scholarly journals Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human Cells

2005 ◽  
Vol 16 (6) ◽  
pp. 2597-2604 ◽  
Author(s):  
Rut Valgardsdottir ◽  
Ilaria Chiodi ◽  
Manuela Giordano ◽  
Fabio Cobianchi ◽  
Silvano Riva ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Human Cells ◽  
Heat Shock Factor 1 ◽  
Small Interfering Rnas ◽  
Argonaute 2 ◽  
Specific Subset ◽  
Nuclear Structures ◽  
Sirna Knockdown ◽  
Processing Factors

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs.

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

scholarly journals miR-125-chinmo pathway regulates dietary restriction dependent enhancement of lifespan in Drosophila

2020 ◽  
Author(s):  
Manish Pandey ◽  
Sakshi Bansal ◽  
Sudipta Bar ◽  
Nicholas S. Sokol ◽  
Jason M. Tennessen ◽  
...  
Keyword(s):  
Dietary Restriction ◽  
Healthy Aging ◽  
Fat Body ◽  
Late Onset ◽  
Functional Characterization ◽  
Drug Candidates ◽  
Downstream Effectors ◽  
Processing Factors

AbstractDietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA mediated networks in DR dependent enhancement of lifespan remains largely unexplored. We show that two neuronally enriched and highly conserved microRNAs, miR-125 and let-7 mediate the DR response in Drosophila melanogaster. Functional characterization of miR-125 demonstrates its role in neurons while its target chinmo acts both in neurons and the fat body to modulate fat metabolism and longevity. Proteomic analysis revealed that Chinmo exerts its DR effects by regulating expression of FATP, CG2017, CG9577, CG17554, CG5009, CG8778, CG9527 and FASN1. Our findings identify miR-125 as a conserved effector of DR pathway and open up the avenue for this small RNA molecule and its downstream effectors to be considered as potential drug candidates for treatment of late-onset diseases and biomarkers for healthy aging in humans.

scholarly journals Genetic and Functional Characterization of ANGPTL7 as a Therapeutic Target for Glaucoma

2021 ◽  
Author(s):  
Kavita Praveen ◽  
Gaurang Patel ◽  
Lauren Gurski ◽  
Ariane Ayer ◽  
Trikaladarshi Persaud ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Human Genetics ◽  
Functional Characterization ◽  
Pathological Changes ◽  
Loss Of Function ◽  
Wild Type ◽  
Adult Mice ◽  
Sirna Knockdown ◽  
Coding Variants

Abstract Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing mAngptl7 levels via injection into mouse eyes increases the IOP. We also show that acute gene silencing via siRNA knockdown of Angptl7 in adult mice lowers the IOP (~2-4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.

scholarly journals Identification and functional characterization of transcriptional activators in human cells

2022 ◽  
Author(s):  
Nader Alerasool ◽  
He Leng ◽  
Zhen-Yuan Lin ◽  
Anne-Claude Gingras ◽  
Mikko Taipale
Keyword(s):  
Functional Characterization ◽  
Human Cells ◽  
Transcriptional Activators

Design and chemoproteomic functional characterization of a chemical probe targeted to bromodomains of BET family proteins

MedChemComm ◽  
2014 ◽  
Vol 5 (12) ◽  
pp. 1871-1878 ◽  
Author(s):  
Jiang Wu ◽  
Julia Shin ◽  
Cara M. M. Williams ◽  
Kieran F. Geoghegan ◽  
Stephen W. Wright ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Human Cells ◽  
Chemical Probe ◽  
Affinity Capture ◽  
Nuclear Extracts

Selectivity of a PFI-1 based BET bromodomain probe was demonstrated using affinity capture in nuclear extracts from human cells.

scholarly journals Functional characterization of ribosomal P1/P2 proteins in human cells

2008 ◽  
Vol 413 (3) ◽  
pp. 527-534 ◽  
Author(s):  
Francisco Martinez-Azorin ◽  
Miguel Remacha ◽  
Juan P. G. Ballesta
Keyword(s):  
Functional Characterization ◽  
Ribosomal Subunit ◽  
Human Cell Line ◽  
Human Cells ◽  
Translation Efficiency ◽  
Ribosomal Stalk ◽  
P Proteins ◽  
Subunit Joining

The ‘stalk’ is a large ribosomal subunit domain that regulates translation. In the present study the role of the ribosomal stalk P proteins in modulating ribosomal activity has been investigated in human cells using RNA interference. A strong down-regulation of P2 mRNA and a drastic decrease in P2 protein in a stable human cell line was achieved using a doxycycline-inducible system. Interestingly, the amount of P1 protein was similarly decreased in these cells, in contrast with the expression of P1 mRNA. The loss of P1/P2 proteins produced a decrease in the growth rate of these cells, as well as an altered polysome pattern with reduced translation efficiency, but without affecting the free 40 S/60 S subunit ratio. A decrease in the ribosomal-subunit joining capacity was also observed. These data indicate that P1/P2 proteins modulate cytoplasmic translation by influencing the interaction between subunits, thereby regulating the rate of cell proliferation.

scholarly journals miR-125-chinmo pathway regulates dietary restriction dependent enhancement of lifespan in Drosophila

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Manish Pandey ◽  
Sakshi Bansal ◽  
Sudipta Bar ◽  
Amit Kumar Yadav ◽  
Nicholas S Sokol ◽  
...  
Keyword(s):  
Dietary Restriction ◽  
Healthy Aging ◽  
Fat Body ◽  
Late Onset ◽  
Functional Characterization ◽  
Drug Candidates ◽  
Downstream Effectors ◽  
Processing Factors

Dietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA-mediated networks in DR-dependent enhancement of lifespan remains largely unexplored. We show that two neuronally enriched and highly conserved microRNAs, miR-125 and let-7 mediate the DR response in Drosophila melanogaster. Functional characterization of miR-125 demonstrates its role in neurons while its target chinmo acts both in neurons and the fat body to modulate fat metabolism and longevity. Proteomic analysis revealed that Chinmo exerts its DR effects by regulating the expression of FATP, CG2017, CG9577, CG17554, CG5009, CG8778, CG9527, and FASN1. Our findings identify miR-125 as a conserved effector of the DR pathway and open the avenue for this small RNA molecule and its downstream effectors to be considered as potential drug candidates for the treatment of late-onset diseases and biomarkers for healthy aging in humans.

scholarly journals Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

2005 ◽  
Vol 280 (16) ◽  
pp. 15880-15887 ◽  
Author(s):  
Stephen A. Baldwin ◽  
Sylvia Y. M. Yao ◽  
Ralph J. Hyde ◽  
Amy M. L. Ng ◽  
Sophie Foppolo ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Functional Characterization ◽  
Human Cells ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Transporter Family ◽  
Human And Mouse

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and inXenopusoocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

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