Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Emerging evidence suggests that Ago/Piwi proteins function in the nucleus as well as the cytoplasm to control gene expression, but the mechanisms they employ in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. We show that Twi12 interacts with the exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2 in vivo, as well as activate its exonuclease activity in vitro. When Twi12 or Xrn2 are depleted, pre-rRNA processing intermediates accumulate and mature rRNA levels decline. RNA polymerase I and II (RNAP I and II) transcripts also accumulate. Twi12 function depends on small RNA (sRNA) binding, which is required for its nuclear import. Twi12-bound sRNAs are Dicer-independent 3' tRNA fragments that we propose may not be involved in sequence-specific base pairing to targets. Our findings suggest a role for tRNA fragments and an Ago/Piwi protein in global control of gene expression through interaction with a conserved exonuclease.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression. Two-condition experiment, Normoxic MSCs vs. Hypoxic MSCs.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs. Two-condition experiment, KP MSCs vs. 3A6 MSCs.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:The Lupus autoantigen (La) is a single-stranded RNA-binding protein that stabilizes RNA polymerase III (pol III) transcripts and supports RNA folding. In addition, La has been implicated in different steps of the mammalian small RNA pathway. Here, we have analyzed effects of La depletion on the Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago protein complexes and our data suggests that La prevents the production and loading of such tRNA fragments. However, one specific isoleucine tRNA escapes this regulation and produces both a functional tRNA as well as a microRNA (miRNA). We demonstrate that fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin 5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading. Thus, La functions as gatekeeper to ensure correct tRNA generation and to protect the miRNA pathway from potentially functional tRNA fragments.
Project description:The Lupus autoantigen (La) is a single-stranded RNA-binding protein that stabilizes RNA polymerase III (pol III) transcripts and supports RNA folding. In addition, La has been implicated in different steps of the mammalian small RNA pathway. Here, we have analyzed effects of La depletion on the Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago protein complexes and our data suggests that La prevents the production and loading of such tRNA fragments. However, one specific isoleucine tRNA escapes this regulation and produces both a functional tRNA as well as a microRNA (miRNA). We demonstrate that fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin 5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading. Thus, La functions as gatekeeper to ensure correct tRNA generation and to protect the miRNA pathway from potentially functional tRNA fragments.