TRAF6 E3 ligase activity contributes to L166P-mediated exclusion of TTRAP from nucleolar cavities.

<p>(A) Expression of TRAF6 DN partially rescues TTRAP nucleolar localization in L166P cells. SH-SY5Y cells stably expressing mutant DJ-1 were transfected with GFP-tagged TRAF6 (wt or deleted of the N-terminus, DN), or with empty vector control, as indicated. After 24 h from transfection, cells were treated for 16 h with MG132. TTRAP localization was followed by indirect immunofluorescence coupled with GFP autofluorescence. Bars, 10 µm. (B) Quantification of TTRAP nucleolar localization. Cells were as in A. At least 100 transfected cells with comparable GFP levels from two independent experiments were counted and scored for TTRAP in the nucleolus (*, p<0.05). (C) Knock-down endogenous TRAF6 expression partially rescues TTRAP nucleolar localization. SH-SY5Y cells stably transfected with mutant DJ-1 L166Pwere transfected with siRNA oligonucleotides targeting endogenous TRAF6 (siTRAF6) or with a scramble control siRNA (Scr). After 72 h from transfection, cells were treated for 16 h with MG132. Immunofluorescence was performed with anti-TTRAP (green), anti-TRAF6 (red), anti-NPM (blue). Bars, 15 µm. (D) Quantification of TTRAP nucleolar localization. Cells were as in C. Analysis was performed as in B on cells with reduced TRAF6 expression (*, p<0.05). (E) Total cell lysates were prepared from SH-SY5Y cells transfected as in C. Cells were treated with MG132 for 16 h or left untreated. Expression of endogenous TRAF6 and TTRAP was measured with specific antibodies. Protein loading was controlled by beta-actin. Molecular weight markers (MWM) are indicated for each gel (kDa). Images are representative of two independent experiments.</p

Mutant DJ-1 L166P inhibits TTRAP nucleolar localization after proteasome inhibition.

<p>(A) TTRAP nucleolar localization is inhibited in L166P expressing cells. SH-SY5Y cells stably transfected with empty vector (c), FLAG-DJ-1 wt (W) or L166P (L) were treated with 5 µM MG132 for 16 h. Triple immunofluorescence was performed with anti-NPM (blue), anti-TTRAP (green) and anti-FLAG (red) antibodies. Images are representatives of three independent experiments. Data have been confirmed on two independent clones for each cell line. Bars, 10 µm. (B) Quantification of TTRAP nucleolar localization. Cells as in A. At least 200 cells from two independent experiments were counted and scored for TTRAP in the nucleolus (*, p<0.05). (C) Localization of TTRAP is unaffected in cells depleted of endogenous DJ-1. SH-SY5Y cells stably expressing an inducible short-hairpin RNA targeting DJ-1 (siDJ-1, clones A and B) or a scramble shRNA control (scramble, clones a and b) were treated with doxycycline for 10 days to induce silencing of DJ-1 expression. Then cells were treated with 5 µM MG132 for 16 h. Immunofluorescence was performed with anti-TTRAP (green) and anti-NCL (red) antibodies. Nuclei were visualized with DAPI (blue). Images are representatives of two independent experiments. Bars, 10 µm. (D) Quantification of TTRAP nucleolar localization in siDJ-1 cells. Data were collected as in B. (NS, not-significant). (E) DJ-1 is efficiently depleted in siDJ-1 cells. Total protein lysates were prepared from cells as in C. Levels of endogenous DJ-1 were measured by western blot with anti-DJ-1 antibody. Beta-actin was detected as loading control. (F) TTRAP nucleolar localization is altered by mutant huntingtin. SH-SY5Y cells were transfected with huntingtin N-terminal fragment fused to GFP with WT (Gln²¹, Q21) or mutated (Gln¹⁵⁰, Q150) polyglutamine expansion. Endogenous TTRAP was visualized by indirect immunofluorescence (red). N-HTT was visible by GFP autofluorescence. Nuclei were visualized with DAPI (blue). Bars, 10 µm. (F) Quantification of TTRAP nucleolar localization in N-HTT expressing cells. Data were collected as in B on GFP-positive cells (*, p<0.05; NS, not-significant).</p

Processing of rRNA is altered in L166P overexpressing cells.

