Small regulatory RNAs may sharpen spatial expression patterns

The precise establishment of gene expression patterns is a crucial step in development. Formation of a sharp boundary between high and low spatial expression domains requires a genetic mechanism that exhibits sensitivity, yet is robust to fluctuations, a demand that may not be easily achieved by morphogens alone. Recently, it has been demonstrated that small RNAs (and, in particular, microRNAs) play many roles in embryonic development. Whereas some RNAs are essential for embryogenesis, others are limited to fine-tuning a predetermined gene expression pattern. Here, we explore the possibility that small RNAs participate in sharpening a gene expression profile that was crudely established by a morphogen. To this end, we study a model in which small RNAs interact with a target gene and diffusively move from cell to cell. Though diffusion generally smoothens spatial expression patterns, we find that intercellular mobility of small RNAs is actually critical in sharpening the interface between target expression domains in a robust manner. This sharpening occurs as small RNAs diffuse into regions of low mRNA expression and eliminate target molecules therein, but cannot affect regions of high mRNA levels. We discuss the applicability of our results, as examples, to the case of leaf polarity establishment in maize and Hox patterning in the early Drosophila embryo. Our findings point out the functional significance of some mechanistic properties, such as mobility of small RNAs and the irreversibility of their interactions. These properties are yet to be established directly for most classes of small RNAs. An indirect yet simple experimental test of the proposed mechanism is suggested in some detail.     

An approach to evaluate the topological significance of motifs and other patterns in regulatory networks

Background  

The identification of network motifs as statistically over-represented topological patterns has become one of the most promising topics in the analysis of complex networks. The main focus is commonly made on how they operate by means of their internal organization. Yet, their contribution to a network's global architecture is poorly understood. However, this requires switching from the abstract view of a topological pattern to the level of its instances. Here, we show how a recently proposed metric, the pairwise disconnectivity index, can be adapted to survey if and which kind of topological patterns and their instances are most important for sustaining the connectivity within a network.     

Behavioral states may be associated with distinct spatial patterns in electrocorticogram

To determine if behavioral states are associated with unique spatial electrocorticographic (ECoG) patterns, we obtained recordings with a microgrid electrode array applied to the cortical surface of a human subject. The array was constructed with the intent of extracting maximal spatial information by optimizing interelectrode distances. A 34-year-old patient with intractable epilepsy underwent intracranial ECoG monitoring after standard methods failed to reveal localization of seizures. During the 8-day period of invasive recording, in addition to standard clinical electrodes a square 1 × 1 cm microgrid array with 64 electrodes (1.25 mm separation) was placed on the right inferior temporal gyrus. Careful review of video recordings identified four extended naturalistic behaviors: reading, conversing on the telephone, looking at photographs, and face-to-face interactions. ECoG activity recorded with the microgrid that corresponded to these behaviors was collected and ECoG spatial patterns were analyzed. During periods of ECoG selected for analysis, no electrographic seizures or epileptiform patterns were present. Moments of maximal spatial variance are shown to cluster by behavior. Comparisons between conditions using a permutation test reveal significantly different spatial patterns for each behavior. We conclude that ECoG recordings obtained on the cortical surface with optimal high spatial frequency resolution reveal distinct local spatial patterns that reflect different behavioral states, and we predict that similar patterns will be found in many if not most cortical areas on which a microgrid is placed.     

Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI.

The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.     

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.     

From components to regulatory motifs in signalling networks.

The developments in biochemistry and molecular biology over the past 30 years have produced an impressive parts list of cellular components. It has become increasingly clear that we need to understand how components come together to form systems. One area where this approach has been growing is cell signalling research. Here, instead of focusing on individual or small groups of signalling proteins, researchers are now using a more holistic perspective. This approach attempts to view how many components are working together in concert to process information and to orchestrate cellular phenotypic changes. Additionally, the advancements in experimental techniques to measure and visualize many cellular components at once gradually grow in diversity and accuracy. The multivariate data, produced by experiments, introduce new and exciting challenges for computational biologists, who develop models of cellular systems made up of interacting cellular components. The integration of high-throughput experimental results and information from legacy literature is expected to produce computational models that would rapidly enhance our understanding of the detail workings of mammalian cells.     

