Tailing-dependent trimming of RISC-associated miRNAs by th HuR protein

HuR is a ubiquitously expressed AU-rich element (ARE) binding protein. AREs are regulatory, typically destabilizing sequences found in the 3' UTR of many mRNAs in eukaryotes. However, binding of HuR acts to stabilize these messages. Another type of regulatory elements, miRNA-binding sequences are also found on the mRNA 3'UTR. miRNAs are short, (~22 nt) non-coding RNAs which guide the RISC complex to regulate the expression of proteins involved in the regulation of numerous biological processes. HuR and miRNA sites have been shown to often co-localize on target mRNAs, and several studies have shown that there is a cross-talk between HuR and miRNAs. For example, HuR can counteract miR-122-mediated repression of CAT-1 mRNA, a process which is accompanied by Ago displacement from mRNA. It was previously shown that HuR possesses an RNA 3'-terminal adenosyl transferase activity; however, the physiological substrates were not determined. In this thesis, we report that miRNAs are bound by HuR and act as substrates for the HuR-mediated transferase activity and that HuR can polyadenylate miRNAs. We further describe different type of evidence strongly suggesting that HuR also has a 3'-5' exonuclease activity acting on miRNAs, and describe and test in vitro a model of how HuR antagonizes the Ago-bound miRNA associated with mRNA. To access the miRNA-Ago complex associated in cis with the same target RNA, HuR recognizes the miRNA 3' end and adds to it a poly(A) tail, thus potentially weakening the Ago-miRNA interaction and creating a landing pad for the nuclease, which in turn leads to exonucleolytic degradation of miRNA turnover and displacement of Ago from RNA. We further show that HuR knockdown in HCT116 colon carcinoma cells has a differential effect on mature miRNAs as compared to miRNA isoforms containing 3'-terminal non-templated additions of A residues. The levels of these isoforms are reduced upon HuR depletion, suggesting that HuR promotes miRNA tailing and degradation also in vivo

Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli

The bacteriostatic aminoglycoside antibiotic kasugamycin inhibits protein synthesis at an initial step without affecting translation elongation. It binds to the mRNA track of the ribosome and prevents formation of the translation initiation complex on canonical mRNAs. In contrast, translation of leaderless mRNAs continues in the presence of the drug in vivo. Previously, we have shown that kasugamycin treatment in E. coli stimulates the formation of protein-depleted ribosomes that are selective for leaderless mRNAs. Here, we provide evidence that prolonged kasugamycin treatment leads to selective synthesis of specific proteins. Our studies indicate that leaderless and short-leadered mRNAs are generated by different molecular mechanisms including alternative transcription and RNA processing. Moreover, we provide evidence for ribosome heterogeneity in response to kasugamycin treatment by alteration of the modification status of the stalk proteins bL7/L12

Loss of tapasin correlates with diminished CD8(+) T-cell immunity and prognosis in colorectal cancer.

BACKGROUND Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival. METHODS A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging. RESULTS A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85). CONCLUSIONS Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC

mRNA stability alterations mediated by HuR are necessary to sustain the fast growth of glioma cells

Regulation of mRNA decay is an important mechanism controlling gene expression. Steady-state levels of mRNAs can be markedly altered by changes in the decay rate. The control of mRNA stability depends on sequences in the transcript itself and on RNA-binding proteins that dynamically bind to these sequences. A well characterized sequence motif, which has been shown to be present in many short-lived mRNAs, is the de-stabilizing AU-rich element (ARE) located at the 3’untranslated region (3’UTR) of mRNAs. HuR is an RNA-binding protein, which binds to AREs and in doing so, increases the half-life and steady state levels of the corresponding mRNA. Using Tissue Microarray technology, we found that HuR is over-expressed in human gliomas. We also found that there is a change in HuR localization from being solely in the nucleus to being expressed at high levels in the cytosol. Moreover, a positive correlation was found between total HuR levels, cytosolic localization and tumor grade. We also studied the decay rate of several HuR target mRNAs and found that these mRNAs have a slower rate of decay in glioma cell lines than in astrocytes. Finally, we have been able to decrease both the stability and steady state level of these transcripts in glioma cells using an RNA decoy. More importantly, the decoy transfected cells and cells exposed to a HuR inhibitor have reduced cell growth. In addition, pharmacological inhibition of HuR also resulted in glioma cell growth inhibition. In conclusion, our data suggest that post-transcriptional control abnormalities mediated by HuR are necessary to sustain the rapid growth of this devastating type of cancer

Loss of tapasin correlates with diminished CD8+ T-cell immunity and prognosis in colorectal cancer

BACKGROUND: Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival. METHODS: A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging. RESULTS: A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85). CONCLUSIONS: Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC

Phenotyping of tumor-associated macrophages in colorectal cancer: Impact on single cell invasion (tumor budding) and clinicopathological outcome.