<p>(A) Schematic diagram showing pre-rRNA structure and processing steps. Positioning of A0, 1 and 4 cleavage sites on rRNA processing intermediates is shown. ETS, external transcribed spacer; ITS, internal transcribed spacer. (B) Analysis of steady-state levels of rRNA precursor and processing intermediates. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) and empty vector (c) were treated with Bars, 5 µM MG132 for 16 h, or left untreated. Total RNA was extracted and levels of pre-rRNA and processing intermediates were analyzed by qPCR with primers targeting A0 , 1 and 4 cleavage sites. Standard deviations are calculated on four replicas from two independent experiments. *, p<0.05. (C) Analysis of rRNA processing by metabolic labeling. L166P (L) and control (c) cells were treated as in A. After treatment, cells were pulse-labeled with ³²P-orthophosphate for 1 h and chased with cold medium for 3 h. RNA was extracted and equal quantities were separated on denaturing agarose gel. Nascent rRNA was visualized by autoradiography after gel drying (upper panel). rRNA processing intermediates and mature forms are indicated on the right. Loading was verified with ethidium bromide staining (lower panel).</p

TTRAP is present in cytoplasmic Lewy bodies and in the nucleolus in surviving dopaminergic neurons in PD <i>post-mortem</i> brains.

<p>(A) TTRAP localization in normal and PD dopaminergic neurons. Cryo-sections of post-mortem brain tissues were taken from Substantia Nigra of healthy individuals and PD patients, as indicated. Immunohistochemistry was performed with anti-TTRAP antibody (green). Nuclei were stained with DAPI (blue). Dopaminergic neurons were identified by neuromelanin (black) in transmitted light (TL). Overlays of fluorescence (merge) and fluorescence with TL (TL merge) are shown. Panels are representative images showing TTRAP nuclear, cytoplasmic diffused, cytoplasmic aggregated and nucleolar localization. The percentage of dopaminergic neurons with nuclear, cytoplasmic or nucleolar TTRAP staining is indicated on the right. Bars, 15 µM. (B) TTRAP-containing cytoplasmic aggregates are Lewy bodies. Immunohistochemistry of Substantia Nigra from PD brains was performed with anti-TTRAP (green) and anti-alpha-synuclein (red) antibodies. Images at low (1×) and high (10×) magnification are shown.</p

Mutant DJ-1 L166P does not inhibit nucleolar localization of PML and p53 upon proteotoxic stress.

<p>(A) Nucleolar localization of PML. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) or empty vector (c) were treated with 5 µM MG132 for 16 h. Endogenous TTRAP (green), PML (red) and NPM (blue) localization was analyzed by triple immunofluorescence. Images are representatives of two separate experiments performed on two independent clones for each cell line. (B) Quantification of PML nucleolar localization. Cells were treated as in A. 200 cells from two independent experiments were counted and scored for PML in the nucleolus (*, p<0.05). (C) Mutant DJ-1 L166P does not affect levels of endogenous PML. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) or empty vector (c) were treated with 5 µM MG132 for 16 h or left untreated, as indicated. The expression of endogenous PML and overexpressed FLAG-DJ-1 were measured with anti-PML and anti-FLAG antibodies, respectively. Beta-actin was detected as loading control. (D) Quantification of PML levels. Densitometric analysis of protein bands was performed on two independent experiments. Relative expression of endogenous PML was normalized to beta-actin. Data are expressed as the percentage of untreated condition in empty cells (c). (E) Nucleolar localization of p53. Cells were exactly as in A. Localization of endogenous p53 (green), NPM (blue) and FLAG-DJ-1 (red) were analyzed by triple immunofluorescence. Bars, 10 µm. (F) Quantification of p53 nucleolar localization. Analysis was performed as in B (NS, non significant).</p

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation

<div><p>SINEUPs are antisense long noncoding RNAs, in which an embedded <u>SINE</u> B2 element <u>UP</u>-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.</p></div

Establishment of semi-automated SINEUP high-throughput detection system by Celigo S.

