Conditioned spin and charge dynamics of a single electron quantum dot

In this article we describe the incoherent and coherent spin and charge dynamics of a single electron quantum dot. We use a stochastic master equation to model the state of the system, as inferred by an observer with access to only the measurement signal. Measurements obtained during an interval of time contribute, by a past quantum state analysis, to our knowledge about the system at any time $t$ within that interval. Such analysis permits precise estimation of physical parameters, and we propose and test a modification of the classical Baum-Welch parameter re-estimation method to systems driven by both coherent and incoherent processes.Comment: 9 pages, 9 figure

Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs

The heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in the regulation of mRNA metabolism by specifically recognizing G-tract RNA sequences. We have determined the solution structures of the three quasi–RNA-recognition motifs (qRRMs) of hnRNP F in complex with G-tract RNA. These structures show that qRRMs bind RNA in a very unusual manner, with the G-tract ‘encaged’, making the qRRM a novel RNA binding domain. We defined a consensus signature sequence for qRRMs and identified other human qRRM-containing proteins that also specifically recognize G-tract RNAs. Our structures explain how qRRMs can sequester G-tracts, maintaining them in a single-stranded conformation. We also show that isolated qRRMs of hnRNP F are sufficient to regulate the alternative splicing of the Bcl-x pre-mRNA, suggesting that hnRNP F would act by remodeling RNA secondary and tertiary structures

Model to explain how a decrease in hnRNP A1/A2 proteins reduces transcription elongation of P-TEFb-dependent genes but stimulates expression of P-TEFb-independent genes.

<p>In panel A, normally high levels of nuclear hnRNP A1/A2 proteins ensure the release of P-TEFb from the repressor 7SK RNP which is used for the efficient transcription of P-TEFb-dependent genes, such as <i>Kitlg</i> and many genes encoding ribosomal proteins. A reduction in the levels of nuclear hnRNP A1/A2 proteins (panel B) would prevent the efficient release of P-TEFb from 7SK, eliciting promoter-proximal pausing of RNA polymerase II on P-TEFb-dependent genes, and a decrease in their expression. The stalling of polymerases at promoter-proximal locations would reduce the average number of polymerases associating with P-TEFb-dependent genes, thereby increasing the level of free polymerases which would then associate preferentially with, and stimulate the expression of, P-TEFb-independent genes, such as the mycUP1 reporter gene and <i>Egr1</i>.</p

Changes in RNA polymerase II occupancy on ribosomal protein-encoding genes elicited by DRB and the depletion of A1/A2.

<p>Density of ChIP-seq reads for RNA polymerase II (H-224 antibody) on genes encoding ribosomal proteins (major RefSeq transcripts indicated). Genes are transcribed from left to right. The size in bp of the first exon is indicated. Red dashed boxes indicate the promoter-proximal regions displaying an increase in pol II occupancy upon treatment with DRB and the siRNA-mediated knockdown of A1/A2.</p

Impact of A1/A2 depletion and DRB on gene expression.

<p><b>A</b>, The transcriptome of untreated (NS), A1/A2-depleted by RNAi and DRB-treated HCT116 cells was sequenced and reads were assigned. The Venn diagram depicts the number of genes in each sample that display a difference in expression relative to the NS sample, and only includes genes displaying a log2 (fold change) ≥ 2 with a correction for genes expressed at lower level using the following equation: corrected-log2(fold change) = log2(fold change) + 1/gamma^(exp-exp.offset), where gamma = 5 and exp.offset = 0, exp is the average expression level expressed as base 10 log, and fold change is the measured fold change in expression. The <i>p</i>-value of the DRB and ΔA1/A2 overlap is 1.4e-12 (Fisher’s exact test). <b>B</b>, Correlation map plotting the expression changes for the overlapping set of reactive genes in the DRB-treated and siA1/A2-depleted samples. Only genes with fold changes ≥ 2 are considered. The correlation coefficient is 0.692 (Pearson correlation ^2).</p

mycUP1 expression is stimulated by the depletion of nuclear of hnRNP A1 and A2.

