33 results on '"Pitchiaya S"'
Search Results
2. A0425 - Spatial transcriptomic profiling of prostate cancer reveals zone specific androgen receptor signaling and immune infiltration.
- Author
-
Patel, P., Nallandhighal, S., Scoville, D., Tran, L., Cotta, B., Udager, A., Rao, A., Palapattu, G., Dadhania, V., Pitchiaya, S., and Salami, S.
- Subjects
- *
ANDROGEN receptors , *PROSTATE cancer , *TRANSCRIPTOMES - Published
- 2022
- Full Text
- View/download PDF
3. Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate Cancer.
- Author
-
Wang X, Qiao Y, Asangani IA, Ateeq B, Poliakov A, Cieślik M, Pitchiaya S, Chakravarthi BVSK, Cao X, Jing X, Wang CX, Apel IJ, Wang R, Ching-Yi Tien J, Juckette KM, Yan W, Jiang H, Wang S, Varambally S, and Chinnaiyan AM
- Published
- 2024
- Full Text
- View/download PDF
4. Biomolecular condensates in kidney physiology and disease.
- Author
-
Gao G, Sumrall ES, Pitchiaya S, Bitzer M, Alberti S, and Walter NG
- Subjects
- Humans, Animals, Water, Mammals, Biomolecular Condensates, Kidney
- Abstract
The regulation and preservation of distinct intracellular and extracellular solute microenvironments is crucial for the maintenance of cellular homeostasis. In mammals, the kidneys control bodily salt and water homeostasis. Specifically, the urine-concentrating mechanism within the renal medulla causes fluctuations in extracellular osmolarity, which enables cells of the kidney to either conserve or eliminate water and electrolytes, depending on the balance between intake and loss. However, relatively little is known about the subcellular and molecular changes caused by such osmotic stresses. Advances have shown that many cells, including those of the kidney, rapidly (within seconds) and reversibly (within minutes) assemble membraneless, nano-to-microscale subcellular assemblies termed biomolecular condensates via the biophysical process of hyperosmotic phase separation (HOPS). Mechanistically, osmotic cell compression mediates changes in intracellular hydration, concentration and molecular crowding, rendering HOPS one of many related phase-separation phenomena. Osmotic stress causes numerous homo-multimeric proteins to condense, thereby affecting gene expression and cell survival. HOPS rapidly regulates specific cellular biochemical processes before appropriate protective or corrective action by broader stress response mechanisms can be initiated. Here, we broadly survey emerging evidence for, and the impact of, biomolecular condensates in nephrology, where initial concentration buffering by HOPS and its subsequent cellular escalation mechanisms are expected to have important implications for kidney physiology and disease., (© 2023. Springer Nature Limited.)
- Published
- 2023
- Full Text
- View/download PDF
5. Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.
- Author
-
Hosono Y, Niknafs YS, Prensner JR, Iyer MK, Dhanasekaran SM, Mehra R, Pitchiaya S, Tien J, Escara-Wilke J, Poliakov A, Chu SC, Saleh S, Sankar K, Su F, Guo S, Qiao Y, Freier SM, Bui HH, Cao X, Malik R, Johnson TM, Beer DG, Feng FY, Zhou W, and Chinnaiyan AM
- Published
- 2023
- Full Text
- View/download PDF
6. Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.
- Author
-
Duran E, Schmidt A, Welty R, Jalihal AP, Pitchiaya S, and Walter NG
- Subjects
- Cell Extracts, RNA Splicing, Biology, Ribonucleoproteins metabolism, RNA metabolism
- Abstract
Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts. This strategy enables elucidation of the interaction and kinetic behavior of specific fluorescently labeled biomolecules within RNPs under conditions that approximate native cellular environments. In this review, we describe single molecule fluorescence microcopy approaches that dissect RNP-driven processes within cellular extracts, highlighting general strategies used in these methods. We further survey biological advances in the areas of pre-mRNA splicing and transcription regulation that have been facilitated through this approach. Finally, we conclude with a summary of practical considerations for the implementation of the featured approaches to facilitate their broader future implementation in dissecting the mechanisms of RNP-driven cellular processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems., (© 2023 Wiley Periodicals LLC.)
- Published
- 2023
- Full Text
- View/download PDF
7. Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging.
