Cooperativity boosts affinity and specificity of proteins with multiple RNA-binding domains

Numerous cellular processes rely on the binding of proteins with high affinity to specific sets of RNAs. Yet most RNA-binding domains display low specificity and affinity in comparison to DNA-binding domains. The best binding motif is typically only enriched by less than a factor 10 in high-throughput RNA SELEX or RNA bind-n-seq measurements. Here, we provide insight into how cooperative binding of multiple domains in RNA-binding proteins (RBPs) can boost their effective affinity and specificity orders of magnitude higher than their individual domains. We present a thermodynamic model to calculate the effective binding affinity (avidity) for idealized, sequence-specific RBPs with any number of RBDs given the affinities of their isolated domains. For seven proteins in which affinities for individual domains have been measured, the model predictions are in good agreement with measurements. The model also explains how a two-fold difference in binding site density on RNA can increase protein occupancy 10-fold. It is therefore rationalized that local clusters of binding motifs are the physiological binding targets of multi-domain RBPs

Direct observation of a condensate effect on super-enhancer controlled gene bursting

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 μm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 μm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting

PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila

Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance

A national survey of managed honey bee 2011-12 winter colony losses in the United States: results from the Bee Informed Partnership

Estimates of winter loss for managed honey bee (Apis mellifera) colonies are an important measure of honey bee health and productivity. We used data from 5,500 US beekeepers (5,244 backyard, 189 sideline and 67 commercial beekeepers) who responded to the April 2012 Bee Informed Partnership Winter Colony Loss Survey and calculated loss as the difference in the number of colonies between October 1, 2011 and April 1, 2012, adjusting for increases and decreases over that period. In the US, the total colony loss was 22.5% for the 2011-12 winter; 45.1% (n = 2,482) of respondents reported no colony loss. Total loss during 2011-12 was substantially lower than loss during 2010-11 (29.9%). Of the 4,484 respondents who kept bees in 2010-11 and 2011-12, 72.0% reported that the loss during 2011-12 was smaller or similar to the loss during 2010-11. There was substantial variation in total loss by state (range 6.2% to 47.7%). The average loss per beekeeping operation was 25.4%, but the average loss was not significantly different by operation type (backyard, sideline, commercial). The average self-reported acceptable loss per respondent was 13.7%; 46.8% (n = 2,259) of respondents experienced winter colony losses in excess of the average acceptable loss. Of beekeepers who reported losing at least one colony during 2011-12, the leading self-identified causes of mortality were weak condition in the fall and queen failure. Respondents who indicated poor wintering conditions, CCD, or pesticides as a leading cause of mortality suffered a higher average loss when compared to beekeepers who did not list these as potential causes.Keywords: Mortality, Colony losses, USA, Honey bee, Overwinter, 2011-1

Abstract

Fine Bruhat intersections for reductive groups have been studied by several au-thors in connection with Kazhdan-Lusztig theory, canonical bases and Lie The-ory. The purpose of this thesis is to study the analogous intersections for reductive monoids. We determine the conditions under which the following intersections BσB ∩ B − θB BσB ∩ B − θB − are nonempty. First we study these intersections for the monoid Mn(K). This work gives rise to two new orderings, ≤1 and ≤2, on the monoid of partial permutation matrices. More precisely, ≤1 and ≤2 are orderings that exist within a particular J-class for a reductive monoid M. The J-classes of Mn(K) consist of matrices of the same rank. Combinatorial descriptions of the orderings are given and their relation to the Bruhat-Chevalley order is discussed. These results are then generalized to an arbitrary reductive monoid. We show that ≤1 is a partial order on R, the Renner monoid, and that ≤2 is in general not a partial order on elements of R, but rather on equivalence classes of elements in R. We describe the equivalence classes for the matrices and conclude with theorems for the partial permutation matrices of rank r &lt; n

Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen <i>Nosema ceranae</i>

<div><p>Recent declines in honey bee populations and increasing demand for insect-pollinated crops raise concerns about pollinator shortages. Pesticide exposure and pathogens may interact to have strong negative effects on managed honey bee colonies. Such findings are of great concern given the large numbers and high levels of pesticides found in honey bee colonies. Thus it is crucial to determine how field-relevant combinations and loads of pesticides affect bee health. We collected pollen from bee hives in seven major crops to determine 1) what types of pesticides bees are exposed to when rented for pollination of various crops and 2) how field-relevant pesticide blends affect bees’ susceptibility to the gut parasite <i>Nosema ceranae</i>. Our samples represent pollen collected by foragers for use by the colony, and do not necessarily indicate foragers’ roles as pollinators. In blueberry, cranberry, cucumber, pumpkin and watermelon bees collected pollen almost exclusively from weeds and wildflowers during our sampling. Thus more attention must be paid to how honey bees are exposed to pesticides outside of the field in which they are placed. We detected 35 different pesticides in the sampled pollen, and found high fungicide loads. The insecticides esfenvalerate and phosmet were at a concentration higher than their median lethal dose in at least one pollen sample. While fungicides are typically seen as fairly safe for honey bees, we found an increased probability of <i>Nosema</i> infection in bees that consumed pollen with a higher fungicide load. Our results highlight a need for research on sub-lethal effects of fungicides and other chemicals that bees placed in an agricultural setting are exposed to.</p></div