 {micro}g/mL for S. brasiliensis and S. schenckii. Posaconazole ECVs were 4 {micro}g/mL for all three species (tentative for S. globosa), while the terbinafine ECV for S. brasiliensis was 0.12 {micro}g/mL. Olorofim demonstrated good in vitro activity, particularly against S. brasiliensis. Overall, this study establishes validated ECVs for key antifungals against Sporothrix species and identifies a low prevalence of non-wild type (NWT) isolates, supporting ongoing antifungal resistance monitoring.

microbiology↗

bioRxiv2025

A chemically inducible multimerization system for tunable and background-free RTK activation

Receptor tyrosine kinases (RTKs) are key regulators of diverse cellular processes, including differentiation, migration, proliferation, survival, and intracellular communications, by transducing extracellular cues into intracellular responses. Upon oligomerization at the plasma membrane, RTKs become activated and initiate major downstream signaling cascades, such as the ERK pathway, which modulates cytoskeletal dynamics through phosphorylation of cytoskeletal regulators, regulation of actin-binding proteins, and transcriptional activation of immediate-early genes involved in cell structure and motility. Light-inducible RTK systems have been developed to achieve spatiotemporal control of RTK clustering and activation for both basic cell biology research, and engineered applications, such as controlling cell migration, proliferation, or differentiation. However, these systems are limited by high basal RTK activation, where substantial RTK activation occurs even before induction, leading to unintended ERK activation and downstream effects. Here, we report a chemically inducible RTK platform that minimizes basal activation while enabling direct visualization of RTK clustering at the plasma membrane upon induction. Single-cell imaging reveals visible RTK clusters after induction, with total RTK abundance in the clusters correlating with ERK phosphorylation levels. Using this system, we achieved precise and rapid control over multiple ERK-dependent cellular processes, including disassembly of spectrin-based membrane skeleton and nuclear entry of transcription factors STAT3 and CREB, while maintaining minimal basal activity before induction. In contrast to previously developed inducible RTK systems, which can perturb cytoskeletal structures or transcription factor dynamics even without stimulation, our design preserves native cellular architecture and nuclear signaling until activation is intentionally triggered. Collectively, these results establish our system as a robust and versatile platform for dissecting RTK signaling dynamics and engineering cell behaviors with precise, on-demand spatiotemporal control.

bioengineering↗

bioRxiv2025

The experimentally evolved fluconazole-resistant Clade II isolates of Candidozyma auris exhibit a distinct lipid compositional landscape, highlighting intra-clade sphingolipid heterogeneity

The intrinsic resistance of Candidozyma auris to antifungal drugs poses a major therapeutic challenge, with conventional resistance mechanisms providing only partial explanations. Sphingolipids (SLs), known for their interclade heterogeneity, play a crucial role in antifungal resistance. This study examined the SL landscape in two drug-susceptible clade II isolates, C-line and P-line, from distinct geographical origins, which were experimentally evolved to develop stable fluconazole (FLC) resistance. The progenitors displayed distinct SL profiles, P1 had higher PhytoCer and OHPhytoCer, indicating a more active acidic SL biosynthesis branch, whereas C1 exhibited elevated OHGlcCer, OHCer, and LCBs, reflecting a greater role of the neutral biosynthesis branch. The principal component analysis (PCA) also confirmed distinct segregation of the two progenitors. Upon evolution, P1.1 and C1.1 adaptors showed significant SL alterations. P1.1 exhibited PhytoCer enrichment, while C1.1 showed reduced OHGlcCer alongside increased PhytoCer, dhCer and OHPhytoCer levels. Notably, OHGlcCer remained unchanged in P1.1, whereas LCBs and OHPhytoCer decreased compared to P1. Despite these lineage-specific differences between the progenitors, both evolved replicates exhibited increased PhytoCer as a common denominator like what is also observed in clinical FLC-resistant isolates. These findings highlight intra-clade SL variability and suggest that specific SLs contribute to FLC resistance in C. auris.

microbiology↗

bioRxiv2024

Circadian rhythm disruption alters mammary gland morphology and accelerates cold aggressive tumorigenesis through a LILRB4-dependent pathway