- Author
-
Larouche JA, Mohiuddin M, Choi JJ, Ulintz PJ, Fraczek P, Sabin K, Pitchiaya S, Kurpiers SJ, Castor-Macias J, Liu W, Hastings RL, Brown LA, Markworth JF, De Silva K, Levi B, Merajver SD, Valdez G, Chakkalakal JV, Jang YC, Brooks SV, and Aguilar CA
- Subjects
- Animals, Female, Male, Mice, Knockout, Mice, Aging, Muscle, Skeletal injuries, Myoblasts, Skeletal physiology, Neuromuscular Junction physiology, Superoxide Dismutase-1 deficiency
- Abstract
During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1
-/- ), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors., Competing Interests: JL, MM, JC, PU, PF, KS, SP, SK, JC, WL, RH, LB, JM, KD, BL, SM, GV, JC, YJ, SB, CA No competing interests declared, (© 2021, Larouche et al.)- Published
- 2021
- Full Text
- View/download PDF
8. Single-cell analyses of renal cell cancers reveal insights into tumor microenvironment, cell of origin, and therapy response.
- Author
-
Zhang Y, Narayanan SP, Mannan R, Raskind G, Wang X, Vats P, Su F, Hosseini N, Cao X, Kumar-Sinha C, Ellison SJ, Giordano TJ, Morgan TM, Pitchiaya S, Alva A, Mehra R, Cieslik M, Dhanasekaran SM, and Chinnaiyan AM
- Subjects
- Carcinoma, Renal Cell immunology, Cell Survival, Endothelial Cells pathology, Epithelial Cells pathology, Humans, Immunotherapy, Kidney pathology, Kidney Neoplasms immunology, Lymphocytes, Tumor-Infiltrating immunology, Myeloid Cells pathology, Treatment Outcome, Carcinoma, Renal Cell pathology, Carcinoma, Renal Cell therapy, Kidney Neoplasms pathology, Kidney Neoplasms therapy, Single-Cell Analysis
- Abstract
Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy., Competing Interests: Competing interest statement: A.A. and P.M. are coauthors on an article [S. M. Esagian et al. BJU International, 10.1111/bju.15324 (2021)]., (Copyright © 2021 the Author(s). Published by PNAS.)
- Published
- 2021
- Full Text
- View/download PDF
9. Antisense oligonucleotides and nucleic acids generate hypersensitive platelets.
- Author
-
Zaslavsky A, Adams M, Cao X, Yamaguchi A, Henderson J, Busch-Østergren P, Udager A, Pitchiaya S, Tourdot B, Kasputis T, Church SJ, Lee SK, Ohl S, Patel S, Morgan TM, Alva A, Wakefield TW, Reichert Z, Holinstat M, and Palapattu GS
- Subjects
- Animals, Blood Platelets, Humans, Mice, Oligonucleotides, Antisense, Phosphorothioate Oligonucleotides, Nucleic Acids, Pharmaceutical Preparations
- Abstract
Introduction: Despite the great promise for therapies using antisense oligonucleotides (ASOs), their adverse effects, which include pro-inflammatory effects and thrombocytopenia, have limited their use. Previously, these effects have been linked to the phosphorothioate (PS) backbone necessary to prevent rapid ASO degradation in plasma. The main aim of this study was to assess the impact of the nucleic acid portion of an ASO-type drug on platelets and determine if it may contribute to thrombosis or thrombocytopenia., Methods: Platelets were isolated from healthy donors and men with advanced prostate cancer. Effects of antisense oligonucleotides (ASO), oligonucleotides, gDNA, and microRNA on platelet activation and aggregation were evaluated. A mouse model of lung thrombosis was used to confirm the effects of PS-modified oligonucleotides in vivo., Results: Platelet exposure to gDNA, miRNA, and oligonucleotides longer than 16-mer at a concentration above 8 mM resulted in the formation of hypersensitive platelets, characterized by an increased sensitivity to low-dose thrombin (0.1 nM) and increase in p-Selectin expression (6-8 fold greater than control; p < 0.001). The observed nucleic acid (NA) effects on platelets were toll-like receptor (TLR) -7 subfamily dependent. Injection of a p-Selectin inhibitor significantly (p = 0.02) reduced the formation of oligonucleotide-associated pulmonary microthrombosis in vivo., Conclusion: Our results suggest that platelet exposure to nucleic acids independent of the presence of a PS modification leads to a generation of hypersensitive platelets and requires TLR-7 subfamily receptors. ASO studies conducted in cancer patients may benefit from testing the ASO effects on platelets ex vivo before initiation of patient treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
10. RNA-seq of human T cells after hematopoietic stem cell transplantation identifies Linc00402 as a regulator of T cell alloimmunity.