Tumor-associated macrophages (TAM) play a controversial role in epithelial-mesenchymal transition (EMT) and prognosis of colorectal cancer (CRC). In particular, the microlocalization, polarization and prognostic impact of TAM in the immediate environment of invading CRC cells has not yet been established. To address this clinically relevant question, intraepithelial (iCD68) and stromal macrophages (sCD68), M1-macrophages (iNOS), M2-macrophages (CD163), cytokeratin-positive cancer cells (tumor buds) and expression of the anti-phagocytic marker CD47 were investigated in primary tumors of 205 well-characterized CRC patients. Cell-to-cell contacts between tumor buds and TAM were detected using high-resolution digital scans. The composition of the tumor microenvironment was analyzed with clinicopathological and molecular features. High CD68 counts predicted long term overall survival independent of microlocalization (iCD68 p=0.0016; sCD68 p=0.03), pT, pN, pM and post-operative therapy. CD68 infiltration correlated with significantly less tumor budding (iCD68 p=0.0066; sCD68 p=0.0091) and absence of lymph node metastasis (sCD68 p=0.0286). Cell-to-cell contact of sCD68 and invading cancer cells was frequent and ameliorated the detrimental prognostic effect of the tumor budding phenotype. Subgroup analysis identified long-term survival with CD47 loss and predominance of CD163(+) M2 macrophages (p = 0.0366). CD163(+) macrophages represented 40% of the total population, and positively correlated with total CD68 macrophage numbers (r[CD68/CD163] = 0.32; p = 0.0001). Strong CD163 infiltration predicted lower tumor grade (p = 0.0026) and less lymph node metastasis (p = 0.0056). This study provides direct morphological evidence of an interaction between TAM and infiltrating cancer cells. The prognostic impact of TAM is modulated by phenotype, microlocalization and the expression of anti-phagocytic markers in CRC

Digital analysis and epigenetic regulation of the signature of rejection in colorectal cancer.

The immune system plays a pivotal role in the development and progression of colorectal cancer (CRC). Tumor immune rejection has been previously linked to the activation of the interferon-stimulated genes (ISG) STAT1, IRF-5 and IRF-1. Specific immunoregulatory microRNAs (miRNAs) may impact the expression of these ISG in the tumor microenvironment. In this translational study, we develop a digital image analysis protocol to identify the ISG-gene expression signature and investigate miRNA expression in the immediate environment of invading cancer cells. Digital immunophenotyping was performed using next generation tissue microarrays from 241 well-characterized CRC patients and analyzed with clinicopathological and molecular information. Active ISG signaling in the tumor stroma differentiated an immune-activated (n = 178) and a quiescent (n = 43) phenotype. The activated phenotype was associated with high counts of intratumoral CD8+ cytotoxic T-lymphocytes (CTL; p = 0.007) and expression of the immune effector molecules granzyme B (p < 0.001) and perforin (p = 0.020). Immune-activated tumors also showed an elevated expression of the intercellular adhesion molecule-1 (ICAM-1, p = 0.006) which may facilitate CTL infiltration. Patients with immune-activated CRC had a considerably reduced risk of developing distant metastases (p = 0.001, OR = 0.034, 95%CI = 0.006-0.183). High expression of the immunoregulatory miR-34a and miR-93 corresponded to a 2-2.5-fold decrease of STAT1 (p = 0.006) and IRF-1 (p = 0.058), a feature more commonly seen in a quiescent microenvironment. Analysis of a combined ISG marker profile by digital pathology stratifies CRC patients into diametrically opposed immune phenotypes. Targeted inhibition of miRNAs within the tumor microenvironment may form a new strategy to stimulate the anti-tumoral immune response