Rapid microRNA (miRNA) detection and classification via surface-enhanced Raman spectroscopy (SERS).

microRNAs (miRNA) are recognized as regulators of gene expression during development and cell differentiation as well as biomarkers of disease. Development of rapid and sensitive miRNA profiling methods is essential for evaluating the pattern of miRNA expression that varies across normal and diseased states. The ability to identify miRNA expression patterns is limited to cumbersome assays that often lack sensitivity and specificity to distinguish between different miRNA families and members. We evaluated a surface-enhanced Raman scattering (SERS) platform for detection and classification of miRNAs. The strength of the SERS-based sensor is its sensitivity to detect extremely low levels of analyte and specificity to provide the molecular fingerprint of the analyte. We show that the SERS spectra of related and unrelated miRNAs can be detected in near-real time, that detection is sequence dependent, and that SERS spectra can be used to classify miRNA patterns with high accuracy.

c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

Human Taf(II)130 is a coactivator for NFATp.

NFATp is one member of a family of transcriptional activators that regulate the expression of cytokine genes. To study mechanisms of NFATp transcriptional activation, we established a reconstituted transcription system consisting of human components that is responsive to activation by full-length NFATp. The TATA-associated factor (TAF(II)) subunits of the TFIID complex were required for NFATp-mediated activation in this transcription system, since TATA-binding protein (TBP) alone was insufficient in supporting activated transcription. In vitro interaction assays revealed that human TAF(II)130 (hTAF(II)130) and its Drosophila melanogaster homolog dTAF(II)110 bound specifically and reproducibly to immobilized NFATp. Sequences contained in the C-terminal domain of NFATp (amino acids 688 to 921) were necessary and sufficient for hTAF(II)130 binding. A partial TFIID complex assembled from recombinant hTBP, hTAF(II)250, and hTAF(II)130 supported NFATp-activated transcription, demonstrating the ability of hTAF(II)130 to serve as a coactivator for NFATp in vitro. Overexpression of hTAF(II)130 in Cos-1 cells inhibited NFATp activation of a luciferase reporter. These studies demonstrate that hTAF(II)130 is a coactivator for NFATp and represent the first biochemical characterization of the mechanism of transcriptional activation by the NFAT family of activators.

Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp.

BACKGROUND: NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. RESULTS: We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. CONCLUSIONS: We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle.

Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers. In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components. Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing. Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex. These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits. We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication.

Two protein-tyrosine phosphatases inactivate the osmotic stress response pathway in yeast by targeting the mitogen-activated protein kinase, Hog1.

Protein phosphatases inactivate mitogen-activated protein kinase (MAPK) signaling pathways by dephosphorylating components of the MAPK cascade. Two genes encoding protein-tyrosine phosphatases, PTP2, and a new phosphatase, PTP3, have been isolated in a genetic selection for negative regulators of an osmotic stress response pathway called HOG, for high osmolarity glycerol, in budding yeast. PTP2 and PTP3 were isolated as multicopy suppressors of a severe growth defect due to hyperactivation of the HOG pathway. Phosphatase activity is required for suppression since mutation of the catalytic Cys residue in Ptp2 and Ptp3, destroys suppressor function and biochemical activity. The substrate of these phosphatases is likely to be the MAPK, Hog1. Catalytically inactive Ptp2 and Ptp3 coprecipitate with Hog1 from yeast extracts. In addition, strains lacking PTP2 and PTP3 do not dephosphorylate Hog1-phosphotyrosine as well as wild type. The latter suggests that PTP2 and PTP3 play a role in adaptation. Consistent with this role, osmotic stress induces expression of PTP2 and PTP3 transcripts in a Hog1-dependent manner. Thus Ptp2 and Ptp3 likely act in a negative feedback loop to inactivate Hog1.