Functional Analysis of Arabidopsis Postprenylation CaaX Processing Enzymes and Their Function in Subcellular Protein Targeting1[C][W][OA]

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase β-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins

In Vivo Direct Molecular Imaging of Early Tumorigenesis and Malignant Progression Induced by Transgenic Expression of GFP-Met

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression

Site-directed mutagenesis of miR-192 binding sites on 3' UTR of Dicer1.

<div><p>Three mutations were introduced into the three potential binding sites of miR-192 on 3' UTR of pI DICER1 using Multi Site-Drected Mutagenesis Kit .</p> <p>The nucleotides that were mutated are circled and were changed to the nucleotides in bold. </p></div

3′UTR of Dicer 1 is directly targeted by miR-192.

<div><p>The dual luciferase assay detected that Dicer1 is modulated by miR-192 in NB cell lines. The relative luciferase unit (RLU) was measured in SHEP (A) or NUB (B) cells.</p> <p>A. The dual-luciferase assay resulted in a significant reduction of RLU of WT Dicer1 (3' UTR of Dicer1 wild type) following transfection with miR-192-vec (*p=0.049).</p> <p>B. Following transfection with miR-192 mimic, WT Dicer1 RLU was significantly decreased (*p=0.0003). Following mutagenesis, cells were transfected with Dicer1 plasmid in which mutations were introduced in all three BSs of Dicer1 (MUT ALL); active BS1 (mutated at BS2+BS3)(* p=0.004); active BS2 (mutated at BS1+BS3) (* p=0.04) and active BS3 (mutated at BS1+BS2). </p> <p>Values are expressed as the mean ± SE of combined results from three independent experiments. </p></div

Kaplan Meier analysis by Dicer1 expression.

<p>Kaplan Meier analysis for PFS by Dicer1 expression (n=47): high and low expression levels of Dicer1 were determined as above (n=24) or below (n=23) the median expression level.</p

Kaplan Meier analysis by miR-192 expression.

<p>Kaplan Meier analysis for PFS by miR-192 expression: high and low miR-192 expression levels were determined as above or below the median expression level and were analyzed in (A) a whole cohort (n=43) and in (B) a cohort following exclusion of MYCNA (n= 36) .</p