In the dogma of molecular biology cap addition only occurs in the nucleus and its loss in the cytoplasm is irreversible. There are numerous reasons why this made sense, the most compelling of which is the concentration of the responsible protein (capping enzyme( in the nucleus and the biochemistry of cap addition, which requires a substrate with 2 phosphate groups, not the single phosphate that is left after the cap is removed. I will present work from my lab describing a new mechanism by which the cap can be restored onto cytoplasmic mRNAs after it has been removed by decapping or endonuclease cleavage. This work began with re-examination of results published in 1992 and never followed up describing a cap or cap-like structure on decay products of ÃŸ-globin mRNA in patients with ÃŸ-thalassemia (Cooley's anemia), a fatal disorder of hemoglobin production that is caused by inheriting two copies of this gene with a premature termination codon. I will describe how we validated those results, some of the basic biochemistry behind the re-capping process, and the identification and properties of a cytoplasmic complex that contains the enzymes that are responsible for mRNA re-capping. The loss of the cap is one of the key steps by which microRNAs repress translation and silence gene expression, and my talk will cover the cycle by which cytoplasmic re-capping may function in re-activating these silenced mRNAs. I will also touch on the possible links between cytoplasmic capping and the activation of neuronal or maternal mRNAs that must be kept in a silenced state until their translation is required. Although at this point it is highly speculative, cytoplasmic capping may also expand the proteome by enabling the translation of different forms of a protein from mRNAs that have lost the cap and sequences from their 5' ends, and the challenges the complexity of this process presents for bioinformatics, molecular and cell biology.
The experimental process is very slow because of
Intrinsic low sensitivity of method
Buggy whip approaches to data analysis and use of prior known information -over-focus on graphical interfaces and link to spectroscopy
There is no 'master equation'
Need improved, faster methods which incorporate chemical information appropriately, use probability methods in an integrated way, and make reasonable assumptions about averaging and motions.
Incorporate known information into experiment design for assignment and data collection
Break separation of assignment and structure calculation
Identify region of conformational spaces available from NMR data
Complete the loop of analysis and place complete analysis in a proper statistical framework
Use predictive power of integrated approach for
Speed up for structural genomics
Synthetic reconstruction and analysis of muilti-domain/ complexes for therapeutic target evaluation
Predictive structure/ function relationships for newly engineered systems (including de novo biology)
Despite more than 50 years of research, the etiology of depressive illness remains unknown. A hypothesis that has been central to much work in pharmacology and electrophysiology is that depression is caused by dysfunction in the serotonergic signaling system. In recent work, with Janet Best (OSU) and H. Frederik Nijhout (Duke), a mathematical model of a serotonergic synapse was created to study regulatory mechanisms in the serotonin system. After an introduction to the serotonin system, the model will be described as well as comparisons to experimental results. We will discuss why it is so difficult to understand the mechanism of efficacy of selective serotonin reuptake inhibitors (SSRIs). We will present predictions of the model as well as a new hypothesis for the mechanism of action of the SSRIs.
The results of these studies, which have involved collaborations with many other laboratories in the Lewis-Sigler Institute, include the following:
1) Expression of a substantial fraction (ca. 1/4) of the yeast genes is strongly correlated with the growth rate regardless of the limiting nutrient. Some genes are expressed more as growth rate increases (positive slope) and others are expressed more as the growth rate decreases (negative slope). These slopes are related to the periodic expression of the same genes in the metabolic cycle, which we have shown, by counting individual mRNAs by fluorescence in situ hybridization (FISH), is an intrinsic feature of yeast cell metabolism.
2) The levels of intracellular metabolites, in contrast, depend strongly on the limiting nutrient and relatively little on the growth rate. Only two (glutathione and trehalose) show strong negative slopes and a handful (e.g. ribose phosphate and fructose bis-phosphate) show strong positive slopes.
3) Starvation for phosphorus, sulfur or nitrogen ("natural nutrients") results in cell-cycle arrest, long-term (weeks) survival and sparing of residual glucose. Starvation, in auxotrophs, for leucine, uracil or histidine, in contrast, fail to arrest the cell cycle promptly, die much more rapidly and waste residual glucose. The glucose wasting is reminiscent of the Warburg effect seen in tumor cells.
4) Mutants that suppress starvation lethality and glucose wasting appear in genes already implicated in nutrient sensing. Genome-scale assessment of fitness during starvation provides a quantitative assessment of the contribution of each of the non-essential yeast genes to nutrient sensing and/or starvation survival.
It seems to us that much of what has been described as "stress response" would better be described as the consequence of slowing growth. The metabolic cycle, which separates oxidative and fermentative metabolism, appears to play a central role in growth-rate regulation. We are testing models in which metabolite levels, position in the metabolic cycle, external nutrient sensing as well as cell size are used to gate entry into the S-phase of the cell division cycle.
In this talk, we will first introduce a new quantity called Signed Activation Time (SAT), which is found to be critical in determining noise attenuation capability of a feedback system. We will next study how noise amplification rates of several biological examples may depend on SAT and investigate strategies for noise attenuation in systems involving both extra-cellular and intra-cellular components. In particular, we will study boundary sharpening during Zebrafish embryonic development.
We discuss diffuse interface (phase field) models of both single-component and multi-component vesicle membranes. We also consider models for the interactions of vesicles with an adhesive substrate and those with a background fluid. We present the mathematical derivations and compare results of numerical simulations with experimental findings.
I will first present recent developments on the Dissipative particle Dynamics (DPD) -- a Lagrangian method that bridges the gap between continuum and atomistic scales. In particular, I will first discuss theoretical foundations of DPD, its relation to molecular dynamics (MD), and its use in modeling seamlessly blood flow interacting with blood cells (platelets, white cells and red blood cells (RBCs). Specific examples will be given for cerebral malaria and sickle cell anemia.
This work is supported by NIH and by the DOE/INCITE program and NSF/NICS for computations.