The landscape of PrPC biosynthesis revealed by an arrayed genome-wide interference screen

AbstractThe availability of the cellular prion protein PrPC is limiting to prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the proteins and the biochemical pathways controlling the biosynthesis and the degradation of PrPC may represent therapeutic targets. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC. We cultured human U251-MG glioblastoma cells in the presence of 64’752 unique siRNAs targeting 21’584 annotated human genes, and measured PrPC using a one-pot fluorescence resonance energy transfer immunoassay in 51’128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC, which were then filtered through multiple secondary screens. Recursive candidate attrition yielded 54 novel regulators of PrPC, nine of which emerged as robust regulators of PrPC biosynthesis and degradation by transcriptional suppression in a CRISPR-interference validation screen. Six candidates were found to regulate PrPC in the opposite direction when transcriptionally activated using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent modulator of PrPC through the degradation of PRNP mRNA. Because of its hypothesis-free design, the present listing paints an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

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Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10894-10904.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10894-10904 ◽

Cited By ~ 32

Author(s):

Robert V. Intine ◽

Miroslav Dundr ◽

Alex Vassilev ◽

Elena Schwartz ◽

Yingmin Zhao ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Affinity Purification ◽

Close Association ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Rna Recognition Motif ◽

Translational Machinery ◽

Terminal Domain

ABSTRACT La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D. J. Kenan and J. D. Keene, Nat. Struct. Mol. Biol. 11 :303-305, 2004). While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known. In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366. We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation. Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin. Moreover, La lacking the SBM does not localize to nucleoli. Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay. The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

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RNA binding activates RIG-I by releasing an autorepressed signaling domain

Science Advances ◽

10.1126/sciadv.aax3641 ◽

2019 ◽

Vol 5 (10) ◽

pp. eaax3641 ◽

Cited By ~ 2

Author(s):

T. H. Dickey ◽

B. Song ◽

A. M. Pyle

Keyword(s):

Rna Binding ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Fluorescent Labeling ◽

Minor Role ◽

Signaling Domain ◽

A Minor ◽

Dual Site

The retinoic acid–inducible gene I (RIG-I) innate immune receptor is an important immunotherapeutic target, but we lack approaches for monitoring the physical basis for its activation in vitro. This gap in our understanding has led to confusion about mechanisms of RIG-I activation and difficulty discovering agonists and antagonists. We therefore created a novel fluorescence resonance energy transfer–based method for measuring RIG-I activation in vitro using dual site-specific fluorescent labeling of the protein. This approach enables us to measure the conformational change that releases the signaling domain during the first step of RIG-I activation, making it possible to understand the role of stimulatory ligands. We have found that RNA alone is sufficient to eject the signaling domain, ejection is reversible, and adenosine triphosphate plays but a minor role in this process. These findings help explain RIG-I dysfunction in autoimmune disease, and they inform the design of therapeutics targeting RIG-I.

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One Pot Bio-Synthesis of Palladium Nanoneedles and Its Bioavailability

10.21203/rs.3.rs-632642/v1 ◽

2021 ◽

Author(s):

Dnyaneshwar K. Kulal ◽

Shubham V. Pansare ◽

Amol A. Shedge ◽

Shhyam Khairkkar ◽

Shraddha Y. Chhatre ◽

...

Keyword(s):

Energy Transfer ◽

Radical Scavenging ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Critical Distance ◽

One Pot ◽

One Dimensional ◽

Transmission Electron ◽

Interaction Interaction ◽

Electron Microscopes

Abstract Bioinorganic chemistry has achieved great importance by considering environmental and health issues. Here we present anticancer, antioxidant, good protein quencher single pot biosynthesized one dimensional palladium Nanoneedles (PdNNs) as a negative catalyst, where water plays a role of safer solvent. Needle-shaped one-dimensional PdNNs was synthesized using filamentous fungal stain of Aspergillus oryzae (biomass) was successively applied as a suppressant for the growth of human breast, colon and leukemia cancer Cell Lines. Quenching process of bovine serum albumin by PdNNs was spontaneous with hydrogen bonding and hydrophilic interaction. Interaction of protein and PdNNs showed binding constant in the range of 104 M-1 and one binding site. Forster's resonance energy transfer (FRET) theory applied to find out distance between the interaction of PdNNs and protein, where critical distance and energy transfer distance varies with change in concentrations of PdNNs 4.84 x 10−6 M to 9.69 x 10-7 M from 2.9 to 3.7 nm and 3.2 to 5.4 nm respectively. Radical scavenging method was applied to find out an antioxidant activity which of nanoneedles. Needle-shaped palladium nanoparticles and particle size found to be ̴ 3.0 nm using high-resolution transmission electron microscopes.

