Smarajit Mondal

All Publications

• The Adhesion GPCR ADGRL2 engages Ga13 to Enable Epidermal Differentiation. bioRxiv : the preprint server for biology Yang, X., He, F., Porter, D. F., Garbett, K., Meyers, R. M., Reynolds, D. L., Bui, D. L., Hong, A., Ducoli, L., Siprashvili, Z., Lopez-Pajares, V., Mondal, S., Ko, L., Jing, Y., Tao, S., Singal, B., Sando, R., Skiniotis, G., Khavari, P. A. 2025

  Abstract

  Homeostasis relies on signaling networks controlled by cell membrane receptors. Although G-protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors, their specific roles in the epidermis are not fully understood. Dual CRISPR-Flow and single cell Perturb-seq knockout screens of all epidermal GPCRs were thus performed, uncovering an essential requirement for adhesion GPCR ADGRL2 (latrophilin 2) in epidermal differentiation. Among potential downstream guanine nucleotide-binding G proteins, ADGRL2 selectively activated Gα13. Perturb-seq of epidermal G proteins and follow-up tissue knockouts verified that Gα13 is also required for epidermal differentiation. A cryo-electron microscopy (cryo-EM) structure in lipid nanodiscs showed that ADGRL2 engages with Gα13 at multiple interfaces, including via a novel interaction between ADGRL2 intracellular loop 3 (ICL3) and a Gα13-specific QQQ glutamine triplet sequence in its GTPase domain. In situ gene mutation of this interface sequence impaired epidermal differentiation, highlighting an essential new role for an ADGRL2-Gα13 axis in epidermal differentiation.

  View details for DOI 10.1101/2025.02.19.639154

  View details for PubMedID 40060693

  View details for PubMedCentralID PMC11888183

• Functional analysis of cancer-associated germline risk variants. Nature genetics Kellman, L. N., Neela, P. H., Srinivasan, S., Siprashvili, Z., Shanderson, R. L., Hong, A. W., Rao, D., Porter, D. F., Reynolds, D. L., Meyers, R. M., Guo, M. G., Yang, X., Zhao, Y., Wozniak, G. G., Donohue, L. K., Shenoy, R., Ko, L. A., Nguyen, D. T., Mondal, S., Garcia, O. S., Elcavage, L. E., Elfaki, I., Abell, N. S., Tao, S., Lopez, C. M., Montgomery, S. B., Khavari, P. A. 2025

  Abstract

  Single-nucleotide variants (SNVs) in regulatory DNA are linked to inherited cancer risk. Massively parallel reporter assays of 4,041 SNVs linked to 13 neoplasms comprising >90% of human malignancies were performed in pertinent primary human cell types and then integrated with matching chromatin accessibility, DNA looping and expression quantitative trait loci data to nominate 380 potentially regulatory SNVs and their putative target genes. The latter highlighted specific protein networks in lifetime cancer risk, including mitochondrial translation, DNA damage repair and Rho GTPase activity. A CRISPR knockout screen demonstrated that a subset of germline putative risk genes also enables the growth of established cancers. Editing one SNV, rs10411210 , showed that its risk allele increases rhophilin RHPN2 expression and stimulus-responsive RhoA activation, indicating that individual SNVs may upregulate cancer-linked pathways. These functional data are a resource for variant prioritization efforts and further interrogation of the mechanisms underlying inherited risk for cancer.

  View details for DOI 10.1038/s41588-024-02070-5

  View details for PubMedID 39962238

  View details for PubMedCentralID 3934208

• Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases. Nature genetics Guo, M. G., Reynolds, D. L., Ang, C. E., Liu, Y., Zhao, Y., Donohue, L. K., Siprashvili, Z., Yang, X., Yoo, Y., Mondal, S., Hong, A., Kain, J., Meservey, L., Fabo, T., Elfaki, I., Kellman, L. N., Abell, N. S., Pershad, Y., Bayat, V., Etminani, P., Holodniy, M., Geschwind, D. H., Montgomery, S. B., Duncan, L. E., Urban, A. E., Altman, R. B., Wernig, M., Khavari, P. A. 2023

  Abstract

  Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

  View details for DOI 10.1038/s41588-023-01533-5

  View details for PubMedID 37857935

  View details for PubMedCentralID 4112379

• PROBER: overcoming the high noise of DNA-protein interactions studies NATURE METHODS Mondal, S., Khavari, P. 2022; 19 (9): 1044

  View details for DOI 10.1038/s41592-022-01553-9

  View details for Web of Science ID 000857686400013

  View details for PubMedID 35945455

• BEHIND THE PAPER NATURE METHODS Mondal, S. 2022; 19 (9): 1045

  View details for Web of Science ID 000857686400025

• PROBER identifies proteins associated with programmable sequence-specific DNA in living cells. Nature methods Mondal, S., Ramanathan, M., Miao, W., Meyers, R. M., Rao, D., Lopez-Pajares, V., Siprashvili, Z., Reynolds, D. L., Porter, D. F., Ferguson, I., Neela, P., Zhao, Y., Meservey, L. M., Guo, M., Yang, Y., Li, L., Wang, Y., Khavari, P. A. 2022; 19 (8): 959-968

