Correlating SHAPE signatures with three-dimensional RNA structures

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a facile technique for quantitative analysis of RNA secondary structure. In general, low SHAPE signal values indicate Watson-Crick base-pairing, and high values indicate positions that are single-stranded within the RNA structure. However, the relationship of SHAPE signals to structural properties such as non-Watson-Crick base-pairing or stacking has thus far not been thoroughly investigated. Here, we present results of SHAPE experiments performed on several RNAs with published three-dimensional structures. This strategy allows us to analyze the results in terms of correlations between chemical reactivities and structural properties of the respective nucleotide, such as different types of base-pairing, stacking, and phosphate-backbone interactions. We find that the RNA SHAPE signal is strongly correlated with cis-Watson-Crick/Watson-Crick base-pairing and is to a remarkable degree not dependent on other structural properties with the exception of stacking. We subsequently generated probabilistic models that estimate the likelihood that a residue with a given SHAPE score participates in base-pairing. We show that several models that take SHAPE scores of adjacent residues into account perform better in predicting base-pairing compared with individual SHAPE scores. This underscores the context sensitivity of SHAPE and provides a framework for an improved interpretation of the response of RNA to chemical modification.     

Golgi Calcium Pump Secretory Pathway Calcium ATPase 1 (SPCA1) Is a Key Regulator of Insulin-like Growth Factor Receptor (IGF1R) Processing in the Basal-like Breast Cancer Cell Line MDA-MB-231

Calcium signaling is a key regulator of pathways important in tumor progression, such as proliferation and apoptosis. Most studies assessing altered calcium homeostasis in cancer cells have focused on alterations mediated through changes in cytoplasmic free calcium levels. Here, we show that basal-like breast cancers are characterized by an alteration in the secretory pathway calcium ATPase 1 (SPCA1), a calcium pump localized to the Golgi. Inhibition of SPCA1 in MDA-MB-231 cells produced pronounced changes in cell proliferation and morphology in three-dimensional culture, without alterations in sensitivity to endoplasmic reticulum stress induction or changes in global calcium signaling. Instead, the effects of SPCA1 inhibition in MDA-MB-231 cells reside in altered regulation of calcium-dependent enzymes located in the secretory pathway, such as proprotein convertases. Inhibition of SPCA1 produced a pronounced alteration in the processing of insulin-like growth factor receptor (IGF1R), with significantly reduced levels of functional IGF1Rβ and accumulation of the inactive trans-Golgi network pro-IGF1R form. These studies identify for the first time a calcium transporter associated with the basal-like breast cancer subtype. The pronounced effects of SPCA1 inhibition on the processing of IGF1R in MDA-MB-231 cells independent of alterations in global calcium signaling also demonstrate that some calcium transporters can regulate the processing of proteins important in tumor progression without major alterations in cytosolic calcium signaling. Inhibitors of SPCA1 may offer an alternative strategy to direct inhibitors of IGF1R and attenuate the processing of other proprotein convertase substrates important in basal breast cancers.     

HIV-1 Transmission by Dendritic Cell-specific ICAM-3-grabbing Nonintegrin (DC-SIGN) Is Regulated by Determinants in the Carbohydrate Recognition Domain That Are Absent in Liver/Lymph Node-SIGN (L-SIGN)

In this study, we identify determinants in dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) necessary for human immunodeficiency virus, type 1 (HIV-1), transmission. Although human B cell lines expressing DC-SIGN efficiently capture and transmit HIV-1 to susceptible target cells, cells expressing the related molecule liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) do not. To understand the differences between DC-SIGN and L-SIGN that affect HIV-1 interactions, we developed Raji B cell lines expressing different DC-SIGN/L-SIGN chimeras. Testing of the chimeras demonstrated that replacement of the DC-SIGN carbohydrate-recognition domain (CRD) with that of L-SIGN was sufficient to impair virus binding and prevent transmission. Conversely, the ability to bind and transmit HIV-1 was conferred to L-SIGN chimeras containing the DC-SIGN CRD. We identified Trp-258 in the DC-SIGN CRD to be essential for HIV-1 transmission. Although introduction of a K270W mutation at the same position in L-SIGN was insufficient for HIV-1 binding, an L-SIGN mutant molecule with K270W and a C-terminal DC-SIGN CRD subdomain transmitted HIV-1. These data suggest that DC-SIGN structural elements distinct from the oligosaccharide-binding site are required for HIV-1 glycoprotein selectivity.     

Potential impact of grey squirrels Sciurus carolinensis on woodland bird populations in England

Several woodland bird species have declined markedly in abundance in England over the past 40 years, whilst the grey squirrel Sciurus carolinensis, a non-native nest predator, has increased. Given the timing, there has been concern that grey squirrels have driven these declines, although there is little data to support this view. Using novel analytical methods and extensive national bird and grey squirrel monitoring data, we examine whether there is evidence that woodland bird populations in England have been depressed by grey squirrels and whether there is a relationship between nest failure and squirrel numbers. Our results indicate that grey squirrels are very unlikely to have driven observed declines of woodland birds in recent years, although the number of associations, positive as well as negative, between grey squirrels and woodland birds is greater than expected by chance. For this reason, we cannot exclude the possibility that the populations of a small number of bird species, principally increasing species, have been depressed to some degree at sites where a greater number of grey squirrels were present. Of these species, perhaps the most convincing evidence is for Common Blackbird Turdus merula and Eurasian Collared Dove Streptopelia decaocto where nest record data also identified a positive relationship between nest failure at the egg stage and squirrel abundance.     

Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.     

Genome-wide expression analysis of a spinal muscular atrophy model: towards discovery of new drug targets

Spinal Muscular Atrophy is a recessive genetic disease and affects lower motor neurones and muscle tissue. A single gene is disrupted in SMA: SMN1 activity is abolished but a second copy of the gene (SMN2) provides limited activity. While the SMN protein has been shown to function in the assembly of RNA-protein complexes, it is unclear how the overall reduction in SMN activity specifically results in the neuromuscular phenotypes. Similar to humans, reduced smn activity in the fly causes earliest phenotypes in neuromuscular tissues. To uncover the effects of reduced SMN activity, we have studied gene expression in control and diseased fly tissues using whole genome micro-arrays. A number of gene expression changes are recovered and independently validated. Identified genes show trends in their predicted function: several are consistent with the function of SMN, in addition some uncover novel pathways. This and subsequent genetic analysis in the fly indicates some of the identified genes could be taken for further studies as potential drug targets for SMA and other neuromuscular disorders.