Genome-Wide Mapping of RNA-Protein Associations via Sequencing.

RNA-protein interactions are crucial for regulating gene expression and cellular functions, with their dysregulation potentially impacting disease progression. Systematically mapping these interactions is resource-intensive due to the vast number of potential RNA and protein interactions. Here, we introduce PRIM-seq (Protein-RNA Interaction Mapping by sequencing), a method for the concurrent identification of RNA-binding proteins (RBPs) and the elucidation of their associated RNAs.

Single-cell multiplex chromatin and RNA interactions in ageing human brain.

Dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA. Chromatin complex compositions change during cellular differentiation and ageing, and are expected to be highly heterogeneous among terminally differentiated single cells. Here we introduce the multinucleic acid interaction mapping in single cells (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression and RNA-chromatin associations within individual nuclei.

Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization in human cells.

The interphase genome is dynamically organized in the nucleus and decorated with chromatin-associated RNA (caRNA). It remains unclear whether the genome architecture modulates the spatial distribution of caRNA and vice versa. Here, we generate a resource of genome-wide RNA-DNA and DNA-DNA contact maps in human cells.

Single-cell multiplex chromatin and RNA interactions in aging human brain.

The dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA (caRNA) . Chromatin complex compositions change during cellular differentiation and aging, and are expected to be highly heterogeneous among terminally differentiated single cells . Here we introduce the Multi-Nucleic Acid Interaction Mapping in Single Cell (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations within individual nuclei.

ALCAM: A Novel Surface Marker on EpCAM Circulating Tumor Cells.

Current strategies in circulating tumor cell (CTC) isolation in pancreatic cancer heavily rely on the EpCAM and cytokeratin cell status. EpCAM is generally not considered a good marker given its transitory change during Epithelial to Mesenchymal Transition (EMT) or reverse EMT. There is a need to identify other surface markers to capture the complete repertoire of PDAC CTCs.

Modulation of Early Neutrophil Granulation: The Circulating Tumor Cell-Extravesicular Connection in Pancreatic Ductal Adenocarcinoma.

Tumor cells dissociate from the primary site and enter into systemic circulation (circulating tumor cells, CTCs) either alone or as tumor microemboli (clusters); the latter having an increased predisposition towards forming distal metastases than single CTCs. The formation of clusters is, in part, created by contacts between cell-cell junction proteins and/or cytokine receptor pairs with other cells such as neutrophils, platelets, fibroblasts, etc. In the present study, we provide evidence for an extravesicular (EV) mode of communication between pancreatic cancer CTCs and neutrophils.

SRSF9 selectively represses ADAR2-mediated editing of brain-specific sites in primates.

Adenosine-to-inosine (A-to-I) RNA editing displays diverse spatial patterns across different tissues. However, the human genome encodes only two catalytically active editing enzymes (ADAR1 and ADAR2), suggesting that other regulatory factors help shape the editing landscape. Here, we show that the splicing factor SRSF9 selectively controls the editing of many brain-specific sites in primates.

Platforms for Investigating LncRNA Functions.

Prior to the sequencing of the human genome, it was presumed that most of the DNA coded for proteins. However, with the advent of next-generation sequencing, it has now been recognized that most complex eukaryotic genomes are in fact transcribed into noncoding RNAs (ncRNAs), including a family of transcripts referred to as long noncoding RNAs (lncRNAs). LncRNAs have been implicated in many biological processes ranging from housekeeping functions such as transcription to more specialized functions such as dosage compensation or genomic imprinting, among others.

Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies.

RAF kinase inhibitors are clinically active in patients with BRAF (V600E) mutant melanoma. However, rarely do tumors regress completely, with the majority of responses being short-lived. This is partially mediated through the loss of negative feedback loops after MAPK inhibition and reactivation of upstream signaling.

Deciphering the roles of lncRNAs in breast development and disease.

Breast cancer is the second leading cause of cancer related deaths in women. It is therefore important to understand the mechanisms underlying breast cancer development as well as raises the need for enhanced, non-invasive strategies for novel prognostic and diagnostic methods. The emergence of long non-coding RNAs (lncRNAs) as potential key players in neoplastic disease has received considerable attention over the past few years.

The roles of ubiquitin modifying enzymes in neoplastic disease.

The initial experiments performed by Rose, Hershko, and Ciechanover describing the identification of a specific degradation signal in short-lived proteins paved the way to the discovery of the ubiquitin mediated regulation of numerous physiological functions required for cellular homeostasis. Since their discovery of ubiquitin and ubiquitin function over 30years ago it has become wholly apparent that ubiquitin and their respective ubiquitin modifying enzymes are key players in tumorigenesis. The human genome encodes approximately 600 putative E3 ligases and 80 deubiquitinating enzymes and in the majority of cases these enzymes exhibit specificity in sustaining either pro-tumorigenic or tumour repressive responses.

Xist RNA repeat E is essential for ASH2L recruitment to the inactive X and regulates histone modifications and escape gene expression.

Long non-coding RNA Xist plays a crucial role in establishing and maintaining X-chromosome inactivation (XCI) which is a paradigm of long non-coding RNA-mediated gene regulation. Xist has Xist-specific repeat elements A-F which are conserved among eutherian mammals, underscoring their functional importance. Here we report that Xist RNA repeat E, a conserved Xist repeat element in the Xist exon 7, interacts with ASH2L and contributes to maintenance of escape gene expression level on the inactive X-chromosome (Xi) during XCI.

FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family.

Dynamic interplay and function of multiple noncoding genes governing X chromosome inactivation.

There is increasing evidence for the emergence of long noncoding RNAs (lncRNAs) as important components, especially in the regulation of gene expression. In the event of X chromosome inactivation, robust epigenetic marks are established in a long noncoding Xist RNA-dependent manner, giving rise to a distinct epigenetic landscape on the inactive X chromosome (Xi). The X inactivation center (Xic) is essential for induction of X chromosome inactivation and harbors two topologically associated domains (TADs) to regulate monoallelic Xist expression: one at the noncoding Xist gene and its upstream region, and the other at the antisense Tsix and its upstream region.

Understanding the Complex Circuitry of lncRNAs at the X-inactivation Center and Its Implications in Disease Conditions.

Balanced gene expression is a high priority in order to maintain optimal functioning since alterations and variations could result in acute consequences. X chromosome inactivation (X-inactivation) is one such strategy utilized by mammalian species to silence the extra X chromosome in females to uphold a similar level of expression between the two sexes. A functionally versatile class of molecules called long noncoding RNA (lncRNA) has emerged as key regulators of gene expression and plays important roles during development.

Quick fluorescent in situ hybridization protocol for Xist RNA combined with immunofluorescence of histone modification in X-chromosome inactivation.

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation.

Binding of NF-κB to nucleosomes: effect of translational positioning, nucleosome remodeling and linker histone H1.

NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates.

The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling.

Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome.