Structure-function analysis of human Integrator subunit-4

Structure-function analysis of human Integrator subunit 4 Anupama Sataluri Advisor: Eric. J. Wagner, Ph.D. Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent discovery of the machinery that mediates this process. In 2005, a complex of 14 subunits (the Integrator complex) associated with RNA Polymerase-II was discovered. The 14subunits were annotated Integrator 1-14 based on their size. The subunits of this complex together were found to facilitate 3′ end processing of snRNA. Identification of the Integrator complex propelled research in the direction of understanding the events of snRNA 3’end processing. Recent studies from our lab confirmed that Integrator subunit (IntS) 9 and 11 together perform the endonucleolytic cleavage of the nascent snRNA 3′ end to generate mature snRNA. However, the role of other members of the Integrator complex remains elusive. Current research in our lab is focused on deciphering the role of each subunit within the Integrator complex This work specifically focuses on elucidating the role of human Integrator subunit 4 (IntS4) and understanding how it facilitates the overall function of the complex. IntS4 has structural similarity with a protein called “Symplekin”, which is part of the mRNA 3’end processing machinery. Symplekin has been thoroughly researched in recent years and structure-function correlation studies in the context of mRNA 3’end processing have reported a scaffold function for Symplekin due to the presence of HEAT repeat motifs in its N-terminus. Based upon the structural similarity between IntS4 and Symplekin, we hypothesized that Integrator subunit 4 may be behaving as a Symplekin-like scaffold molecule that facilitates the interaction between other members of the Integrator Complex. To answer this question, the two important goals of this study were to: 1) identify the region of IntS4, which is important for snRNA 3′ end processing and 2) determine binding partners of IntS4 which promote its function as a scaffold. IntS4 structurally consists of a highly conserved N-terminus with 8 HEAT repeats, followed by a nonconserved C- terminus. A series of siRNA resistant N and C-terminus deletion constructs as well as specific point mutants within its N-terminal HEAT repeats were generated for human IntS4 and, utilizing a snRNA transcriptional readthrough GFP-reporter assay, we tested their ability to rescue misprocessing. This assay revealed a possible scaffold like property of IntS4. To probe IntS4 for interaction partners, we performed co-immunoprecipitation on nuclear extracts of IntS4 expressing stable cell lines and identified IntS3 and IntS5 among other Integrator subunits to be binding partners which facilitate the scaffold like function of hIntS4. These findings have established a critical role for IntS4 in snRNA 3′ end processing, identified that both its N and C termini are essential for its function, and mapped putative interaction domains with other Integrator subunits.

Analysis of VirB4, a membrane-associated ATPase subunit essential for T -complex transport in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a plant pathogen with the unique ability to export oncogenic DNA-protein complexes (T-complexes) to susceptible plant cells and cause crown gall tumors. Delivery of the T-complexes across the bacterial membranes requires eleven VirB proteins and VirD4, which are postulated to form a transmembrane transporter. This thesis examines the subcellular localization and oligomeric structure of the 87-kDa VirB4 protein, which is one of three essential ATPases proposed to energize T-complex transport and/or assembly. Results of subcellular localization studies showed that VirB4 is tightly associated with the cytoplasmic membrane, suggesting that it is a membrane-spanning protein. The membrane topology of VirB4 was determined by using a nested deletion strategy to generate random fusions between virB4 and the periplasmically-active alkaline phosphatase, $\sp\prime phoA$. Analysis of PhoA and complementary $\beta$-galactosidase reporter fusions identified two putative periplasmically-exposed regions in VirB4. A periplasmic exposure of one of these regions was further confirmed by protease susceptibility assays using A. tumefaciens spheroplasts. To gain insight into the structure of the transporter, the topological configurations of other VirB proteins were also examined. Results from hydropathy analyses, subcellular localization, protease susceptibility, and PhoA reporter fusion studies support a model that all of the VirB proteins localize at one or both of the bacterial membranes. Immunoprecipitation and Co$\sp{2+}$ affinity chromatography studies demonstrated that native VirB4 (87-kDa) and a functional N-terminally tagged HIS-VirB4 derivative (89-kDa) interact and that the interaction is independent of other VirB proteins. A $\lambda$ cI repressor fusion assay supplied further evidence for VirB4 dimer formation. A VirB4 dimerization domain was localized to the N-terminal third of the protein, as judged by: (i) transdominance of an allele that codes for this region of VirB4; (ii) co-retention of a His-tagged N-terminal truncation derivative and native VirB4 on Co$\sp{2+}$ affinity columns; and (iii) dimer formation of the N-terminal third of VirB4 fused to the cI repressor protein. Taken together, these findings are consistent with a model that VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains and that VirB4 assembles as homodimers via an N-terminal dimerization domain. Dimer formation is postulated to be essential for stabilization of VirB4 monomers during T-complex transporter assembly. ^