- Author
-
Peltier D, Radosevich M, Ravikumar V, Pitchiaya S, Decoville T, Wood SC, Hou G, Zajac C, Oravecz-Wilson K, Sokol D, Henig I, Wu J, Kim S, Taylor A, Fujiwara H, Sun Y, Rao A, Chinnaiyan AM, Goldstein DR, and Reddy P
- Subjects
- Animals, Graft vs Host Disease genetics, Histocompatibility, Humans, Mice, RNA-Seq, Transplantation, Homologous, Hematopoietic Stem Cell Transplantation, RNA, Long Noncoding genetics, T-Lymphocytes
- Abstract
Mechanisms governing allogeneic T cell responses after solid organ and allogeneic hematopoietic stem cell transplantation (HSCT) are incompletely understood. To identify lncRNAs that regulate human donor T cells after clinical HSCT, we performed RNA sequencing on T cells from healthy individuals and donor T cells from three different groups of HSCT recipients that differed in their degree of major histocompatibility complex (MHC) mismatch. We found that lncRNA differential expression was greatest in T cells after MHC-mismatched HSCT relative to T cells after either MHC-matched or autologous HSCT. Differential expression was validated in an independent patient cohort and in mixed lymphocyte reactions using ex vivo healthy human T cells. We identified Linc00402 , an uncharacterized lncRNA, among the lncRNAs differentially expressed between the mismatched unrelated and matched unrelated donor T cells. We found that Linc00402 was conserved and exhibited an 88-fold increase in human T cells relative to all other samples in the FANTOM5 database. Linc00402 was also increased in donor T cells from patients who underwent allogeneic cardiac transplantation and in murine T cells. Linc00402 was reduced in patients who subsequently developed acute graft-versus-host disease. Linc00402 enhanced the activity of ERK1 and ERK2, increased FOS nuclear accumulation, and augmented expression of interleukin-2 and Egr-1 after T cell receptor engagement. Functionally, Linc00402 augmented the T cell proliferative response to an allogeneic stimulus but not to a nominal ovalbumin peptide antigen or polyclonal anti-CD3/CD28 stimulus. Thus, our studies identified Linc00402 as a regulator of allogeneic T cell function., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2021
- Full Text
- View/download PDF
11. Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2 .
- Author
-
Qiao Y, Wang XM, Mannan R, Pitchiaya S, Zhang Y, Wotring JW, Xiao L, Robinson DR, Wu YM, Tien JC, Cao X, Simko SA, Apel IJ, Bawa P, Kregel S, Narayanan SP, Raskind G, Ellison SJ, Parolia A, Zelenka-Wang S, McMurry L, Su F, Wang R, Cheng Y, Delekta AD, Mei Z, Pretto CD, Wang S, Mehra R, Sexton JZ, and Chinnaiyan AM
- Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, employs two key host proteins to gain entry and replicate within cells, angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease serine 2 (TMPRSS2). TMPRSS2 was first characterized as an androgen-regulated gene in the prostate. Supporting a role for sex hormones, males relative to females are disproportionately affected by COVID-19 in terms of mortality and morbidity. Several studies, including one employing a large epidemiological cohort, suggested that blocking androgen signaling is protective against COVID-19. Here, we demonstrate that androgens regulate the expression of ACE2 , TMPRSS2 , and androgen receptor (AR) in subsets of lung epithelial cells. AR levels are markedly elevated in males relative to females greater than 70 y of age. In males greater than 70 y old, smoking was associated with elevated levels of AR and ACE2 in lung epithelial cells. Transcriptional repression of the AR enhanceosome with AR or bromodomain and extraterminal domain (BET) antagonists inhibited SARS-CoV-2 infection in vitro. Taken together, these studies support further investigation of transcriptional inhibition of critical host factors in the treatment or prevention of COVID-19.
- Published
- 2021
- Full Text
- View/download PDF
12. Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles.
- Author
-
Jalihal AP, Schmidt A, Gao G, Little SR, Pitchiaya S, and Walter NG
- Subjects
- Cytoplasmic Granules metabolism, Inclusion Bodies metabolism, Organelles metabolism, Osmosis
- Abstract
Biological liquid-liquid phase separation has gained considerable attention in recent years as a driving force for the assembly of subcellular compartments termed membraneless organelles. The field has made great strides in elucidating the molecular basis of biomolecular phase separation in various disease, stress response, and developmental contexts. Many important biological consequences of such "condensation" are now emerging from in vivo studies. Here we review recent work from our group and others showing that many proteins undergo rapid, reversible condensation in the cellular response to ubiquitous environmental fluctuations such as osmotic changes. We discuss molecular crowding as an important driver of condensation in these responses and suggest that a significant fraction of the proteome is poised to undergo phase separation under physiological conditions. In addition, we review methods currently emerging to visualize, quantify, and modulate the dynamics of intracellular condensates in live cells. Finally, we propose a metaphor for rapid phase separation based on cloud formation, reasoning that our familiar experiences with the readily reversible condensation of water droplets help understand the principle of phase separation. Overall, we provide an account of how biological phase separation supports the highly intertwined relationship between the composition and dynamic internal organization of cells, thus facilitating extremely rapid reorganization in response to internal and external fluctuations., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