<p><b>A</b>, Western analysis of A1, A2 and mycUP1 from four selected HCT116 clones engineered to express a transfected myc-tagged mouse UP1. Stable transformants were transfected with various siRNA mixtures and proteins were extracted 72 hours later. Only siRNA A1₂ also targets the mouse mycUP1. <b>B</b>, Cytoplasmic accumulation of hnRNP A1 and hnRNP A2 in HCT116 cells upon osmotic shock. HCT116 cells growing on glass coverslips were left untreated (0 mM) or were exposed to 600 mM sorbitol. After 1 hour, cells were fixed and immunostained with anti-hnRNP A1 and anti-hnRNP A2 antibodies. <b>C</b>, Sorbitol stimulates mycUP1 expression in a p38 kinase-dependent manner. The p38 kinase inhibitor SB203580 was added to H55-34 cells for 2 hours (8 μg/ml) before treating cells with 600 mM sorbitol for 1 hour. Cells were then washed with PBS and incubated in complete media for 24 hours. Endogenous A1/A2 and mycUP1 proteins were detected simultaneously using the anti-A1/A2 antibody.</p

Transcription elongation recovery assay.

<p>Change in the rate of transcription of different portions of <i>Kitlg</i> upon depletion of A1 and A2 by RNAi. Seventy-two hours after transfecting a control siRNA against <i>luciferase</i> (Luc) or siA1₆ + siA2₁ (A1/A2) in HCT116 cells (H55-34 clone), DRB was applied, washed out, and the recovery of transcription was assessed at different positions. Each graph illustrates the position analyzed and the level of RNA at each time after the DRB block was released. The graphs represent averages of three independent experiments and standard deviations are provided. The positions analyzed on <i>Kitlg</i> are shown schematically in the map provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126654#pone.0126654.g003" target="_blank">Fig 3C</a>.</p

The depletion of nuclear hnRNP A1 and A2 stimulates the transcription of mycUP1.

<p><b>A</b>, Steady-state levels of mycUP1 transcripts measured by semi-quantitative or real-time quantitative RT-PCR (sqRT-PCR or qRT-PCR). At least 5 independent depletion experiments (A1₆+A2₁ vs Luc) and sorbitol treatments (0 or 600 mM) were analyzed by sqRT-PCR (examples are shown in Fig B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126654#pone.0126654.s001" target="_blank">S1 File</a>). Fold increases are represented by white diamonds over an averaged logarithmic histogram. Numbers below the histograms indicate averaged fold stimulation in mycUP1 transcripts. <b>B</b>, Actinomycin D (ActD) abrogates the increase in mycUP1 protein and RNA expression following osmotic shock. The top panel represents a western with the A1/A2 antibody and the results of sqRT-PCR for mycUP1 and actin (ACTB) are shown below. <b>C</b>, ActD abrogates the increase in mycUP1 protein expression associated with the knockdown of A1 and A2. Western analysis of mycUP1 protein expression is shown at different times after siRNA treatment with or without ActD for the last 24 hours of each period. <b>D</b>, HCT116 cells expressing mycUP1 were treated with 1 or 10 μg/ml of cycloheximide 1 hour prior to sorbitol treatment. The mycUP1 protein and RNA levels are shown. Duplicate experiments with quantitated mycUP1 transcript levels are provided in Fig C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126654#pone.0126654.s001" target="_blank">S1 File</a>.</p

Depleting nuclear hnRNP A1/A2 mimics the effect of the transcription elongation inhibitor DRB.

<p><b>A</b>, The P-TEFb kinase CDK9 was inactivated by treating cells with DRB (100 μM) for 24 hours. The impact on mycUP1 protein expression was compared to the impact of a 1 hour treatment with sorbitol (cells harvested 24 hours later), treatment with siA1/A2 for 72 hours or untreated (mock). <b>B</b>, Quantitative RT-PCR was used to measure the impact of siA1/A2, sorbitol or DRB on the steady state levels of mycUP1 transcripts from the reporter, and endogenous <i>Egr1</i> and <i>Kitlg</i> transcripts. Levels are expressed relative to the mock treatment. <b>C</b>, Occupancy profiling of RNA polymerase II on the mycUP1, <i>Egr1</i>, <i>Kitlg</i> and genes. ChIP assays were performed with extracts of H55-34 cells using the pol II antibody H-224. A linear map of each gene locus is provided. Exons are represented by boxes and coding sequences are in black. The location of amplicons used in qPCR is shown below and numbers indicate the relative position of the center of the amplicon relative to the transcription start site. Pol II-ChIP performed on extract produced after DRB treatment (100 μM) for 8 hours. <b>D</b>, Pol II-ChIP performed on extract prepared 8 hours following the application of sorbitol for 1 hour. <b>E</b>, Pol II/ChIP performed on extract from a 72 hours of RNAi treatment targeting hnRNP A1 and A2. For panels C–E, the DNA recovered after ChIP was quantitated by qPCR using the indicated amplicons. Values are expressed as percentage of input DNA. Error bars indicate standard deviations and are based on three independent experiments.</p