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model that recapitulates the human basal type of breast cancer was used for this study. The effect of CRD on mammary gland morphology was investigated using wild-type mice model. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA seq. ScRNA seq was substantiated by multiplexing immunostaining, flow cytometry, and realtime PCR. The effect of LILRB4 immunotherapy on CRD-induced tumorigenesis was also investigated. Here we identified the impact of CRD on basal tumorigenesis and mammary gland morphology and identified the role of LILRB4 on CRD-induced lung metastasis. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden, and lung metastasis and induced an immunosuppressive tumor microenvironment by enhancing LILRB4a expression. Moreover, CRD increased the M2-macrophage and regulatory T-cell populations but decreased the M1-macrophage, and dendritic cell populations. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis. This study also establishes the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

cancer biology↗

bioRxiv2023

In Silico Therapeutic Intervention on Cytokine Storm in COVID-19

The recent global COVID-19 outbreak, attributed by the World Health Organization to the rapid spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), underscores the need for an extensive exploration of virological intricacies, fundamental pathophysiology, and immune responses. This investigation is vital to unearth potential therapeutic avenues and preventive strategies. Our study delves into the intricate interaction between SARS-CoV-2 and the immune system, coupled with exploring therapeutic interventions to counteract dysfunctional immune responses like the cytokine storm (CS), a driver of disease progression. Understanding these immunological dimensions informs the design of precise multiepitopetargeted peptide vaccines using advanced immunoinformatics and equips us with tools to confront the cytokine storm. Employing a control theory-based approach, we scrutinize the perturbed behavior of key proteins associated with cytokine storm during COVID-19 infection. Our findings support ACE2 activation as a potential drug target for CS control and confirm AT1R inhibition as an alternative strategy. Leveraging deep learning, we identify potential drugs to individually target ACE2 and AT1R, with Lomefloxacin and Fostamatinib emerging as standout options due to their close interaction with ACE2. Their stability within the protein-drug complex suggests superior efficacy among many drugs from our deep-learning analysis. Moreover, there is a significant scope for optimization in fine-tuning protein-drug interactions. Strong binding alone may not be the sole determining factor for potential drugs; precise adjustments are essential. The application of advanced computational power offers novel solutions, circumventing time-consuming lab work. In scenarios necessitating both ACE2 and AT1R targeting, optimal drug combinations can be derived from our analysis of drug-drug interactions, as detailed in the manuscript.

bioinformatics↗

bioRxiv2023

Microevolution of clade II isolates of Candida auris highlights multifaceted intra-clade heterogeneity in acquiring resistance towards amphotericin B

Candida auris exhibits high-level resistance to amphotericin B (AmB). Mechanisms such as ergosterol biosynthesis malfunction, oxidative damage mismanagement, and increased drug efflux contribute to AmB resistance in C. auris. In this study, we experimentally evolved two East Asian drug-susceptible clade II isolates of C. auris (P2428 and CBS10913T) isolated from different geographical locations to develop resistance against AmB. We analysed alterations in karyotype, genome sequence, and gene expression profiles to uncover the mechanisms driving AmB resistance. The independently evolved clade II adaptors displayed up to 4-16-fold higher MIC50, as compared to the parental cells. AOX2 (alternative oxidase) and the cell wall integrity pathway have been identified as critical in the development of AmB resistance. However, we noted certain intra-clade heterogeneity in the associated mechanisms. While in P2428 adaptors (P-lines), the ergosterol and sphingolipid pathways appear to play a crucial role, this was not the case for CBS10913T adaptors (A-lines), which acquired resistance independent of lipid-associated changes. The transcriptomic, WGS, and phenotypic analyses also confirm that the evolved AmB-resistant isolates follow distinct trajectories for adaptation, Furthermore, unlike the fluconazole-resistant isolates, as reported previously, changes in ploidy do not seem to contribute to the differential mechanisms of AmB resistance. Overall, this study not only provides insights into the mechanisms and pathways involved in AmB resistance but also highlights intra-clade-heterogeneity that exists within clade II of C. auris. ImportanceCandida auris demonstrates significant resistance to amphotericin B (AmB) that stems from factors like alteration of ergosterol biosynthesis, perturbation of the oxidative damage response, etc. A comprehensive understanding of underlying mechanisms can be studied in a holistic manner by subjecting resistant as well as susceptible clinical isolates to a comparative genome-level analysis. An alternate and more dynamic approach is to expose susceptible isolates to a certain concentration of drug which is not lethal but can trigger the resistance mechanisms. In the present study, we evolved C. auris towards AmB and observed novel and differential mechanisms of resistance development, in two different isolates despite belonging to the same clade. This study provides insights into the intra-clade heterogeneous behavior of C. auris towards AmB.