Geochemical composition of gabbroic rocks from ODP Hole 179-1105A, southwest Indian Ridge

Oxide-free olivine gabbro and gabbro, and oxide olivine gabbro and gabbro make up the bulk of the gabbroic suite recovered from Ocean Drilling Program (ODP) Leg 179 Hole 1105A, which lies 1.2 km away from Hole 735B on the eastern transverse ridge of the Atlantis II Fracture Zone, Southwest Indian Ridge. The rocks recovered during Leg 179 show striking similarities to rocks recovered from the uppermost 500 m of Hole 735B during ODP Leg 118. The rocks of the Atlantis platform were likely unroofed as part of the footwall block of a large detachment fault on the inside corner of the intersection of the Southwest Indian Ridge and the Atlantis II Transform at ~11.5 Ma. We analyzed the lithologic, geochemical, and structural stratigraphy of the section. Downhole lithologic variation allowed division of the core into 141 lithologic intervals and 4 main units subdivided on the basis of predominance of oxide gabbroic vs. oxide-free gabbroic rocks. Detailed analyses of whole-rock chemistry, mineral chemistry, microstructure, and modes of 147 samples are presented and clearly show that the gabbroic rocks are of cumulate origin. These studies also indicate that geochemistry results correlate well with downhole magnetic susceptibility and Formation MicroScanner (FMS) resistivity measurements and images. FMS images show rocks with a well-layered structure and significant numbers of mappable layer contacts or compositional contrasts. Downhole cryptic mineral and whole-rock chemical variations depict both "normal" and inverse fine-scale variations on a scale of 10 m to <2 m with significant compositional variation over a short distance within the 143-m section sampled. A Mg# shift in whole-rock or Fo contents of olivine of as much as 20-30 units over a few meters of section is not atypical of the extreme variation in downhole plots. The products of the earliest stages of basaltic differentiation are not represented by any cumulates, as the maximum Fo content was Fo78. Similarly, the extent of fractionation represented by the gabbroic rocks and scarce granophyres in the section is much greater than that represented in the Atlantis II basalts. The abundance of oxide gabbros is similar to that in Hole 735B, Unit IV, which is tentatively correlated as a similar unit or facies with the oxide gabbroic units of Hole 1105A. Oxide phases are generally present in the most fractionated gabbroic rocks and lacking in more primitive gabbroic rocks, and there is a definite progression of oxide abundance as, for example, the Mg# of clinopyroxene falls below 73-75. Coprecipitation of oxide at such early Mg#s cannot be modeled by perfect fractional crystallization. In situ boundary layer fractionation may offer a more plausible explanation for the complex juxtaposition of oxide- and nonoxide-bearing more primitive gabbroic rocks. The geochemical signal may, in part, be disrupted by the presence of mylonitic shear zones, which strike east-west and dip both to the south and north, but predominantly to the south away from the northern rift valley where they formed. Downhole deformation textures indicate increasing average strain and crystal-plastic deformation in units that contain oxides. Oxide-rich zones may represent zones of rheologic weakness in the cumulate section along which mylonitic and foliated gabbroic shear zones nucleate in the solid state at high temperature, or the oxide may be a symptom of former melt-rich zones and hypersolidus flow, as predicted during study of Hole 735B.