13. Multivalent Proteins Rapidly and Reversibly Phase-Separate upon Osmotic Cell Volume Change.
- Author
-
Jalihal AP, Pitchiaya S, Xiao L, Bawa P, Jiang X, Bedi K, Parolia A, Cieslik M, Ljungman M, Chinnaiyan AM, and Walter NG
- Subjects
- Animals, Cell Size, Cell Survival genetics, Humans, Osmotic Pressure physiology, Proteome genetics, Endoribonucleases genetics, RNA Precursors genetics, Stress, Physiological genetics, Trans-Activators genetics, Transcription Termination, Genetic
- Abstract
Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membraneless compartments that are observed under physiological and stress conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ∼10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ∼100 s) with minimal effect on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS sequesters pre-mRNA cleavage factor components from actively transcribing genomic loci, providing a mechanism for hyperosmolarity-induced global impairment of transcription termination. Our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
14. An essential role for Argonaute 2 in EGFR-KRAS signaling in pancreatic cancer development.
- Author
-
Shankar S, Tien JC, Siebenaler RF, Chugh S, Dommeti VL, Zelenka-Wang S, Wang XM, Apel IJ, Waninger J, Eyunni S, Xu A, Mody M, Goodrum A, Zhang Y, Tesmer JJ, Mannan R, Cao X, Vats P, Pitchiaya S, Ellison SJ, Shi J, Kumar-Sinha C, Crawford HC, and Chinnaiyan AM
- Subjects
- Alleles, Animals, Cell Line, Tumor, Cell Membrane metabolism, Cellular Senescence, Disease Progression, ErbB Receptors metabolism, Female, Gene Expression Regulation, Neoplastic, Genotype, Humans, Male, Mice, Mice, Transgenic, Neoplasm Transplantation, Pancreatic Neoplasms pathology, Phosphorylation, Protein Binding, Signal Transduction, Tumor Suppressor Protein p53 metabolism, Argonaute Proteins metabolism, Pancreatic Neoplasms metabolism, Proto-Oncogene Proteins p21(ras) metabolism
- Abstract
Both KRAS and EGFR are essential mediators of pancreatic cancer development and interact with Argonaute 2 (AGO2) to perturb its function. Here, in a mouse model of mutant KRAS-driven pancreatic cancer, loss of AGO2 allows precursor lesion (PanIN) formation yet prevents progression to pancreatic ductal adenocarcinoma (PDAC). Precursor lesions with AGO2 ablation undergo oncogene-induced senescence with altered microRNA expression and EGFR/RAS signaling, bypassed by loss of p53. In mouse and human pancreatic tissues, PDAC progression is associated with increased plasma membrane localization of RAS/AGO2. Furthermore, phosphorylation of AGO2
Y393 disrupts both the wild-type and oncogenic KRAS-AGO2 interaction, albeit under different conditions. ARS-1620 (G12C-specific inhibitor) disrupts the KRASG12C -AGO2 interaction, suggesting that the interaction is targetable. Altogether, our study supports a biphasic model of pancreatic cancer development: an AGO2-independent early phase of PanIN formation reliant on EGFR-RAS signaling, and an AGO2-dependent phase wherein the mutant KRAS-AGO2 interaction is critical for PDAC progression.- Published
- 2020
- Full Text
- View/download PDF
15. Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer.
- Author
-
Parolia A, Cieslik M, Chu SC, Xiao L, Ouchi T, Zhang Y, Wang X, Vats P, Cao X, Pitchiaya S, Su F, Wang R, Feng FY, Wu YM, Lonigro RJ, Robinson DR, and Chinnaiyan AM
- Subjects
- Cell Line, Tumor, Chromatin genetics, Chromatin metabolism, Gene Expression Regulation, Neoplastic, Genome, Human genetics, Hepatocyte Nuclear Factor 3-alpha chemistry, Hepatocyte Nuclear Factor 3-alpha metabolism, Humans, Male, Models, Molecular, Neoplasm Metastasis genetics, Protein Domains, Receptors, Androgen metabolism, Wnt Signaling Pathway, Hepatocyte Nuclear Factor 3-alpha genetics, Mutation genetics, Prostatic Neoplasms genetics, Prostatic Neoplasms pathology
- Abstract
Abtract: Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland
1,2 . FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours3-8 . However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive9-11 and oncogenic12-14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and-through TLE3 inactivation-promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element-herein denoted FOXA1 mastermind (FOXMIND)-to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies.- Published