microbiology↗

bioRxiv2022

Federated analysis in COINSTAC reveals functional network connectivity and spectral links to smoking and alcohol consumption in nearly 2,000 adolescent brains

With the growth of decentralized/federated analysis approaches in neuroimaging, the opportunities to study brain disorders using data from multiple sites has grown multi-fold. One such initiative is the Neuromark, a fully automated spatially constrained independent component analysis (ICA) that is used to link brain network abnormalities among different datasets, studies, and disorders while leveraging subject-specific networks. In this study, we implement the neuromark pipeline in COINSTAC, an open-source neuroimaging framework for collaborative/decentralized analysis. Decentralized analysis of nearly 2000 resting-state functional magnetic resonance imaging datasets collected at different sites across two cohorts and co-located in different countries was performed to study the resting brain functional network connectivity changes in adolescents who smoke and consume alcohol. Results showed hypoconnectivity across the majority of networks including sensory, default mode, and subcortical domains, more for alcohol than smoking, and decreased low frequency power. These findings suggest that global reduced synchronization is associated with both tobacco and alcohol use. This work demonstrates the utility and incentives associated with large-scale decentralized collaborations spanning multiple sites.

bioinformatics↗

bioRxiv2021

Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins

Hemoglobin oxidation due to oxidative stress and disease conditions leads to generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the RBC membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2, 2-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-{beta}-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.

biochemistry↗

bioRxiv2021

Ethology of morphogenesis reveals the design principles of cnidarian size and shape development

During development, organisms interact with their natural habitats while undergoing morphological changes, yet it remains unclear whether the interplay between developing systems and their environments impacts animal morphogenesis. Here, we use the cnidarian Nematostella vectensis as a developmental model to uncover a mechanistic link between organism size, shape and behavior. Using quantitative live imaging, including extensive behavioral profiling, combined with molecular and biophysical experiments, we demonstrate that the muscular hydraulic machinery that controls body movement directly drives larva-polyp morphogenesis. Unexpectedly, size and shape development are differentially controlled by antagonistic muscles. A simple theoretical model shows how a combination of slow-priming and fast-pumping pressures generated by muscular hydraulics acts as a global mechanical regulator that coordinates tissue remodeling. Altogether, our findings illuminate how dynamic behavioral modes in the environment can be harnessed to drive morphogenetic trajectories, establishing ethology as a critical component of organismal morphogenesis - termed ethology of morphogenesis.

developmental biology↗

bioRxiv2021

Functional and comparative genome analysis reveals clade-specific genome innovations in the killer fungus Candida auris

The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 and simultaneously evolved in different geographical zones worldwide, causing superficial as well as systemic infections. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional centromere landscape lacking pericentromeres. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Inter-species genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris. Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug susceptible isolates of C. auris.

genomics↗

bioRxiv2020

Discovery of tREP-18, a novel class of tRNA encoded peptide with potent leishmanicidal activity

In the post genomic era, tRNA-derived fragments have emerged as a new class of non-coding gene regulators, those play crucial roles both at the transcriptional and translational levels, in different cellular biogenesis. However, none of the studies has ever asked whether tRNAs can also be translated into peptides with any biological significance. Thus, we present a novel hypothesis which suggested that; design and synthesis of tRNA-derived peptides from prokaryotic genome can be exploited for developing unique chemotherapeutics against neglected tropical diseases, like Visceral leishmaniasis (VL) and its aggressive form known as post kalazar dermal leishmaniasis (PKDL). To achieve this aim, we have used a novel system biology-based strategy, which involved; i) mining of unique tRNAs from E. coli genome and their translation into peptide in silico, ii) designing of theoretical 3D models to evaluate their stability, iii) prediction of their biological activity by screening against anti-parasitic database to filter the lead peptide. Based on this strategy, a unique tRNA-derived peptide (tREP-18) was selected, chemically synthesized, and used in vitro for elucidating its therapeutic significance against L. donovani, a causative agent of VL and PKDL. Our findings demonstrated that, tREP-18 can impose high level toxicity to L. donovani promastigotes, by disrupting the ultrastructural cellular architect, destabilizing the mitochondrial membrane potential ({Delta}{Psi}m), thus leading to drastic reduction in cell viability and proliferation. It also imparted high level of toxicity to BS12 a clinical isolate of PKDL. Conceivably, we for the first time reports a novel tRNA-derived peptide "tREP18" with excellent anti-leishmanial property, which can be further utilized for developing it as antileishmanial drug. One Sentence SummaryHere we report a novel first-in-class tRNA encoded peptide and its super anti-leishmanial characteristics.

