Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq

Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria

Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis

The Notch signaling pathway is a critical player in embryogenesis but also plays various roles in tumorigenesis, with both tumor suppressor and oncogenic activities. Mutations, deletions, amplifications, or over-expression of Notch receptors, ligands, and a growing list of downstream Notch-activated genes have by now been described for most human cancer types. Yet, it often remains unclear what may be the functional impact of these changes for tumor biology, initiation, and progression, for cancer therapy, and for personalized medicine. Emerging data indicate that Notch signaling can also contribute to increased aggressive properties such as invasion, tumor heterogeneity, angiogenesis, or tumor cell dormancy within solid cancer tissues; especially in epithelial cancers, which are in the center of this review. Notch further supports the "stemness" of cancer cells and helps define the stem cell niche for their long-term survival, by integrating the interaction between cancer cells and the cells of the tumor microenvironment (TME). The complexity of Notch crosstalk with other signaling pathways and its roles in cell fate and trans-differentiation processes such as epithelial-to-mesenchymal transition (EMT) point to this pathway as a decisive player that may tip the balance between tumor suppression and promotion, differentiation and invasion. Here we not only review the literature, but also explore genomic databases with a specific focus on Notch signatures, and how they relate to different stages in tumor development. Altered Notch signaling hereby plays a key role for tumor cell survival and coping with a broad spectrum of vital issues, contributing to failed therapies, poor patient outcome, and loss of lives

miRNA Multiplayers in glioma. From bench to bedside

Glioblastoma multiforme (GBM) is the most common type of malignant gliomas, characterized by genetic instability, intratumoral histopathological variability and unpredictable clinical behavior. Disappointing results in the treatment of gliomas with surgery, radiation and chemotherapy have fuelled a search for a new therapeutic targets and treatment modalities. A novel small non-coding RNA molecules, microRNAs (miRNAs), appear to represent one of the most attractive target molecules contributing to the pathogenesis of various types of tumors. They play crucial roles in tumorigenesis, angiogenesis, invasion and apoptosis. Some miRNAs are also associated with clinical outcome and chemo- and radiotherapy resistance. Moreover, miRNA have the potential to affect the responses to molecular-targeted therapies and they also might be associated with cancer stem cell properties, affecting tumor maintenance and progression. The expression profiles of miRNAs are also useful for subclassification of GBM, what underscores the heterogeneity of diseases that all share the same WHO histopathological grade. Importantly, molecular subtypes of GBM appear to correlate with clinical phenotypes, tumor characteristic and treatment outcomes. miRNAs are then biological markers with possible diagnostic and prognostic potential. They could also serve as one of the promising treatment targets in human glioblastoma

Günter Blobel — a Great Scientist and a Great Patron of Art and Culture

Günter Blobel jest wybitnym niemieckim naukowcem, profesorem Uniwersytetu Rockefellera w Nowym Jorku, który za swoje osiągnięcia i prace nad transportem białek przez błony wewnątrzkomórkowe został wyróżniony w 1999 roku Nagrodą Nobla w dziedzinie fizjologii lub medycyny. Wyniki doświadczeń przez niego prowadzonych zaowocowały sformułowaniem tzw. hipotezy sygnałowej, zgodnie z którą białka zawierają wbudowane sygnały, kierujące je do układu błon i pozwalające na przemieszczenie się na zewnątrz komórki. Odkrycia Güntera Blobela okazały się kluczowe i uniwersalne dla biologii komórki, pozwalając zrozumieć podłoże wielu chorób, a tym samym przyczyniając się do ratowania życia ludzkiego. Poza niezwykłą pasją, z jaką Günter Blobel podchodzi do nauki, jest on także wielkim entuzjastą kultury i sztuki, a najlepszym na to dowodem było przekazanie przez niego honorarium noblowskiego na odbudowę synagogi drezdeńskiej.Günter Blobel, a professor of Rockefeller University, is a leading German scientist, who has been awarded the 1999 Nobel Prize in Physiology or Medicine for his achievements and research on the transport of proteins through intracellular membranes. The results of his experiments resulted in the formulation of the “signal hypothesis”, whereby the proteins contain signals directing them to the membrane and allowing to travel outside the cell. Günter Blobel’s discovery proved to be crucial and universal for cell biology, helpful to understand the basis of many diseases. Besides having an incredible passion of science, Günter Blobel is also a great enthusiast of art and culture. The best proof of this was to give his Nobel Prize fee for the reconstruction of the Dresden synagogue

Down-regulation of tenascin-C inhibits breast cancer cells development by cell growth, migration, and adhesion impairment.