Community structure and biodiversity in marine benthic communities during experiments, 2011

A mesocosm experiment was conducted to quantify the effects of reduced pH and elevated temperature on an intact marine invertebrate community. Standardised faunal communities, collected from the extreme low intertidal zone using artificial substrate units, were exposed to one of eight nominal treatments (four pH levels: 8.0, 7.7, 7.3 and 6.7, crossed with two temperature levels: 12 and 16°C). After 60 days exposure communities showed significant changes in structure and lower diversity in response to reduced pH. The response to temperature was more complex. At higher pH levels (8.0 and 7.7) elevated temperature treatments contained higher species abundances and diversity than the lower temperature treatments. In contrast, at lower pH levels (7.3 and 6.7), elevated temperature treatments had lower species abundances and diversity than lower temperature treatments. The species losses responsible for these changes in community structure and diversity were not randomly distributed across the different phyla examined. Molluscs showed the greatest reduction in abundance and diversity in response to low pH and elevated temperature, whilst annelid abundance and diversity was mostly unaffected by low pH and was higher at the elevated temperature. The arthropod response was between these two extremes with moderately reduced abundance and diversity at low pH and elevated temperature. Nematode abundance increased in response to low pH and elevated temperature, probably due to the reduction of ecological constraints, such as predation and competition, caused by a decrease in macrofaunal abundance. This community-based mesocosm study supports previous suggestions, based on observations of direct physiological impacts, that ocean acidification induced changes in marine biodiversity will be driven by differential vulnerability within and between different taxonomical groups. This study also illustrates the importance of considering indirect effects that occur within multispecies assemblages when attempting to predict the consequences of ocean acidification and global warming on marine communities.

Transverse drainages of the Susquehanna River basin, Pennsylvania, USA

While large-scale transverse drainages (TDs) such as those of the Susquehanna River above Harrisburg, PA, have been recognized since the 19th century, there have been no systematic surveys done of TDs since that of Ver Steeg's in 1930. Here, the results are presented of a topographic and statistical analysis of TDs in the Susquehanna River basin using Google Earth and associated overlays. 653 TDs were identified in the study area, 95% of which contain streams with discharges of less than 10 m3/s. TD depths ranged from a 23 m deep water gap near Blain, PA, to the 539 m deep gorge of the Juniata River through Jacks Mountain. Although TD depth tended to increase with stream size, many small streams were located in deep gaps, and eight streams with discharges of 10 m3/s or less were found in gorges whose depths matched or exceeded the deepest TD of the Susquehanna, the largest stream in the basin. Streams of less than 10 m3/s made up the majority of TDs regardless of the rock type capping the breached structure. Overall, TDs through sandstone-capped ridges were deeper than those topped by shales, and TDs in both sandstones and shales displayed a lognormal distribution of depths, which may be indicative of a preferred value. Stream flow direction was primarily perpendicular to local structural strike, with 47% of streams flowing NW and 53% flowing SE. 19% of the TDs were found to be in alignment with at least one other TD, with aligned segment lengths ranging from .5 to 14.8 km. The majority of TDs were in rocks of Paleozoic age. The techniques described here allow the frequency and distribution of TDs to be quantified so that they can be integrated into models of basin evolution.

Competition may explain the fine-scale spatial patterns and genetic structure of two co-occurring plant congeners

1. The spatial distribution of individual plants within a population and the population’s genetic structure are determined by several factors, like dispersal, reproduction mode or biotic interactions. The role of interspecific interactions in shaping the spatial genetic structure of plant populations remains largely unknown. 2. Species with a common evolutionary history are known to interact more closely with each other than unrelated species due to the greater number of traits they share. We hypothesize that plant interactions may shape the fine genetic structure of closely related congeners. 3. We used spatial statistics (georeferenced design) and molecular techniques (ISSR markers) to understand how two closely related congeners, Thymus vulgaris (widespread species) and T. loscosii (narrow endemic) interact at the local scale. Specific cover, number of individuals of both study species and several community attributes were measured in a 10 × 10 m plot. 4. Both species showed similar levels of genetic variation, but differed in their spatial genetic structure. Thymus vulgaris showed spatial aggregation but no spatial genetic structure, while T. loscosii showed spatial genetic structure (positive genetic autocorrelation) at short distances. The spatial pattern of T. vulgaris’ cover showed significant dissociation with that of T. loscosii. The same was true between the spatial patterns of the cover of T. vulgaris and the abundance of T. loscosii and between the abundance of each species. Most importantly, we found a correlation between the genetic structure of T. loscosii and the abundance of T. vulgaris: T. loscosii plants were genetically more similar when they were surrounded by a similar number of T. vulgaris plants. 5. Synthesis. Our results reveal spatially complex genetic structures of both congeners at small spatial scales. The negative association among the spatial patterns of the two species and the genetic structure found for T. loscosii in relation to the abundance of T. vulgaris indicate that competition between the two species may account for the presence of adapted ecotypes of T. loscosii to the abundance of a competing congeneric species. This suggests that the presence and abundance of close congeners can influence the genetic spatial structure of plant species at fine scales.