synthetic biology↗

bioRxiv2020

Spectrin Interactome under normal and HbE-disease conditions

Spectrin, the major component of the erythrocyte membrane skeleton is a key player in red cell biology. It has a significant role in signalling pathways and as such knowledge of spectrin interactors becomes crucial. Here we report the cytosolic interactome of human erythroid spectrin (ProteomeXchange id: PXD021525). This is to the best of our knowledge the first report of the interactome of human erythroid spectrin. We have further investigated the spectrin interactome under HbE disease conditions. Our findings indicate that there is no difference in the identity of the proteins interacting with spectrin between normal and disease conditions. However relative abundance of the interacting partners is seen to change. Very interestingly the interacting chaperone proteins, heme-containing proteins and redox active proteins are seen to be up-regulated in HbE disease state. This is consistent with our previous observation that presence of oxidation prone hemoglobin variants leads to an increase of redox regulators and chaperones in the red cell proteome. Spectrin can also interact with horse radish peroxidase and oxidatively crosslink hemoglobin, which has possible implications in oxidative stress management. Since a large fraction of spectrin interacting proteins are chaperones and redox active proteins, it is possible that spectrin may have a broader role in redox regulation, especially in cases where there are unstable hemoglobin variants present.

biochemistry↗

bioRxiv2020

Metalloprotease Gp63 targeting novel glycoside exhibits potential antileishmanial activity

Visceral Leishmaniasis (VL) and its aggressive cutaneous exacerbation known as Post Kala-azar Dermal Leishmaniasis (PKDL) cause a huge disease burden in tropics and sub-tropic endemic zones worldwide. Contemporary treatment modalities have been associated with various complications. Encouraged from the recent marked antimalarial effects from plant derived glycosides; here we have chemically synthesized a library of diverse Glycoside derivatives (Gly 1-12) and evaluated their inhibitory efficacy against Ag83 strain of Leishmania donovani. In vitro activity of Glycoside-2 (Gly 2) on promastigote form of Ag83 strain, unravelled its prominent anti-leishmanial property with IC50 value of 1.13M. In-silico studies also unveiled the efficacy of Gly 2 to bind to the membrane surface of parasite. The toxic effect of Gly 2 causes necrosis like death in promastigote by abrogating its proliferation leading to imbalanced redox homeostasis by disruption of mitochondrial membrane potential. Additionally, Gly 2 treatment demonstrated increased susceptibility of parasites towards complement mediated lysis and displayed strong lethal effect on amastigote-macrophage infection model mimicking pathophysiological condition of body. This lead molecule was quite effective against the clinical on promastigotes form of PKDL strain BS12 with IC50 value of 1.97 M making it the most suitable drug so far which can target both VL and PKDL simultaneously. Based on the above experimental validations we narrowed our thoughts regarding the potent role of Gly 2 targeting surface protein of L. donovani such as Gp63, a zinc metalloprotease. Further analysis of structure activity relationship (SAR) of these glycoside derivatives, demonstrated exceptional binding affinity of Gly 2 towards Gp63, a zinc metalloprotease of L. donovani; with strong H-bond interactions of Gly 2 with catalytic domain in the -helix B region of Gp63. The strong confined interactions between Gly 2 and the target protein Gp63 in a physiologically relevant cellular environment was further assessed by Cellular Thermal Shift Assay (CETSA) which corroborated with our previous results. Taken together, this study reports the serendipitous discovery of glycoside derivative Gly 2 with enhanced leishmanicidal activity and proves to be novel chemotherapeutic prototype against VL and PKDL. HighlightsO_LIA novel glycoside derivative (Gly 2) targets Gp63 functioning in L. donovani promastigotes, resulting in its abrogated proliferation and severely detabilized redox homeostasis, leading to parasitic death. C_LIO_LIStructure activity relationship (SAR) analysis revealed exceptional ligandability of Gly 2 towards Gp63 catalytic domain both in silico and in Cellular Thermal Shift Assay (CETSA) based in vitro analysis. C_LIO_LIGly 2 treatment exhibited increased parasite susceptibility towards complement mediated lysis and reduced macrophage infection in vitro mimicking the pathophysiological conditions. C_LIO_LIGly 2 showed profound antileishmanial activity against the clinical isolates of Post Kala-azar Dermal Leishmaniasis (PKDL). C_LI

pharmacology and toxicology↗

bioRxiv2020

A series of five population-specific Indian brain templates and atlases spanning ages 6 to 60 years

