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

Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for Individuals with Neurofibromatosis Type 1 and Legius Syndrome

Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase-activating protein (GAP) for RAS that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Obtaining a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci, and uncertainty over the effects of some NF1 and SPRED1 variants on pre-mRNA splicing and/or protein expression and function. To improve NF1 and SPRED1 variant classification and establish pathogenicity for NF1 and SPRED1 variants identified in individuals with NF1 or LS, we analyzed patient RNA by RT-PCR and performed in vitro exon trap experiments and estimated NF and SPRED1 protein expression, RAS GAP activity, and interaction. We obtained evidence to support pathogenicity according to American College of Medical Genetics guidelines for 73/114 variants tested, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.</p

Increased expression of aggrecan and biglycan mRNA in Achilles tendinopathy

To determine the expression of mRNA encoding the proteoglycans aggrecan, versican, biglycan and decorin in mid-tendon samples of chronic painful Achilles tendinopathy and ruptured Achilles tendons, compared with normal tendons. Total RNA isolated from frozen tendon samples (14 normal, 13 painful, 14 ruptured) was assayed by relative quantitative reverse transcription polymerase chain reaction for aggrecan, versican, biglycan and decorin mRNA, normalized using 18S rRNA. Differences between sample groups were tested by univariate analysis of variance with age as co-variate. In normal tendon samples expression of each of the proteoglycan mRNA decreased with increasing age. Decorin mRNA was the most highly-expressed of the proteoglycan mRNA, while versican mRNA expression was higher (3.8-fold) than that of aggrecan. In painful tendinopathy both aggrecan and biglycan mRNA expression increased (more than 10-fold and 5-fold, respectively) compared with normal tendon samples, but levels of versican and decorin mRNA were not significantly changed. In ruptured tendons the levels of aggrecan, biglycan and versican mRNA were not changed compared with normal tendon samples, but decorin mRNA decreased markedly. Increased aggrecan and biglycan mRNA expression in painful tendinopathy resembles the pattern in fibrocartilaginous regions of tendon, and may reflect an altered mechanical environment at the site of the lesion. Increased aggrecan mRNA expression may underlie the increase in glycosaminoglycan observed in painful tendinopathy

Quantifying mRNA synthesis and decay rates using small RNAs

Regulation of mRNA decay is a critical component of global cellular adaptation to changing environments. The corresponding changes in mRNA lifetimes can be coordinated with changes in mRNA transcription rates to fine-tune gene expression. Current approaches for measuring mRNA lifetimes can give rise to secondary effects due to transcription inhibition and require separate experiments to estimate changes in mRNA transcription rates. Here, we propose an approach for simultaneous determination of changes in mRNA transcription rate and lifetime using regulatory small RNAs to control mRNA decay. We analyze a stochastic model for coupled degradation of mRNAs and sRNAs and derive exact results connecting RNA lifetimes and transcription rates to mean abundances. The results obtained show how steady-state measurements of RNA levels can be used to analyze factors and processes regulating changes in mRNA transcription and decay

Identification of an Alternative Exon in a GABA Receptor Gene

The central dogma of biology states that DNA is transcribed into mRNA, which is then translated into proteins. In order for translation to occur, pre-mRNAs first must be processed. In pre-mRNA processing, parts of the nucleotide sequence called introns are spliced out from the transcript, so the final mRNA is made up entirely of exons. In alternative splicing, an exon is spliced out of the pre-mRNA transcript much like an intron. An mRNA transcript produced as a result of alternative splicing could produce a different protein than the mRNA without alternative splicing. Alternative splicing of an mRNA transcript could also result in a premature termination codon (PTC) within the mRNA sequence. This premature termination codon causes translation to stop before the full transcript has been translated, resulting in a truncated protein. Nonsense Mediated Decay (NMD) functions by degrading mRNA transcripts containing a PTC. NMD occurs during translation by an intricate series of protein-protein and protein-mRNA interactions that detect a PTC and result in the cleavage of PTC-containing mRNAs. We discovered an alternative exon in a zebrafish GABA receptor gene that leads to a PTC when excluded from the final mRNA and investigated the role of NMD in degrading the PTC-containing transcript

An agent-based model for mRNA export through the nuclear pore complex.

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics

Inference of RNA decay rate from transcriptional profiling highlights the regulatory programs of Alzheimer's disease.

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease.

Inhibition of translation by poliovirus: Inactivation of a specific initiation factor

Translation of vesicular stomatitis virus (VSV) mRNA, like host mRNA translation, is inhibited in cells infected with poliovirus. To study the mechanism of poliovirus-induced inhibition of protein synthesis, we prepared extracts from poliovirus-infected and uninfected HeLa cells. Poliovirus mRNA was translated in lysates from both infected and uninfected cells, while VSV mRNA was translated only in the lysate from uninfected cells. Addition of purified translation initiation factors to the extract from infected cells showed that one factor, eIF-4B, could restore VSV mRNA translation in the infected lysate, but did not increase poliovirus mRNA translation. Further experiments involving translation of VSV mRNA in mixed extracts from poliovirus-infected and uninfected cells showed (i) that there was not an excess of an inhibitor of VSV mRNA translation in the infected lysate, but (ii) that an activity that caused a slow inactivation of eIF-4B was present in the infected lysate. Inactivation of eIF-4B appears to be the mechanism by which poliovirus infection causes a selective inhibition of translation

mRNA

mRNA (messenger RNA) is a large class of single-stranded RNA molecules, and a blueprint for protein synthesis, transferring the genetic information from the DNA to the ribosome, where they specify the amino acid sequence of the protein product. During the movement of the ribosome along the mRNA, the base sequence on the mRNA is read and each three-base codon is translated into its corresponding amino acid using the genetic code

tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay.

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR