Spatio-temporal regulation of mRNA decay revealed by a novel single-molecule dual-color imaging method

Discoveries that have been made over past decades emphasized the importance of post-transcriptional control as a means of regulating gene expression. RNA turnover is one of the key aspects of post-transcriptional control that contributes directly towards maintenance of normal cellular homeostasis. Degradation of functional messenger RNAs (mRNAs) is a tightly regulated process and its dysregulation results in either excessive or insufficient amounts of mRNAs within cells that eventually lead to a disease-associated condition. Furthermore, multiple quality control mechanisms eliminate aberrant mRNAs thereby preventing their translation into malfunctioning proteins. The realization of the importance of RNA decay pathways has fueled further research towards understanding the underlying molecular mechanisms in RNA turnover and its regulation. All protein-coding mRNAs, as well as non-coding RNAs, have distinct half-lives and are ultimately degraded. Previously, many of the factors involved in RNA decay pathways have been identified and studied. Two types of enzymes are shared among RNA decay pathways: exonucleases and endonucleases. The former are further divided into 5′-to-3′ and 3′-to-5′ degrading enzymes and their activation is often dependent on prior removal of terminal stability marks from an RNA molecule. The best-studied exonuclease is Xrn1 that degrades an RNA substrate from the 5′-end to 3′-end. On the other hand, endonucleases cleave an RNA strand to expose the resultant fragments to exonucleases, circumventing the requirement of first removing the stability marks. Most of our current appreciation of the molecular mechanisms related to the mRNA decay is attributable to the methods that involve ensemble measurements. However, these measurements often result in an averaged outcome from whole population of cells, wherein information about variability among individual cells is lost. In addition, the possibility to get information on the spatio-temporal regulation of mRNA decay is limited using ensemble methods. Hence, accurate dissection of the spatial and temporal regulation of mRNA decay requires development of a single-molecule method that preserves information on cell-to-cell variability. Single-molecule RNA imaging methods have already been used to study several aspects of the mRNA life cycle and they have helped to uncover in vivo regulations that were not possible to observe before. However, a powerful imaging method allowing for an observation of mRNA turnover in real-time at the level of single cells/molecules has been missing. During my PhD, I established a robust single-molecule imaging technique in order to characterize the spatio-temporal dynamics of RNA turnover within its cellular context. I engineered an mRNA reporter that contains viral tandem pseudo-knots placed between PP7 and MS2 stem-loops. These orthogonal stem-loops can be labeled with spectrally distinct fluorescent proteins. In addition, the viral pseudo-knots block Xrn1-mediated degradation resulting in stabilization of the reporter’s 3′-degradation intermediate that is otherwise inherently instable. This stabilized 3′-end contains only the MS2 stem-loop region. Thus, intact mRNAs are labeled with both fluorophores, while incompletely degraded mRNA fragments are labeled only with a single fluorophore. I used the amounts and positions of intact mRNAs and stabilized 3′-ends as readout of mRNA degradation. Therefore, this technique is called 3(Three)′-RNA End Accumulation during Turnover (TREAT). I applied TREAT to monitor the fates of mRNAs in single fixed and living mammalian cells. Using this method, I measured the kinetics and cell-to-cell variability of mRNA decay in fixed cells. The nuclear export rates and cytoplasmic mRNA half-lives showed that individual degradation events occur independently within the cytoplasm suggesting that there is no burst in mRNA degradation. In addition, I found that transcripts, as well as degradation intermediates, are dispersed throughout cytoplasm and are not enriched within processing bodies in living cells. Imaging of an mRNA biosensor targeted for an endonucleolytic cleavage by the RNA-induced silencing complex (RISC) showed that slicing can be observed in real-time in cytoplasm of living cells but does not occur in nucleus. The slicing events were found to have no spatial preference with respect to the distance from the nucleus. In addition to the rate of synthesis and the rate of turnover, the levels of mRNAs were found to be affected by the rate of translation as well. Indeed, I have also observed that inhibition of translation by several compounds increases mRNA stability, suggesting that the processes of mRNA degradation and translation are globally interconnected. The cross-talk among three processes central to the mRNA life cycle, transcription, degradation and translation, is becoming increasingly apparent. However, further research is required to obtain a detailed understanding of the molecular interplays in eukaryotic cells. As TREAT system visualizes mRNA from its synthesis in the nucleus through export to degradation in cytoplasm, I anticipate that this methodology will provide a framework for investigating the entire life history of individual mRNAs in single cells

Nauczanie przez projekty i efektywne zarządzanie nim w edukacji przedszkolnej

The paper deals with project based learning as an effective approach for acquiring English language at pre-primary level of education. It provides the reader with the theoretical framework and practical samples of this approach emphasizing the role of „driving question which initiates this process“.Artykuł poświęcony jest nauczaniu przez projekty jako skutecznemu podejściu do zdobywania języka angielskiego na etapie edukacji przedszkolnej. Tekst przedstawia teoretyczne ramy i praktyczne próbki tego podejścia, podkreślające rolę „pytań-kluczy, które inicjują ten proces“

Detection and quantification of RNA decay intermediates using XRN1-resistant reporter transcripts

RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression

Decreased methylation in the SNAI2 and ADAM23 genes associated with de-differentiation and haematogenous dissemination in breast cancers

Abstract Background In breast cancer (BC), deregulation of DNA methylation leads to aberrant expressions and functions of key regulatory genes. In our study, we investigated the relationship between the methylation profiles of genes associated with cancer invasivity and clinico-pathological parameters. In detail, we studied differences in the methylation levels between BC patients with haematogenous and lymphogenous cancer dissemination. Methods We analysed samples of primary tumours (PTs), lymph node metastases (LNMs) and peripheral blood cells (PBCs) from 59 patients with sporadic disseminated BC. Evaluation of the DNA methylation levels of six genes related to invasivity, ADAM23, uPA, CXCL12, TWIST1, SNAI1 and SNAI2, was performed by pyrosequencing. Results Among the cancer-specific methylated genes, we found lower methylation levels of the SNAI2 gene in histologic grade 3 tumours (OR = 0.61; 95% CI, 0.39–0.97; P = 0.038) than in fully or moderately differentiated cancers. We also evaluated the methylation profiles in patients with different cancer cell dissemination statuses (positivity for circulating tumour cells (CTCs) and/or LNMs). We detected the significant association between reduced DNA methylation of ADAM23 in PTs and presence of CTCs in the peripheral blood of patients (OR = 0.45; 95% CI, 0.23–0.90; P = 0.023). Conclusion The relationships between the decreased methylation levels of the SNAI2 and ADAM23 genes and cancer de-differentiation and haematogenous dissemination, respectively, indicate novel functions of those genes in the invasive processes. After experimental validation of the association between the lower values of SNAI2 and ADAM23 methylation and clinical features of aggressive BCs, these methylation profiles could improve the management of metastatic disease

miR-205-5p Downregulation and ZEB1 Upregulation Characterize the Disseminated Tumor Cells in Patients with Invasive Ductal Breast Cancer

Background: Dissemination of breast cancer (BC) cells through the hematogenous or lymphogenous vessels leads to metastatic disease in one-third of BC patients. Therefore, we investigated the new prognostic features for invasion and metastasis. Methods: We evaluated the expression of miRNAs and epithelial-to-mesenchymal transition (EMT) genes in relation to CDH1/E-cadherin changes in samples from 31 patients with invasive ductal BC including tumor centrum (TU-C), tumor invasive front (TU-IF), lymph node metastasis (LNM), and CD45-depleted blood (CD45-DB). Expression of miRNA and mRNA was quantified by RT-PCR arrays and associations with clinico-pathological characteristics were statistically evaluated by univariate and multivariate analysis. Results: We did not verify CDH1 regulating associations previously described in cell lines. However, we did detect extremely high ZEB1 expression in LNMs from patients with distant metastasis, but without regulation by miR-205-5p. Considering the ZEB1 functions, this overexpression indicates enhancement of metastatic potential of lymphogenously disseminated BC cells. In CD45-DB samples, downregulated miR-205-5p was found in those expressing epithelial and/or mesenchymal markers (CTC+) that could contribute to insusceptibility and survival of hematogenously disseminated BC cells mediated by increased expression of several targets including ZEB1. Conclusions: miR-205-5p and potentially ZEB1 gene are promising candidates for markers of metastatic potential in ductal BC