Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At ∼96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na⁺ and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.

Background: Trypanosome gene expression is regulated almost exclusively at the post-transcriptional level, with mRNA degradation playing a decisive role. When trypanosomes are transferred from the blood of a mammal to the midgut of a Tsetse fly, they transform to procyclic forms: gene expression is reprogrammed, changing the cell surface and switching the mode of energy metabolism. Within the blood, trypanosomes can pre-adapt for Tsetse transmission, becoming growth-arrested stumpy forms. We describe here the transitions in gene expression that occur during differentiation of in-vitro cultured bloodstream forms to procyclic forms. Results: Some mRNAs showed changes within 30 min of cis- aconitate addition, whereas others responded 12-24 hours later. For the first 12 h after addition of cis-aconitate, cells accumulated at the G1 phase of the cell cycle, and showed decreases in mRNAs required for proliferation, mimicking the changes seen in stumpy forms: many mRNAs needed for ribosomal and flagellar biogenesis showed striking co-regulation. Other mRNAs encoding components of signal transduction pathways and potential regulators were specifically induced only during differentiation. Messenger RNAs encoding proteins required for individual metabolic pathways were often co-regulated. Conclusion: Trypanosome genes form post-transcriptional regulons in which mRNAs with functions in particular pathways, or encoding components of protein complexes, show almost identical patterns of regulation.

Introduction: Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties. Methods: We describe a sub-population expressing the stem-like marker CD44⁺/CD24^-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44⁺/CD24^low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments. Results: Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo. Conclusion: In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.

A solid process for diagnosis could have a substantial impact on the successful treatment of pancreatic cancer, for which currently mortality is nearly identical to incidence. Variations in the abundance of all microRNA molecules from peripheral blood cells and pancreas tissues were analyzed on microarrays and in part validated by real-time PCR assays. In total, 245 samples from two clinical centers were studied that were obtained from patients with pancreatic ductal adenocarcinoma or chronic pancreatitis and from healthy donors. Utilizing the minimally invasive blood test, receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analysis demonstrated very high sensitivity and specificity of a distinction between healthy people and patients with either cancer or chronic pancreatitis; respective AUC values of 0.973 and 0.950 were obtained. Confirmative and partly even more discriminative diagnosis could be performed on tissue samples with AUC values of 1.0 and 0.937, respectively. In addition, discrimination between cancer and chronic pancreatitis was achieved (AUC = 0.875). Also, several miRNAs were identified that exhibited abundance variations in both tissue and blood samples. The results could have an immediate diagnostic value for the evaluation of tumor reoccurrence in patients, who have undergone curative surgical resection, and for people with a familial risk of pancreatic cancer.

Despite recent advances in N⁶-methyladenosine (m⁶A) biology, the regulation of crucial RNA processing steps by the RNA methyltransferase-like 3 (METTL3) in glioma stem-like cells (GSCs) remains obscure. An integrated analysis of m⁶A-RIP (RNA immunoprecipitation) and total RNA-Seq of METTL3-silenced GSCs identified that m⁶A modification in GSCs is principally carried out by METTL3. The m⁶A-modified transcripts showed higher abundance compared to non-modified transcripts. Further, we showed that the METTL3 is essential for the expression of GSC-specific actively transcribed genes. Silencing METTL3 resulted in the elevation of several aberrant alternative splicing events. We also found that putative m⁶A reader proteins play a key role in the RNA stabilization function of METTL3. METTL3 altered A-to-I and C-to-U RNA editing events by differentially regulating RNA editing enzymes ADAR and APOBEC3A. Similar to protein-coding genes, lincRNAs (long intergenic non-coding RNAs) with m⁶A marks showed METTL3-dependent high expression. m⁶A modification of 3′UTRs appeared to result in a conformation-dependent hindrance to miRNA binding to their targets. The integrated analysis of the m⁶A regulome in METTL3-silenced GSCs showed global disruption in tumorigenic pathways that are indispensable for GSC maintenance and glioma progression. We conclude that METTL3 plays a vital role in many steps of RNA processing and orchestrates successful execution of oncogenic pathways in GSCs.