Combining phylogenetic data with co-regulated genes to identify regulatory motifs

MOTIVATION: Discovery of regulatory motifs in unaligned DNA sequences remains a fundamental problem in computational biology. Two categories of algorithms have been developed to identify common motifs from a set of DNA sequences. The first can be called a 'multiple genes, single species' approach. It proposes that a degenerate motif is embedded in some or all of the otherwise unrelated input sequences and tries to describe a consensus motif and identify its occurrences. It is often used for co-regulated genes identified through experimental approaches. The second approach can be called 'single gene, multiple species'. It requires orthologous input sequences and tries to identify unusually well conserved regions by phylogenetic footprinting. Both approaches perform well, but each has some limitations. It is tempting to combine the knowledge of co-regulation among different genes and conservation among orthologous genes to improve our ability to identify motifs. RESULTS: Based on the Consensus algorithm previously established by our group, we introduce a new algorithm called PhyloCon (Phylogenetic Consensus) that takes into account both conservation among orthologous genes and co-regulation of genes within a species. This algorithm first aligns conserved regions of orthologous sequences into multiple sequence alignments, or profiles, then compares profiles representing non-orthologous sequences. Motifs emerge as common regions in these profiles. Here we present a novel statistic to compare profiles of DNA sequences and a greedy approach to search for common subprofiles. We demonstrate that PhyloCon performs well on both synthetic and biological data. AVAILABILITY: Software available upon request from the authors. http://ural.wustl.edu/softwares.html     

Structure of regulatory networks and diversity of gene expression patterns

Complexity of gene regulatory network has been considered to be responsible for diversity of cells. Different types of cells, characterized by the expression patterns of genes, are produced in early development through the dynamics of gene activities based on the regulatory network. However, very little is known about relationship between the structure of regulatory networks and the dynamics of gene activities. In this paper, I introduce new idea of “steady-state compatibility” by which the diversity of possible gene activities can be determined from the topological structure of gene regulatory networks. The basic premise is very simple: the activity of a gene should be a function of the controlling genes. Thus, a gene should always show unique expression activity if the activities of the controlling genes are unique. Based on this, the maximum possible diversity of steady states is determined using only information regarding regulatory linkages without knowing the regulatory functions of genes. By extending this idea, some general properties were derived. For example, multiple loop structures in regulatory networks are necessary for increasing the diversity of gene activity. On the other hand, connected multiple loops sharing the same genes do not increase the diversity. The method was applied to a gene regulatory network responsible for early development in a sea urchin species. A set of important genes responsible for generating diversities of gene activities was derived based on the concept of compatibility of steady states.     

Inheritance patterns of idiotype expression: maternal-fetal immune regulatory networks

The production of Id-1, a cross-reactive idiotype associated with rat anti-group A streptococcal carbohydrate antibodies, by 11 strains of rats indicates that genes coding for Id-1 are in the germline. Its expression, however, follows a complex inheritance pattern. It was our intent in these studies to determine if immune responsiveness of streptococcus (GASV) immunized females could alter Id-1 expression of GASV-immunized progeny, and, in turn, introduce complications in Id-1 inheritance patterns.-We observed that Id-1-specific immune reactivity of GASV-immunized females could induce significant alterations in Id-1 production by progeny. The relationship between maternal and progeny Id-1 was complex, reflecting the complexity of autologous regulation of Id-1 production, and could be the opposite of what one would predict based upon parental transfer of Id-1 regulatory genes. The nongenetic nature of the maternal regulatory influence was confirmed by foster-mother studies.— We conclude that antigen-induced maternal immune responsiveness can exert a permanent regulatory influence on idiotype expression by progeny and consequently introduce error into the interpretations of idiotype inheritance patterns. The recognition of this maternal regulatory influence also lends further support to Jerne's hypothesis that idiotype-specific immune networks play a significant role in the regulation of immune responsiveness in vivo.     

MHC class II expression identifies functionally distinct human regulatory T cells

It has been known for decades that circulating human CD4 cells can express functional MHC class II molecules that induce T cell nonresponsiveness with Ag presentation. Because there is significant expression of MHC class II (MHC-II) determinants (DR) on a subpopulation CD4+ CD25(high) regulatory T cells (Treg), we examined the function of CD4 cells expressing MHC-DR. We demonstrate that MHC-II expression on human CD4+ CD25(high) T cells identifies a functionally distinct population of Treg that induces early contact-dependent suppression that is associated with high Foxp3 expression. In striking contrast, MHC-II- CD4+ CD25(high) Treg induce early IL-4 and IL-10 secretion and a late Foxp3-associated contact-dependent suppression. The DR expressing CD25(high) Treg express higher levels of Foxp3 message and protein, compared with the DR- CD25(high) Treg population. Direct single-cell cloning of CD4+ CD25(high) Treg revealed that, regardless of initial DR expression, ex vivo expression of CD25(high), and not DR, predicted which clones would exhibit contact-dependent suppression, high levels of Foxp3 message, and an increased propensity to become constitutive for DR expression. Thus, the direct ex vivo expression of MHC-II in the context of CD25(high) identifies a mature, functionally distinct regulatory T cell population involved in contact-dependent in vitro suppression.