- 2019
- Full Text
- View/download PDF
16. Dynamic Recruitment of Single RNAs to Processing Bodies Depends on RNA Functionality.
- Author
-
Pitchiaya S, Mourao MDA, Jalihal AP, Xiao L, Jiang X, Chinnaiyan AM, Schnell S, and Walter NG
- Subjects
- Cytoplasmic Granules genetics, Gene Silencing, HeLa Cells, Humans, RNA Processing, Post-Transcriptional genetics, RNA, Untranslated genetics, Single Molecule Imaging, MicroRNAs genetics, RNA, Long Noncoding genetics, RNA, Messenger genetics, Ribonucleoproteins genetics
- Abstract
Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
17. Analysis of the androgen receptor-regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression.
- Author
-
Zhang Y, Pitchiaya S, Cieślik M, Niknafs YS, Tien JC, Hosono Y, Iyer MK, Yazdani S, Subramaniam S, Shukla SK, Jiang X, Wang L, Liu TY, Uhl M, Gawronski AR, Qiao Y, Xiao L, Dhanasekaran SM, Juckette KM, Kunju LP, Cao X, Patel U, Batish M, Shukla GC, Paulsen MT, Ljungman M, Jiang H, Mehra R, Backofen R, Sahinalp CS, Freier SM, Watt AT, Guo S, Wei JT, Feng FY, Malik R, and Chinnaiyan AM
- Subjects
- Androgens genetics, Androgens metabolism, Cell Line, Tumor, Disease Progression, Gene Expression Regulation, Neoplastic, Humans, Male, Prostate physiology, Prostatic Neoplasms metabolism, Prostatic Neoplasms pathology, RNA, Long Noncoding metabolism, Receptors, Androgen metabolism, Signal Transduction, Prostatic Neoplasms genetics, RNA, Long Noncoding genetics, Receptors, Androgen genetics
- Abstract
The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target.
- Published
- 2018
- Full Text
- View/download PDF
18. Oncogenic Role of THOR, a Conserved Cancer/Testis Long Non-coding RNA.
- Author
-
Hosono Y, Niknafs YS, Prensner JR, Iyer MK, Dhanasekaran SM, Mehra R, Pitchiaya S, Tien J, Escara-Wilke J, Poliakov A, Chu SC, Saleh S, Sankar K, Su F, Guo S, Qiao Y, Freier SM, Bui HH, Cao X, Malik R, Johnson TM, Beer DG, Feng FY, Zhou W, and Chinnaiyan AM
- Subjects
- Animals, Cell Line, Tumor, Gene Knockout Techniques, Humans, Male, Mice, RNA-Binding Proteins metabolism, Testis metabolism, Disease Models, Animal, Melanoma metabolism, RNA, Long Noncoding metabolism, Zebrafish
- Abstract
Large-scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Here, we functionally characterize a novel ultraconserved lncRNA, THOR (ENSG00000226856), which exhibits expression exclusively in testis and a broad range of human cancers. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. We discovered a conserved interaction of THOR with IGF2BP1 and show that THOR contributes to the mRNA stabilization activities of IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
19. Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks.
- Author
-
Michelini F, Pitchiaya S, Vitelli V, Sharma S, Gioia U, Pessina F, Cabrini M, Wang Y, Capozzo I, Iannelli F, Matti V, Francia S, Shivashankar GV, Walter NG, and d'Adda di Fagagna F
- Subjects
- ATP-Binding Cassette Transporters metabolism, Acid Anhydride Hydrolases, Animals, Cell Cycle Proteins metabolism, Cell Line, Cell-Free System, DNA-Binding Proteins, MRE11 Homologue Protein metabolism, Mice, Models, Biological, Nuclear Proteins metabolism, Oligonucleotides, Antisense genetics, RNA Polymerase II metabolism, RNA, Long Noncoding genetics, Transcription, Genetic, Tumor Suppressor p53-Binding Protein 1 metabolism, DNA Breaks, Double-Stranded, DNA Damage genetics, DNA Damage physiology, DNA Repair genetics, DNA Repair physiology, RNA, Long Noncoding metabolism
- Abstract
The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.
- Published
- 2017
- Full Text
- View/download PDF
20. Development of Peptidomimetic Inhibitors of the ERG Gene Fusion Product in Prostate Cancer.
- Author
-
Wang X, Qiao Y, Asangani IA, Ateeq B, Poliakov A, Cieślik M, Pitchiaya S, Chakravarthi BVSK, Cao X, Jing X, Wang CX, Apel IJ, Wang R, Tien JC, Juckette KM, Yan W, Jiang H, Wang S, Varambally S, and Chinnaiyan AM
- Published
- 2017
- Full Text
- View/download PDF
21. Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution.