  Abstract

  DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

  View details for DOI 10.1038/s41592-022-01552-w

  View details for PubMedID 35927480

• RNA-protein interaction detection in living cells. Nature methods Ramanathan, M. n., Majzoub, K. n., Rao, D. S., Neela, P. H., Zarnegar, B. J., Mondal, S. n., Roth, J. G., Gai, H. n., Kovalski, J. R., Siprashvili, Z. n., Palmer, T. D., Carette, J. E., Khavari, P. A. 2018

  Abstract

  RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.

  View details for PubMedID 29400715

• Modular Organization of the NusA- and NusG-Stimulated RNA Polymerase Pause Signal That Participates in the Bacillus subtilis trp Operon Attenuation Mechanism JOURNAL OF BACTERIOLOGY Mondal, S., Yakhnin, A. V., Babitzke, P. 2017; 199 (14)

  Abstract

  The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to the nascent transcript and blocks the formation of an antiterminator structure such that the formation of an overlapping intrinsic terminator causes termination in the 5' untranslated region (5' UTR). In the absence of bound TRAP, the antiterminator forms and transcription continues into the trp genes. RNA polymerase pauses at positions U107 and U144 in the 5' UTR. The general transcription elongation factors NusA and NusG stimulate pausing at both positions. NusG-stimulated pausing at U144 requires sequence-specific contacts with a T tract in the nontemplate DNA (ntDNA) strand within the paused transcription bubble. Pausing at U144 participates in a trpE translation repression mechanism. Since U107 just precedes the critical overlap between the antiterminator and terminator structures, pausing at this position is thought to participate in attenuation. Here we carried out in vitro pausing and termination experiments to identify components of the U107 pause signal and to determine whether pausing affects the termination efficiency in the 5' UTR. We determined that the U107 and U144 pause signals are organized in a modular fashion containing distinct RNA hairpin, U-tract, and T-tract components. NusA-stimulated pausing was affected by hairpin strength and the U-tract sequence, whereas NusG-stimulated pausing was affected by hairpin strength and the T-tract sequence. We also determined that pausing at U107 results in increased TRAP-dependent termination in the 5' UTR, implying that NusA- and NusG-stimulated pausing participates in the trp operon attenuation mechanism by providing additional time for TRAP binding.IMPORTANCE The expression of several bacterial operons is controlled by regulated termination in the 5' untranslated region (5' UTR). Transcription attenuation is defined as situations in which the binding of a regulatory molecule promotes transcription termination in the 5' UTR, with the default being transcription readthrough into the downstream genes. RNA polymerase pausing is thought to participate in several attenuation mechanisms by synchronizing the position of RNA polymerase with RNA folding and/or regulatory factor binding, although this has only been shown in a few instances. We found that NusA- and NusG-stimulated pausing participates in the attenuation mechanism controlling the expression of the Bacillus subtilis trp operon by increasing the TRAP-dependent termination efficiency. The pause signal is organized in a modular fashion containing RNA hairpin, U-tract, and T-tract components.

  View details for DOI 10.1128/JB.00223-17

  View details for Web of Science ID 000404968900011

  View details for PubMedID 28507243

  View details for PubMedCentralID PMC5494738

• NusA-dependent transcription termination prevents misregulation of global gene expression NATURE MICROBIOLOGY Mondal, S., Yakhnin, A. V., Sebastian, A., Albert, I., Babitzke, P. 2016; 1 (1)

  Abstract

  Intrinsic transcription terminators consist of an RNA hairpin followed by a U-rich tract, and these signals can trigger termination without the involvement of additional factors. Although NusA is known to stimulate intrinsic termination in vitro, the in vivo targets and global impact of NusA are not known because it is essential for viability. Using genome-wide 3' end-mapping on an engineered Bacillus subtilis NusA depletion strain, we show that weak suboptimal terminators are the principle NusA substrates. Moreover, a subclass of weak non-canonical terminators was identified that completely depend on NusA for effective termination. NusA-dependent terminators tend to have weak hairpins and/or distal U-tract interruptions, supporting a model in which NusA is directly involved in the termination mechanism. Depletion of NusA altered global gene expression directly and indirectly via readthrough of suboptimal terminators. Readthrough of NusA-dependent terminators caused misregulation of genes involved in essential cellular functions, especially DNA replication and metabolism. We further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures. Instead, NusA-stimulated termination in its 5' UTR dictates the extent of transcription into the operon, thereby ensuring tight control of cellular NusA levels.

  View details for DOI 10.1038/NMICROBIOL.2015.7

  View details for Web of Science ID 000381577400013

  View details for PubMedID 27571753

  View details for PubMedCentralID PMC5358096