Binding Sites for hnRNP A1/A2 Stimulate the In Vitro Removal of Enlarged Introns

<div><p>(A) The model pre-mRNAs contain portions of exons 7 or 7B of the hnRNP A1 gene paired with the adenovirus L2 exon. The size of the small introns in 7-Ad and 7B-Ad pre-mRNAs is indicated in nt. The size of lambda inserts A, B, and C are, respectively, 1015, 943, and 1038 nt. The lambda inserts do not contain the sequences U AGGG^U/_A or U AGAG^U/_A, which correspond to high-affinity binding sites for hnRNP A/B proteins [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040021#pbio-0040021-b020" target="_blank">20</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040021#pbio-0040021-b043" target="_blank">43</a>]. The larger intron substrates contain either exon 7 or exon 7B as first exon, and either the adenovirus L2 or the Bcl-x exon 3 as second exon. When no other elements are inserted, the pre-mRNAs correspond to the (−.−) version. The (a.a) versions contain ABS inserted 26 nt downstream of the 5′ splice site and 88 nt upstream of the 3′ splice site. The (→.←) versions contain inverted repeats at the same positions as ABS. </p> <p>(B) The 7-Ad and 7B-Ad pre-mRNAs were co-incubated for the times indicated (in minutes) in a HeLa nuclear extract (lanes 1–6). Additional mixtures were prepared with pre-mRNAs carrying lambda insert A lacking or containing ABS (lanes 7–12 and 13–18, respectively). The concentration of each pre-mRNA was 80 pM. Following RNA extraction, the mRNA products from mixtures were amplified by RT-PCR using a common set of primers (reverse primer complementary to the adenovirus exonic sequence and forward primer corresponding to plasmid sequence upstream of exon 7 or exon 7B sequences). The graph displays the abundance of amplified splicing product at different times for 7-Ad and the different 7-AdA pre-mRNAs. The RT-PCR assay shown here and in other figures was performed in conditions that displayed a linear relationship between the amounts of input RNA and amplified products over a large range of input RNA concentrations (from 10-fold less to at least 6-fold more than the amounts used in the assays [data not shown]).</p> <p>(C) Splicing reactions were set using ³²P-labeled pre-mRNAs and incubated for 0 or 2 h in HeLa nuclear extracts. Total RNA was extracted, and the splicing products were fractionated on a 5% acrylamide/8 M urea gel. The position of the lariat products is indicated.</p> <p>(D) Each of the 7-Ad pre-mRNAs carrying lambda inserts B or C (7-AdB or 7-AdC; 80 pM) was co-incubated with the small-intron 7B-Ad pre-mRNA (8 pM). Versions lacking (−.−) or containing ABS (a.a), as well as carrying inverted repeats (→.←), were used. Following incubation for different times, spliced products were amplified by RT-PCR using a common set of primers. The co-incubated small-intron control is only shown for the 7-AdC pre-mRNA mixture. M indicates molecular-weight markers.</p> <p>(E) Large-intron pre-mRNAs 7-BclA and 7B-BclA (80 pM each) lacking (−.−) or containing ABS (a.a) were co-incubated for the indicated times in a HeLa extract. RT-PCR was performed as described in (B) except that a Bcl-x reverse primer was used. The band amplified at <i>t</i> = 0 (lane 2) is artifactual and does not co-migrate with the 7B/Bcl splicing product. M indicates molecular-weight markers.</p></div