Conceptual Modeling of mRNA Decay Provokes New Hypotheses

Biologists are required to integrate large amounts of data to construct a working model of the system under investigation. This model is often informal and stored mentally or textually, making it prone to contain undetected inconsistencies, inaccuracies, or even contradictions, not much less than a representation in free natural language. Using Object-Process Methodology (OPM), a formal yet visual and humanly accessible conceptual modeling language, we have created an executable working model of the mRNA decay process in Saccharomyces cerevisiae, as well as the import of its components to the nucleus following mRNA decay. We show how our model, which incorporates knowledge from 43 articles, can reproduce outcomes that match the experimental findings, evaluate hypotheses, and predict new possible outcomes. Moreover, we were able to analyze the effects of the mRNA decay model perturbations related to gene and interaction deletions, and predict the nuclear import of certain decay factors, which we then verified experimentally. In particular, we verified experimentally the hypothesis that Rpb4p, Lsm1p, and Pan2p remain bound to the RNA 3′-untralslated region during the entire process of the 5′ to 3′ degradation of the RNA open reading frame. The model has also highlighted erroneous hypotheses that indeed were not in line with the experimental outcomes. Beyond the scientific value of these specific findings, this work demonstrates the value of the conceptual model as an in silico vehicle for hypotheses generation and testing, which can reinforce, and often even replace, risky, costlier wet lab experiments

Transcription in the nucleus and mRNA decay in the cytoplasm are coupled processes

Maintaining appropriate mRNAs levels is vital for any living cell. mRNA synthesis in the nucleus by RNA polymerase II core enzyme (Pol II) and mRNA decay by cytoplasmic machineries determine these levels. Yet, little is known about possible cross-talk between these processes. The yeast Rpb4/7 is a nucleo-cytoplasmic shuttling heterodimer that interacts with Pol II and with mRNAs and is required for mRNA decay in the cytoplasm. Here we show that interaction of Rpb4/7 with mRNAs and eventual decay of these mRNAs in the cytoplasm depends on association of Rpb4/7 with Pol II in the nucleus. We propose that, following its interaction with Pol II, Rpb4/7 functions in transcription, interacts with the transcript cotranscriptionally and travels with it to the cytoplasm to stimulate mRNA decay. Hence, by recruiting Rpb4/7, Pol II governs not only transcription but also mRNA decay

Re-Evaluating the Spherical-Nucleic-Acid Technology

We re-evaluate the evidence presented for the detection of mRNA in cells and tissues with Spherical Nucleic Acids.<br /

Experimental validation of model’s predictions shows that import of some decay factors is independent of Xrn1p exonuclease activity.

<p>XRN1 (WT) PAB1-GFP xpo1-1, mex67-5 cells, or xpo1-1, mex67-5, Δxrn1 cells expressing xrn1D208A-GFP and RFP fusion of the indicated DFs were proliferated at 24°C and then shifted to 37°C for 1 h; images were taken as previously described [<b> </b>10]. (A) Representative images of WT cells expressing the indicated proteins after 1h incubation at 37°C. Pab1-GFP, whose export is dependent on Xpo1p and Mex67p, serves as a nuclear marker, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107085#pone.0107085-Haimovich1" target="_blank">[10]</a>. Arrows point at examples of nuclei carrying both fluorescent proteins. All factors were cytoplasmic at 24°C ([<b> </b>10] and not shown) (B) Percentage of cells with nuclear localization of the indicated DF was determined, as described previously [<b> </b>10]. Mean values ± SD are shown. P-values of any pairwise difference that was <0.05 is indicated.</p

Summary of <i>in vivo</i> observations regarding Xrn1p and Dcp2p import dependencies.

<p>Summary of <i>in vivo</i> observations regarding Xrn1p and Dcp2p import dependencies.</p

<i>In-silico</i> DFs import results and processes activation when deleting one factor/domain at a time.

<p>“+” represent import and “−” represent not-imported. “O” represents actual observation from experiments (see [10]), and “P” represents <i>in-silico</i> predictions that may not be experimentally tested. NA – not applicable.</p><p><i>In-silico</i> DFs import results and processes activation when deleting one factor/domain at a time.</p