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Association of a Novel Preribosomal Complex in Trypanosoma brucei Determined by Fluorescence Resonance Energy Transfer

Eukaryotic Cell ◽

10.1128/ec.00316-12 ◽

2012 ◽

Vol 12 (2) ◽

pp. 322-329 ◽

Cited By ~ 8

Author(s):

Lei Wang ◽

Martin Ciganda ◽

Noreen Williams

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Trypanosoma Brucei ◽

Rna Binding ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

5S Rrna ◽

Rna Recognition Motif ◽

Fluorescence Resonance

ABSTRACT We have previously reported that the trypanosome-specific proteins P34 and P37 form a unique preribosomal complex with ribosomal protein L5 and 5S rRNA in the nucleoplasm. We hypothesize that this novel trimolecular complex is necessary for stabilizing 5S rRNA in Trypanosoma brucei and is essential for the survival of the parasite. In vitro quantitative analysis of the association between the proteins L5 and P34 is fundamental to our understanding of this novel complex and thus our ability to exploit its unique characteristics. Here we used in vitro fluorescence resonance energy transfer (FRET) to analyze the association between L5 and P34. First, we demonstrated that FRET can be used to confirm the association between L5 and P34. We then determined that the binding constant for L5 and P34 is 0.60 ± 0.03 μM, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we used FRET to identify the critical regions of L5 and P34 involved in the protein-protein association. We found that the N-terminal APK-rich domain and RNA recognition motif (RRM) of P34 and the L18 domain of L5 are important for the association of the two proteins with each other. These results provide us with the framework for the discovery of ways to disrupt this essential complex.

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One-Pot Synthesis of Carbon Dots-Based Förster Resonance Energy Transfer Material and its Application in Fe3+ Detection

Journal of Nano Research ◽

10.4028/www.scientific.net/jnanor.45.134 ◽

2017 ◽

Vol 45 ◽

pp. 134-141 ◽

Cited By ~ 2

Author(s):

Yong Qiang Dang ◽

Yu Jing Zhou ◽

Jiang Tao Cai ◽

Guo Yang Liu ◽

Ya Ting Zhang ◽

...

Keyword(s):

Energy Transfer ◽

Carbon Dots ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Maximum Efficiency ◽

Step Method ◽

One Pot ◽

Excitation Time ◽

One Step ◽

Fret Efficiency

In this work, an efficient Förster resonance energy transfer (FRET) system has been prepared using carbon dots (CDs). A one-step method has been developed using β-cyclodextrin and tryptophan to synthesize the novel CT-CDs with FRET properties. The FRET was found to occur within the CT-CDs from the tryptophan moiety to the CDs. The FRET efficiency of the system increased with excitation time, reaching a maximum efficiency after 120 min. The CT-CDs were then evaluated in Fe3+ detection application, and showed selective and sensitive detection of Fe3+ over 14 other metal ions.

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In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer

RNA ◽

10.1261/rna.1678209 ◽

2009 ◽

Vol 15 (11) ◽

pp. 2063-2071 ◽

Cited By ~ 15

Author(s):

M. Huranova ◽

J. A. Jablonski ◽

A. Benda ◽

M. Hof ◽

D. Stanek ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Protein Interactions ◽

Rna Binding ◽

Rna Binding Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Rna Sequences ◽

In Vivo Detection

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A biosensor for MAPK-dependent Lin28 signaling

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0500 ◽

2018 ◽

Vol 29 (10) ◽

pp. 1157-1167 ◽

Cited By ~ 2

Author(s):

Laurel M. Oldach ◽

Kirill Gorshkov ◽

William T. Mills ◽

Jin Zhang ◽

Mollie K. Meffert

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Dynamic Monitoring ◽

Protein Levels ◽

Differentiated Cells

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)–mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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