Ground maritime structure interaction. Recent failures in inner gravity constructions on the Spanish Coastline

A maritime construction is usually a slender line in the ocean.It is usual to see just its narrow surface strip and not analyse the large amount of submerged material the latter is supporting.Without doubt,it is the ground to which a notable load is transmitted in an environment subjected to periodic,alternating stresses,dynamic forces which the sea's media constitute. Both an outer and inner maritime construction works in a complex fashion.A granular solid(breakwater)breathes with the incident wave flow,dissipating part of the wave energy between its gaps.The backflow tries to extract the different items from the solid block,setting a balance between effective and neutral tensions that follow Terzaghui's principle. On some occasions,fluidification of the armour layer has caused the breakwater to collapse(Sines,Portugal,February 1978).On others,siphoning or liquefaction of sand supporting monoliths(vertical breakwaters)lead them to destruction or collapse(New Barcelona Harbour Mouth,Spain,November 2001). This is why the ground-force-structure interaction is a complicated analysis with joint design tools still in an incipient state. The purpose of this article is to describe two singular failures in inner maritime constructions in Spain deriving from ground problems(Malaga,July 2004and Barcelona,January 2007).They occurred recently and the causes are the subject of reflection and analysis.

Rapid sizing tool for a low cost university microsatellite structure subsystem

In recent decays university class small satellites are creating many opportunities for space research and professional trainings while at the same time responding to constrained budgets. In this work the main focus is on developing a simple and rapid structural sizing tool considering the main objectives of a low cost university class microsatellite project. In satellite projects, structure subsystem is one of the influential subsystems as a driver of the cost and acceptance of the final design. At the first steps of such projects there is no confirmed data regarding the launch vehicle or even in some cases there is no data for the satellite payload. Due to these facts, developing simple sizing tools at conceptual design phase for obtaining an over view of the effect of different variables is useful before entering complex calculations in detailed design phases. In this study, after developing a simple analytical model of satellite structure subsystem, a design space is evaluated with practical boundaries considering mass and dimensions constraints of such projects. The results are useful to give initial insight to establish the system level structural sizing

Community Structure in Soil Porous System

Diffusion controls the gaseous transport process in soils when advective transport is almost null. Knowledge of the soil structure and pore connectivity are critical issues to understand and modelling soil aeration, sequestration or emission of greenhouse gasses, volatilization of volatile organic chemicals among other phenomena. In the last decades these issues increased our attention as scientist have realize that soil is one of the most complex materials on the earth, within which many biological, physical and chemical processes that support life and affect climate change take place. A quantitative and explicit characterization of soil structure is difficult because of the complexity of the pore space. This is the main reason why most theoretical approaches to soil porosity are idealizations to simplify this system. In this work, we proposed a more realistic attempt to capture the complexity of the system developing a model that considers the size and location of pores in order to relate them into a network. In the model we interpret porous soils as heterogeneous networks where pores are represented by nodes, characterized by their size and spatial location, and the links representing flows between them. In this work we perform an analysis of the community structure of porous media of soils represented as networks. For different real soils samples, modelled as heterogeneous complex networks, spatial communities of pores have been detected depending on the values of the parameters of the porous soil model used. These types of models are named as Heterogeneous Preferential Attachment (HPA). Developing an exhaustive analysis of the model, analytical solutions are obtained for the degree densities and degree distribution of the pore networks generated by the model in the thermodynamic limit and shown that the networks exhibit similar properties to those observed in other complex networks. With the aim to study in more detail topological properties of these networks, the presence of soil pore community structures is studied. The detection of communities of pores, as groups densely connected with only sparser connections between groups, could contribute to understand the mechanisms of the diffusion phenomena in soils.

