Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

Multicellular architecture of malignant breast epithelia influences mechanics.

Cell-matrix and cell-cell mechanosensing are important in many cellular processes, particularly for epithelial cells. A crucial question, which remains unexplored, is how the mechanical microenvironment is altered as a result of changes to multicellular tissue structure during cancer progression. In this study, we investigated the influence of the multicellular tissue architecture on mechanical properties of the epithelial component of the mammary acinus. Using creep compression tests on multicellular breast epithelial structures, we found that pre-malignant acini with no lumen (MCF10AT) were significantly stiffer than normal hollow acini (MCF10A) by 60%. This difference depended on structural changes in the pre-malignant acini, as neither single cells nor normal multicellular acini tested before lumen formation exhibited these differences. To understand these differences, we simulated the deformation of the acini with different multicellular architectures and calculated their mechanical properties; our results suggest that lumen filling alone can explain the experimentally observed stiffness increase. We also simulated a single contracting cell in different multicellular architectures and found that lumen filling led to a 20% increase in the "perceived stiffness" of a single contracting cell independent of any changes to matrix mechanics. Our results suggest that lumen filling in carcinogenesis alters the mechanical microenvironment in multicellular epithelial structures, a phenotype that may cause downstream disruptions to mechanosensing.

Rapid disorganization of mechanically interacting systems of mammary acini.

Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 µm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.

A mathematical model of fluid and gas flow in nanoporous media.

The mathematical modeling of the flow in nanoporous rocks (e.g., shales) becomes an important new branch of subterranean fluid mechanics. The classic approach that was successfully used in the construction of the technology to develop oil and gas deposits in the United States, Canada, and the Union of Soviet Socialist Republics becomes insufficient for deposits in shales. In the present article a mathematical model of the flow in nanoporous rocks is proposed. The model assumes the rock consists of two components: (i) a matrix, which is more or less an ordinary porous or fissurized-porous medium, and (ii) specific organic inclusions composed of kerogen. These inclusions may have substantial porosity but, due to the nanoscale of pores, tubes, and channels, have extremely low permeability on the order of a nanodarcy (~109-²¹ m² ) or less. These inclusions contain the majority of fluid: oil and gas. Our model is based on the hypothesis that the permeability of the inclusions substantially depends on the pressure gradient. At the beginning of the development of the deposit, boundary layers are formed at the boundaries of the low-permeable inclusions, where the permeability is strongly increased and intensive flow from inclusions to the matrix occurs. The resulting formulae for the production rate of the deposit are presented in explicit form. The formulae demonstrate that the production rate of deposits decays with time following a power law whose exponent lies between -1/2 and -1. Processing of experimental data obtained from various oil and gas deposits in shales demonstrated an instructive agreement with the prediction of the model.