- Author
-
Pitchiaya S, Heinicke LA, Park JI, Cameron EL, and Walter NG
- Subjects
- Animals, Argonaute Proteins metabolism, Cell Count, Cell Line, Tumor, Cell Nucleus metabolism, Humans, Intracellular Space metabolism, Mice, MicroRNAs genetics, Models, Biological, Molecular Probes metabolism, RNA Stability, RNA Transport, RNA, Messenger genetics, RNA, Messenger metabolism, Reproducibility of Results, Subcellular Fractions metabolism, MicroRNAs metabolism, Single Molecule Imaging methods
- Abstract
Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
22. Corrigendum: DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs.
- Author
-
Rossiello F, Aguado J, Sepe S, Iannelli F, Nguyen Q, Pitchiaya S, Carninci P, and d'Adda di Fagagna F
- Published
- 2017
- Full Text
- View/download PDF
23. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs.
- Author
-
Rossiello F, Aguado J, Sepe S, Iannelli F, Nguyen Q, Pitchiaya S, Carninci P, and d'Adda di Fagagna F
- Subjects
- Animals, Mice, Inbred C57BL, Mice, Knockout, Oligonucleotides, Antisense pharmacology, RNA genetics, RNA Processing, Post-Transcriptional drug effects, RNA Processing, Post-Transcriptional genetics, Ribonuclease III metabolism, Transcription, Genetic drug effects, DNA Damage, RNA metabolism, Telomere metabolism
- Abstract
The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs.
- Published
- 2017
- Full Text
- View/download PDF
24. The lncRNA landscape of breast cancer reveals a role for DSCAM-AS1 in breast cancer progression.
- Author
-
Niknafs YS, Han S, Ma T, Speers C, Zhang C, Wilder-Romans K, Iyer MK, Pitchiaya S, Malik R, Hosono Y, Prensner JR, Poliakov A, Singhal U, Xiao L, Kregel S, Siebenaler RF, Zhao SG, Uhl M, Gawronski A, Hayes DF, Pierce LJ, Cao X, Collins C, Backofen R, Sahinalp CS, Rae JM, Chinnaiyan AM, and Feng FY
- Subjects
- Antineoplastic Agents, Hormonal pharmacology, Cell Line, Tumor, Drug Resistance, Neoplasm genetics, Female, Gene Expression Regulation, Neoplastic, Gene Knockdown Techniques, Humans, Neoplasm Invasiveness, RNA, Long Noncoding genetics, Receptors, Estrogen, Tamoxifen pharmacology, Breast Neoplasms metabolism, RNA, Long Noncoding metabolism
- Abstract
Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer., Competing Interests: Oncomine is supported by ThermoFisher, Inc. (Previously Life Technologies and Compendia Biosciences). A.M.C. was a co-founder of Compendia Biosciences and served on the scientific advisory board of Life Technologies before it was acquired. D.F.H. receives research funding from Astra-Zeneca, Eli Lilly, Janssen, Pfizer and Puma. He has stock options in OncImmune and in InBiomotion, and he is named as a primary inventor on two patents issued to the University of Michigan, one of which is licensed to Janssen and for which he receives royalties. These patents are not pertinent to the work presented here. The remaining authors declare no competing financial interests.
- Published
- 2016
- Full Text
- View/download PDF
25. KRAS Engages AGO2 to Enhance Cellular Transformation.
- Author
-
Shankar S, Pitchiaya S, Malik R, Kothari V, Hosono Y, Yocum AK, Gundlapalli H, White Y, Firestone A, Cao X, Dhanasekaran SM, Stuckey JA, Bollag G, Shannon K, Walter NG, Kumar-Sinha C, and Chinnaiyan AM
- Subjects
- Animals, Argonaute Proteins antagonists & inhibitors, Argonaute Proteins metabolism, Base Sequence, Carboxypeptidases genetics, Carboxypeptidases metabolism, Cell Transformation, Neoplastic metabolism, Cell Transformation, Neoplastic pathology, Gene Silencing, Humans, Mice, MicroRNAs genetics, MicroRNAs metabolism, Molecular Sequence Data, Mutation, NIH 3T3 Cells, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Protein Binding, Proto-Oncogene Proteins p21(ras) metabolism, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Transgenes, Argonaute Proteins genetics, Cell Transformation, Neoplastic genetics, Endoplasmic Reticulum metabolism, Gene Expression Regulation, Neoplastic, Proto-Oncogene Proteins p21(ras) genetics
- Abstract
Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRAS(G12V)-mediated transformation. Using AGO2-/- cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