Anatomical brain templates are commonly used as references in neurological MRI studies, for bringing data into a common space for group-level statistics and coordinate reporting. Given the inherent variability in brain morphology across age and geography, it is important to have templates that are as representative as possible for both age and population. A representative-template increases the accuracy of alignment, decreases distortions as well as potential biases in final coordinate reports. In this study, we developed and validated a new set of T1w Indian brain templates (IBT) from a large number of brain scans (total n=466) acquired across different locations and multiple 3T MRI scanners in India. A new tool in AFNI, make_template_dask.py, was created to efficiently make five age-specific IBTs (ages 6-60 years) as well as maximum probability map (MPM) atlases for each template; for each age-groups template-atlas pair, there is both a "population-average" and a "typical" version. Validation experiments on an independent Indian structural and functional-MRI dataset show the appropriateness of IBTs for spatial normalization of Indian brains. The results indicate significant structural differences when comparing the IBTs and MNI template, with these differences being maximal along the Anterior-Posterior and Inferior-Superior axes, but minimal Left-Right. For each age-group, the MPM brain atlases provide reasonably good representation of the native-space volumes in the IBT space, except in a few regions with high inter-subject variability. These findings provide evidence to support the use of age and population-specific templates in human brain mapping studies. This dataset is made publicly available (https://hollabharath.github.io/IndiaBrainTemplates). HighlightsO_LIA new set of age-specific T1w Indian brain templates for ages 6-60 yr are developed and validated. C_LIO_LIA new AFNI tool, make_template_dask.py, for the creation of group-based templates. C_LIO_LIMaximum probability map atlases are also provided for each template. C_LIO_LIResults indicate the appropriateness of Indian templates for spatial normalization of Indian brains C_LI

neuroscience↗

bioRxiv2019

Localizing the chaperone activity of erythroid spectrin

Spectrin, the major protein of the RBC membrane skeleton has canonically been thought to only serve a structural function. We have described a novel chaperone-like property of spectrin and have shown that it is able to prevent the aggregation of other proteins such as alcohol dehydrogenase, insulin and free globin chains. We have tried to localize the molecular origin of chaperone-like activity in multi-domain spectrin by using recombinant spectrin fragments and investigating individual domains. We have characterized the recombinant domains using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Hydrophobic ligands Prodan (6-propionyl-2[dimethylamino]-naphthalene) and ANS (1-anilinonaphthalene-8-sulfonic acid) binding has been used to probe the hydrophobicity of the recombinant domains and it is seen that all domains have surface exposed hydrophobic patches; and in accordance with our previous hypothesis only the reconstituted self-association domain binds Prodan. Recombinant domains display comparable chaperone potential in preventing protein aggregation; and substrate selectivity of -over {beta}-globin is seen. Enzyme refolding studies show alternate pathways of chaperone action. Our current study points to the presence of hydrophobic patches on the surface of these domains as the source of the chaperone activity of spectrin, as notably seen in the self-association domain. There is no one domain largely responsible for the chaperone activity of spectrin; rather all domains appear to contribute equally, such that the chaperone activity of spectrin seems to be a linear sum of the individual activities of the domains.

biochemistry↗

bioRxiv2018

Transcriptome-wide profiling of mammalian spliceosome and branchpoints with iCLIP

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to map human spliceosome engagement with snRNAs and pre-mRNAs. This identified over 50,000 branchpoints (BPs) that have canonical sequence and structural features. Moreover, it revealed 7 binding peaks around BPs and splice sites, each precisely overlapping with binding profiles of specific splicing factors. We show how the binding patterns of these RBPs are affected by the position and strength of BPs. For example, strong or proximally located BPs preferentially bind SF3 rather than U2AF complex. Notably, these effects are partly neutralized during spliceosomal assembly in a way that depends on the core spliceosomal protein PRPF8. These insights exemplify spliceosome iCLIP as a broadly applicable method for transcriptomic studies of splicing mechanisms.

genomics↗