<p>(A) Flow chart of the procedures. (B) Live imaging pictures of EGFP from Celigo S auto-detecting camera. HEK 293T/17 cells were transfected with pEGFP in combination with SINEUP-GFP (Δ5’-32 nt) (right) or empty control plasmid (left). Images are representative of n = 3 independent experiments. (C) Image quantification by Celigo S software. EGFP integrated intensity from cells transfected with control and SINEUP-GFP (Δ5’-32 nt) expressing plasmid. Cell numbers are counted by Hoechst 33342 to normalize integrated intensity. (D) Total proteins were extracted from cells transfected as in B. Proteins were extracted after Celigo S measurement. Western blot analysis was performed with anti-GFP antibody, as indicated. Beta-actin was used as loading control. (E) Quantification of EGFP band intensity normalized to beta-actin in control and SINEUP-GFP (Δ5’-32 nt) expressing cells. (F) GFP mRNA, SINEUP RNA and NeoR mRNA were measured by qRT-PCR. n = 3, ***p<0.0005, two-tailed Student’s t-test; Error bars are STDEV. FOV: field of view.</p

Optimization of SINEUP-GFP binding domain design.

<p>(A) TSS analysis of EGFP and SINEUP-GFP by CAGE. CAGE analysis was performed on RNA extracted from HEK293T/17 cells transfected with pEGFP and pcDNA3.1-SINEUP-GFP. Sequencing reads were mapped on reference EGFP (left) and SINEUP-GFP (right) transcripts. Green graph indicates TSS of EGFP and red graph indicates TSS of SINEUP-GFP. The exact position of EGFP and SINEUP-GFP TSS is numbered relative to translation initiation and SINEUP insertion, respectively. (B) Shorter variants of SINEUP-GFP BD show improved activity. Scheme of the anatomy of sense EGFP (derived from pEGFP-C2 plasmid) and SINEUP-GFP transcripts is shown on top. Details of BD sequences used for the screening are indicated. Underlined pEGFP-C2 sequence indicates AUG-Kozak sequence. HEK 293T/17 cells were transfected with pEGFP in combination with SINEUP-GFP or empty control plasmid. EGFP protein quantities were analyzed by Western Blot. Respective EGFP expressions are normalized by ACTINB (endogenous control) fold changes are normalized by control (empty vector). n = 9, ***p < 0.0005, two-tailed Student’s t-test; Error bars are STDEV. Δ: deletion.</p

SINEUP-GFP does not alter PKR pathways and 4EBP1 phosphorylation in cultured cells.

<p>Effects of SINEUP-GFP on the expression of proteins involved in (A) PKR pathway (B) 4EBP1 phosphorylation.</p

Collision detection on transmission lines with optical interferometer

V diplomski nalogi skušamo ugotoviti, v kolikšni meri je možno zaznavati in klasificirati trke na jeklenicah daljnovodov z optičnim interferometrom. Na začetku predstavimo osnovne pojme interferometrije in opišemo uporabljen optični interferometer. V jedru diplomske naloge natančneje opišemo eksperimentalni protokol in obdelavo signalov. Nadaljujemo z implementacijo algoritmov za segmentacijo in klasifikacijo zajetih signalov ter predstavimo dobljene rezultate. Segmentacijo izvedemo v domeni števila prehodov signala skozi ničlo, za klasifikacijo pa uporabimo večplastno nevronsko mrežo z algoritmom vzvratnega učenja. Rezultati študije nakazujejo, da sta implementirani segmentacija in klasifikacija uspešni v 77 % izvedenih trkov različnih predmetov.We analyse feasibility of collision detection on transmission lines with optical interferometer. We first provide a brief introduction into interferometry, along with a description of the optical interferometer used for measurements in this study. Afterwards, we describe the conducted experimental protocol and signal processing methodology. The focus is on implementation of algorithms for signal segmentation and collision classification. We used zero-crossing algorithm to transform signals into segmentation domain. Classification of collisions is done with a multilayer neural network trained by the backpropagation algorithm. The results demonstrate an average success rate of 77% for segmentation and classification of collision with five different objects