Tenascin-C (TNC) is an extracellular matrix (ECM) glycoprotein that plays an important role in cell proliferation, migration, and tumour invasion in various cancers. TNC is one of the main protein overexpressed in breast cancer, indicating a role for this ECM molecule in cancer pathology. In this study we have evaluated the TNC loss-off-function in breast cancer cells. In our approach, we used dsRNA sharing sequence homology with TNC mRNA, called ATN-RNA. We present the data showing the effects of ATN-RNA in MDA-MB-231 cells both in monolayer and three-dimensional culture. Cells treated with ATN-RNA were analyzed for phenotypic alterations in proliferation, migration, adhesion, cell cycle, multi-caspase activation and the involvement in epithelial to mesenchymal transition (EMT) processes. As complementary analysis the oncogenomic portals were used to assess the clinical implication of TNC expression on breast cancer patient's survival, showing the TNC overexpression associated with a poor survival outcome. Our approach applied first in brain tumors and then in breast cancer cell lines reveals that ATN-RNA significantly diminishes the cell proliferation, migration and additionally, reverses the mesenchymal cells phenotype to the epithelial one. Thus, TNC could be considered as the universal target in different types of tumors, where TNC overexpression is associated with poor prognosis

The Sequence and Structure Determine the Function of Mature Human miRNAs.

Micro RNAs (miRNAs) (19-25 nucleotides in length) belong to the group of non-coding RNAs are the most abundant group of posttranscriptional regulators in multicellular organisms. They affect a gene expression by binding of fully or partially complementary sequences to the 3'-UTR of target mRNA. Furthermore, miRNAs present a mechanism by which genes with diverse functions on multiple pathways can be simultaneously regulated at the post-transcriptional level. However, little is known about the specific pathways through which miRNAs with specific sequence or structural motifs regulate the cellular processes. In this paper we showed the broad and deep characteristics of mature miRNAs according to their sequence and structural motifs. We investigated a distinct group of miRNAs characterized by the presence of specific sequence motifs, such as UGUGU, GU-repeats and purine/pyrimidine contents. Using computational function and pathway analysis of their targeted genes, we were able to observe the relevance of sequence and the type of targeted mRNAs. As the consequence of the sequence analysis we finally provide the comprehensive description of pathways, biological processes and proteins associated with the distinct group of characterized miRNAs. Here, we found that the specific group of miRNAs with UGUGU can activate the targets associated to the interferon induction pathway or pathways prominently observed during carcinogenesis. GU-rich miRNAs are prone to regulate mostly processes in neurogenesis, whereas purine/pyrimidine rich miRNAs could be involved rather in transport and/or degradation of RNAs. Additionally, we have also analyzed the simple sequence repeats (SSRs). Their variation within mature miRNAs might be critical for normal miRNA regular activity. Expansion or contraction of SSRs in mature miRNA might directly affect its mRNA interaction or even change the function of that distinct miRNA. Our results prove that due to the specific sequence features, these molecules can also be involved in well-defined cellular processes depending on their sequence contents. The pathway mapping and theoretical gene target identification allowed us to create a biological framework to show the relevance of the specific miRNAs in regulation the distinct type of targets

Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis

The Notch signaling pathway is a critical player in embryogenesis but also plays various roles in tumorigenesis, with both tumor suppressor and oncogenic activities. Mutations, deletions, amplifications, or over-expression of Notch receptors, ligands, and a growing list of downstream Notch-activated genes have by now been described for most human cancer types. Yet, it often remains unclear what may be the functional impact of these changes for tumor biology, initiation, and progression, for cancer therapy, and for personalized medicine. Emerging data indicate that Notch signaling can also contribute to increased aggressive properties such as invasion, tumor heterogeneity, angiogenesis, or tumor cell dormancy within solid cancer tissues; especially in epithelial cancers, which are in the center of this review. Notch further supports the “stemness” of cancer cells and helps define the stem cell niche for their long-term survival, by integrating the interaction between cancer cells and the cells of the tumor microenvironment (TME). The complexity of Notch crosstalk with other signaling pathways and its roles in cell fate and trans-differentiation processes such as epithelial-to-mesenchymal transition (EMT) point to this pathway as a decisive player that may tip the balance between tumor suppression and promotion, differentiation and invasion. Here we not only review the literature, but also explore genomic databases with a specific focus on Notch signatures, and how they relate to different stages in tumor development. Altered Notch signaling hereby plays a key role for tumor cell survival and coping with a broad spectrum of vital issues, contributing to failed therapies, poor patient outcome, and loss of lives

Mature MiRNAs Form Secondary Structure, which Suggests Their Function beyond RISC

<div><p>The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.</p></div

The KEGG pathway analysis for GU-rich miRNAs.

<p>A. The table illustrating the: list of GU-rich miRNAs (first column); the IDs and KEGG pathways names (second and third column); the number of genes and GU-rich miRNAs involved associated with the pathways (fourth and sixth column). P-value was given in fifth column as a result of statistical analysis. P-value threshold is considered 0.05. B. GU-rich miRNAs in predicted pathway heat map. Significant miRNA-pathway interaction p<0.001.</p

Nucleotide occupancy position in human mature miRNAs.

<p>A Web logo with the distinct nucleotides at all positions of the mature miRNA sequences. B. Nucleotides representation at the first position of miRNAs mature strand at 5’ and 3’ end, in context to their length. The number under the name of nucleotide (e.g. 108 for A) reflects 108 sequences with A as a first nucleoside at 5’ end, and 27 at 3’, respectively.</p