The Gallery 2010

This is a digital copy of the print book produced by the Gallery 2010 team. Contents: p. 1 Life Around Westby, p. 9 Graphic Design, p. 19 Illustration, p. 27 Jewelry, p. 37 Painting, p. 45 Photography, p. 57 Printmaking, p. 67 Three-Dimensional, p. 77 Rowan Gallery Openings. A Compact Disc (CD) included with the print book is not included here. Files for individual sections may be viewed on the detailed metadata page by clicking on the book title.https://rdw.rowan.edu/the_gallery/1006/thumbnail.jp

The Gallery 2009

This is a digital copy of the print content produced by the Gallery 2009 team. It consists of four books. Book 1 contains Introduction, Reel Art, and Gallery Members. Book 2 contains 3D Artwork: Ceramics, Metal Work, and Sculpture. Book 3 contains Graphic Design, Illustration, and Painting. Book 4 contains Photography and Printmaking. The final product also included a Viewfinder with accompanying reels, and time-based media such as animation. This content is not included. Files for individual books may be viewed on the detailed metadata page by clicking on the title.https://rdw.rowan.edu/the_gallery/1011/thumbnail.jp

Breaking Up the C Complex Spliceosome Shows Stable Association of Proteins with the Lariat Intron Intermediate

Spliceosome assembly requires several structural rearrangements to position the components of the catalytic core. Many of these rearrangements involve successive strengthening and weakening of different RNA∶RNA and RNA∶proteins interactions within the complex. To gain insight into the organization of the catalytic core of the spliceosome arrested between the two steps of splicing chemistry (C complex), we investigated the effects of exposing C complex to low concentrations of urea. We find that in the presence of 3M urea C complex separates into at least three sub-complexes. One sub-complex contains the 5′exon, another contains the intron-lariat intermediate, and U2/U5/U6 snRNAs likely comprise a third sub-complex. We purified the intron-lariat intermediate sub-complex and identified several proteins, including U2 snRNP and PRP19 complex (NTC) components. The data from our study indicate that U2 snRNP proteins in C complex are more stably associated with the lariat-intron intermediate than the U2 snRNA. The results also suggest a set of candidate proteins that hold the lariat-intron intermediate together in C complex. This information is critical for further interpreting the complex architecture of the mammalian spliceosome

The Human Nucleolar Protein FTSJ3 Associates with NIP7 and Functions in Pre-rRNA Processing

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A′ to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells

A two-step probing method to compare lysine accessibility across macromolecular complex conformations.

Structural models of large and dynamic molecular complexes are appearing in increasing numbers, in large part because of recent technical advances in cryo-electron microscopy. However, the inherent complexity of such biological assemblies comprising dozens of moving parts often limits the resolution of structural models and leaves the puzzle as to how each functional configuration transitions to the next. Orthogonal biochemical information is crucial to understanding the molecular interactions that drive those rearrangements. We present a two-step method for chemical probing detected by tandem mass-spectrometry to globally assess the reactivity of lysine residues within purified macromolecular complexes. Because lysine side chains often balance the negative charge of RNA in ribonucleoprotein complexes, the method is especially useful for detecting changes in protein-RNA interactions. By probing the E. coli 30S ribosome subunit, we established that the reactivity pattern of lysine residues quantitatively reflects structure models derived from X-ray crystallography. We also used the strategy to assess differences in three conformations of purified human spliceosomes in the context of recent cryo-electron microscopy models. Our results demonstrate that the probing method yields powerful biochemical information that helps contextualize architectural rearrangements of intermediate resolution structures of macromolecular complexes, often solved in multiple conformations

NIP7 associates with FTSJ3 in vivo in an RNA-dependent manner.