26. Meeting report: SMART timing--principles of single molecule techniques course at the University of Michigan 2014.
- Author
-
Bartke RM, Cameron EL, Cristie-David AS, Custer TC, Denies MS, Daher M, Dhakal S, Ghosh S, Heinicke LA, Hoff JD, Hou Q, Kahlscheuer ML, Karslake J, Krieger AG, Li J, Li X, Lund PE, Vo NN, Park J, Pitchiaya S, Rai V, Smith DJ, Suddala KC, Wang J, Widom JR, and Walter NG
- Subjects
- Congresses as Topic, Microscopy, Fluorescence
- Abstract
Four days after the announcement of the 2014 Nobel Prize in Chemistry for "the development of super-resolved fluorescence microscopy" based on single molecule detection, the Single Molecule Analysis in Real-Time (SMART) Center at the University of Michigan hosted a "Principles of Single Molecule Techniques 2014" course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines., (© 2014 Wiley Periodicals, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
27. The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling.
- Author
-
Nyati S, Schinske-Sebolt K, Pitchiaya S, Chekhovskiy K, Chator A, Chaudhry N, Dosch J, Van Dort ME, Varambally S, Kumar-Sinha C, Nyati MK, Ray D, Walter NG, Yu H, Ross BD, and Rehemtulla A
- Subjects
- Animals, Blotting, Western, Cell Line, Tumor, Dimerization, Fluorescent Antibody Technique, Gene Knockdown Techniques, High-Throughput Screening Assays, Humans, Immunohistochemistry, Immunoprecipitation, Mice, Protein Serine-Threonine Kinases genetics, RNA Interference, RNA, Small Interfering genetics, Receptors, Transforming Growth Factor beta chemistry, Signal Transduction genetics, Protein Serine-Threonine Kinases metabolism, Receptors, Transforming Growth Factor beta metabolism, Signal Transduction physiology, Smad Proteins, Receptor-Regulated metabolism, Transforming Growth Factor beta physiology
- Abstract
Transforming growth factor-β (TGF-β) signaling regulates cell proliferation and differentiation, which contributes to development and disease. Upon binding TGF-β, the type I receptor (TGFBRI) binds TGFBRII, leading to the activation of the transcription factors SMAD2 and SMAD3. Using an RNA interference screen of the human kinome and a live-cell reporter for TGFBR activity, we identified the kinase BUB1 (budding uninhibited by benzimidazoles-1) as a key mediator of TGF-β signaling. BUB1 interacted with TGFBRI in the presence of TGF-β and promoted the heterodimerization of TGFBRI and TGFBRII. Additionally, BUB1 interacted with TGFBRII, suggesting the formation of a ternary complex. Knocking down BUB1 prevented the recruitment of SMAD3 to the receptor complex, the phosphorylation of SMAD2 and SMAD3 and their interaction with SMAD4, SMAD-dependent transcription, and TGF-β-mediated changes in cellular phenotype including epithelial-mesenchymal transition (EMT), migration, and invasion. Knockdown of BUB1 also impaired noncanonical TGF-β signaling mediated by the kinases AKT and p38 MAPK (mitogen-activated protein kinase). The ability of BUB1 to promote TGF-β signaling depended on the kinase activity of BUB1. A small-molecule inhibitor of the kinase activity of BUB1 (2OH-BNPP1) and a kinase-deficient mutant of BUB1 suppressed TGF-β signaling and formation of the ternary complex in various normal and cancer cell lines. 2OH-BNPP1 administration to mice bearing lung carcinoma xenografts reduced the amount of phosphorylated SMAD2 in tumor tissue. These findings indicated that BUB1 functions as a kinase in the TGF-β pathway in a role beyond its established function in cell cycle regulation and chromosome cohesion., (Copyright © 2015, American Association for the Advancement of Science.)
- Published
- 2015
- Full Text
- View/download PDF
28. Single molecule fluorescence approaches shed light on intracellular RNAs.
- Author
-
Pitchiaya S, Heinicke LA, Custer TC, and Walter NG
- Subjects
- Fluorescent Dyes chemistry, Microscopy, Fluorescence, RNA chemistry, RNA, Long Noncoding chemistry, RNA, Long Noncoding metabolism, RNA, Messenger metabolism, RNA, Small Untranslated chemistry, RNA, Small Untranslated metabolism, Eukaryotic Cells metabolism, RNA metabolism