Pyramidal cells in prefrontal cortex: comparative observations reveal unparalleled specializations in neuronal structure among primate species

The most ubiquitous neuron in the cerebral cortex, the pyramidal cell, is characterized by markedly different dendritic structure among different cortical areas. The complex pyramidal cell phenotype in granular prefrontal cortex (gPFC) of higher primates endows specific biophysical properties and patterns of connectivity, which differ from those in other cortical regions. However, within the gPFC, data have been sampled from only a select few cortical areas. The gPFC of species such as human and macaque monkey includes more than 10 cortical areas. It remains unknown as to what degree pyramidal cell structure may vary among these cortical areas. Here we undertook a survey of pyramidal cells in the dorsolateral, medial, and orbital gPFC of cercopithecid primates. We found marked heterogeneity in pyramidal cell structure within and between these regions. Moreover, trends for gradients in neuronal complexity varied among species. As the structure of neurons determines their computational abilities, memory storage capacity and connectivity, we propose that these specializations in the pyramidal cell phenotype are an important determinant of species-specific executive cortical functions in primates.

Automated attribute inference in complex service workflows based on sharing analysis

The properties of data and activities in business processes can be used to greatly facilítate several relevant tasks performed at design- and run-time, such as fragmentation, compliance checking, or top-down design. Business processes are often described using workflows. We present an approach for mechanically inferring business domain-specific attributes of workflow components (including data Ítems, activities, and elements of sub-workflows), taking as starting point known attributes of workflow inputs and the structure of the workflow. We achieve this by modeling these components as concepts and applying sharing analysis to a Horn clause-based representation of the workflow. The analysis is applicable to workflows featuring complex control and data dependencies, embedded control constructs, such as loops and branches, and embedded component services.

Diversity and Genetic structure of the Spanish collection of durum wheat (Triticum turgidum L) landraces

The objectives of this study were to assess diversity and genetic structure of a collection of Spanish durum wheat (Triticum turgidum L) landraces, using SSRs, DArTs and gliadin-markers, and to correlate the distribution of diversity with geographic and climatic features, as well as agro-morphological traits. A high level of diversity was detected in the genotypes analyzed, which were separated into nine populations with a moderate to great genetic divergence among them. The three subspecies taxa, dicoccon, turgidum and durum, present in the collection, largely determined the clustering of the populations. Genotype variation was lower in dicoccon (one major population) and turgidum (two major populations) than in durum (five major populations). Genetic differentiation by the agro-ecological zone of origin was greater in dicoccon and turgidum than in durum. DArT markers revealed two geographic substructures, east-west for dicoccon and northeast-southwest for turgidum. The ssp. durum had a more complex structure, consisting of seven populations with high intra-population variation. DArT markers allowed the detection of subgroups within some populations, with agro-morphological and gliadin differences, and distinct agro-ecological zones of origin. Two different phylogenetic groups were detected; revealing that some durum populations were more related to ssp. turgidum from northern Spain, while others seem to be more related to durum wheats from North Africa

Hysteresis phenomena in transverse galloping of triangular cross-section bodies

Transverse galloping is a type of aeroelastic instability characterised by large amplitude, low frequency oscillation of a structure in the direction normal to the mean wind direction. It normally appears in bodies with small stiffness and structural damping, provided the incident flow velocity is high enough. In the simplest approach transverse galloping can be considered as a one-degree-of-freedom oscillator subjected to aerodynamic forces, which in turn can be described by using a quasi-steady description. In this frame it has been demonstrated that hysteresis phenomena in transverse galloping is related to the existence of inflection points in the curve giving the dependence with the angle of attack of the aerodynamic coefficient normal to the incident flow. Aiming at experimentally checking such a relationship between these inflection points and hysteresis, wind tunnel experiments have been conducted. Experiments have been restricted to isosceles triangular cross-section bodies, whose galloping behaviour is well documented. Experimental results show that, according to theoretical predictions, hysteresis takes place at the angles of attack where there are inflection points in the lift coefficient curve, provided that the body is prone to gallop at these angles of attack.