<p>(<b>A</b>) Analysis by western blot of FLAG-FTSJ3 induction in stably transfected HEK293 Flp-In T-Rex cells with increasing concentrations of tetracycline. Both endogenous and recombinant FLAG-FTSJ3 were detected by using antibody specific to FTSJ3. FLAG-FTSJ3 expression was confirmed by using antibody against the FLAG peptide. eIF4AI/II was used as a gel loading control. NIP7 levels are not affected by over-expression of FLAG-FTSJ3. (<b>B</b>) FTSJ3 relative levels as determined by band quantification using ImageJ software and normalized to eIF4AI/II. (<b>C</b>) Coimmunoprecipitation of NIP7 with FLAG-FTSJ3. FLAG-tagged FTSJ3 and 3PGDH were immunoprecipitated from extracts of stably transfected HEK293 Flp-In T-Rex cell lines using anti-FLAG (IP) followed by immunoblotting (IB) with anti-NIP7, anti-FTSJ3 and anti-FLAG. Parallel controls were performed using cells without induction and with cells stably transfected with FLAG-3PGDH. NIP7 is detected in the immunoprecitipation with FLAG-FTSJ3 (panel IP:anti-FLAG, lane + tetracycline) (<b>D</b>) FLAG-FTSJ3 was immunoprecipitated with anti-FLAG from cell extracts of stably transfected HEK293 Flp-In T-Rex treated with increasing concentrations of the RNases A and T1 and immunoblotted with antibodies for NIP7 and FTSJ3. RNase treatment abolishes NIP7 coimmunoprecipitation with FLAG-FTSJ3. (<b>E</b>) Analysis of FTSJ3 sedimentation. Cell extracts of HEK293 Flp-In T-Rex cells were fractionated by sucrose density gradient centrifugation and the fractions analyzed by immunoblotting using antibodies for the indicated proteins. FTSJ3 sedimentation overlaps with NIP7 in the range of the 40S–60S ribosome subunits. GAPDH was used as reference for soluble proteins and RPL26 as reference for 60S, 80S and polysome sedimentation.</p

Analysis of pre-rRNAs intermediates in HeLa and HEK293 Flp-In T-Rex cells transiently transfected with a siRNA targeting the FTSJ3 mRNA.

<p>(<b>A</b>) Western blot analysis to determine FTSJ3 levels 72 h following siRNA transfection. (<b>B</b>) Northern blot using probe P3 complementary to ITS1 upstream site 2b. (<b>C</b>) Graphs representing the ratio between the 34S and 47S pre-rRNA of the Northern blots shown in B. Bands were quantified by using ImageQuant software. Control cells were transfected with siRNA targeting the GFP mRNA.</p

Analysis of cell proliferation over a 5-day period of induction with doxycycline (Dox).

<p>(<b>A</b>) Proliferation rate of cells expressing the shRNA targeting FTSJ3. (<b>B</b>) Proliferation rate of cells expressing the scrambled control SC RNA. (The graphs represent one of four independent assays performed using four technical replicates for each cell treatment). Induction of the shRNA targeting FTSJ3 leads to a reduction in cell proliferation. (<b>C</b>) Cell cycle analysis by fluorescence-activated cell sorting (FACS). FACS analyses were performed with HEK293 Flp-In T-Rex cells stably transfected with the pFRT-U6tetO plasmid expressing either a shRNA targeting FTSJ3 (shRNA) or a scrambled control (SC) and induced (+) or not (−) with 5 µg/ml of doxycycline for 5 days. (<b>D</b>) Relative growth rate differences between cells expressing the scrambled control scRNA (sc+/sc−) and cell expressing the shRNA targeting the FTSJ3 mRNA (sh+/sh−) induced (+) or not (−) with doxycycline for the 120 h time point. (<b>E</b>) Immunoblot analysis showing the levels of the FTSJ3 and p53 proteins in cells expressing either the shRNA targeting the FTSJ3 mRNA or the scrambled control induced (+) or not (−) with doxycycline. GAPDH was used as a gel loading control. The levels of FTSJ3 are reduced as expected and the levels of p53 are not affected.</p