- Published
- 2014
- Full Text
- View/download PDF
29. Dissecting non-coding RNA mechanisms in cellulo by Single-molecule High-Resolution Localization and Counting.
- Author
-
Pitchiaya S, Krishnan V, Custer TC, and Walter NG
- Subjects
- Animals, Base Sequence, Fluorescent Dyes chemistry, Genes, Reporter, HeLa Cells, Humans, Luciferases, Firefly biosynthesis, Luciferases, Firefly genetics, Luciferases, Renilla biosynthesis, Luciferases, Renilla genetics, MicroRNAs chemistry, MicroRNAs genetics, Microinjections, Microscopy, Fluorescence, RNA Interference, Ribonucleoproteins metabolism, MicroRNAs metabolism, Single-Cell Analysis methods
- Abstract
Non-coding RNAs (ncRNAs) recently were discovered to outnumber their protein-coding counterparts, yet their diverse functions are still poorly understood. Here we report on a method for the intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) of microRNAs (miRNAs), a conserved, ubiquitous class of regulatory ncRNAs that controls the expression of over 60% of all mammalian protein coding genes post-transcriptionally, by a mechanism shrouded by seemingly contradictory observations. We present protocols to execute single particle tracking (SPT) and single-molecule counting of functional microinjected, fluorophore-labeled miRNAs and thereby extract diffusion coefficients and molecular stoichiometries of micro-ribonucleoprotein (miRNP) complexes from living and fixed cells, respectively. This probing of miRNAs at the single molecule level sheds new light on the intracellular assembly/disassembly of miRNPs, thus beginning to unravel the dynamic nature of this important gene regulatory pathway and facilitating the development of a parsimonious model for their obscured mechanism of action., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
30. High-resolution three-dimensional mapping of mRNA export through the nuclear pore.
- Author
-
Ma J, Liu Z, Michelotti N, Pitchiaya S, Veerapaneni R, Androsavich JR, Walter NG, and Yang W
- Subjects
- Active Transport, Cell Nucleus, Cell Nucleus metabolism, Diffusion, HeLa Cells, Humans, Microscopy, RNA, Messenger genetics, RNA, Messenger metabolism, Ribonucleoproteins metabolism, Imaging, Three-Dimensional methods, Nuclear Pore metabolism, RNA Transport
- Abstract
The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional (3D) pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms, interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the centre of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.
- Published
- 2013
- Full Text
- View/download PDF
31. Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly.
- Author
-
Pitchiaya S, Androsavich JR, and Walter NG
- Subjects
- Animals, Diffusion, Fluorescent Dyes metabolism, HeLa Cells, Humans, Kinetics, Mice, Microinjections, Models, Biological, Photobleaching, RNA Transport genetics, Time Factors, Intracellular Space metabolism, MicroRNAs metabolism, Microscopy methods, Signal Transduction genetics
- Abstract
MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.
- Published
- 2012
- Full Text
- View/download PDF
32. Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2.
- Author
-
Kuszak AJ, Pitchiaya S, Anand JP, Mosberg HI, Walter NG, and Sunahara RK
- Subjects
- Allosteric Regulation, Amino Acid Sequence, Analgesics, Opioid metabolism, Analgesics, Opioid pharmacology, Animals, Binding, Competitive drug effects, Cell Line, Enkephalin, Ala(2)-MePhe(4)-Gly(5)- metabolism, Enkephalin, Ala(2)-MePhe(4)-Gly(5)- pharmacology, Guanosine 5'-O-(3-Thiotriphosphate) metabolism, Humans, Lipoproteins, HDL chemistry, Lipoproteins, HDL metabolism, Luminescent Proteins genetics, Luminescent Proteins metabolism, Molecular Sequence Data, Opioid Peptides metabolism, Opioid Peptides pharmacology, Protein Binding, Receptors, Opioid, mu genetics, Receptors, Opioid, mu isolation & purification, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Spodoptera, GTP-Binding Protein alpha Subunit, Gi2 metabolism, Receptors, Opioid, mu metabolism
- Abstract
Despite extensive characterization of the mu-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A mu-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [(35)S]guanosine 5'-(gamma-thio)triphosphate binding to Galpha(i), and is allosterically regulated by coupled G(i) protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys(7), Cys(8)]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the mu-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.
- Published
- 2009
- Full Text
- View/download PDF
33. First blueprint, now bricks: DNA as construction material on the nanoscale.
- Author
-
Pitchiaya S and Krishnan Y
- Subjects
- Nanostructures ultrastructure, Nucleic Acid Conformation, DNA chemistry, Nanostructures chemistry
- Abstract
For the most part DNA was considered Nature's instruction manual for life leading to the popular description 'blueprint of life'. However, DNA is now taking on a new aspect where it is finding use as a construction element for architecture on the nanoscale. This tutorial review addresses the importance of building ordered structures with DNA on the nanoscale, the underlying principles and approaches to build such scaffolds, the current limitations and the anticipated trajectory of the area. This is would be of interest to the chemical biology, supramolecular and bioengineering communities in particular.
- Published
- 2006
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.