The NORAD lncRNA assembles a topoisomerase complex critical for genome stability

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA—non-coding RNA activated by DNA damage (NORAD)—has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex—which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)—that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression—which represent phenotypes that are mechanistically linked to TOP1 and PRPF19–CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection

The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remains unclear. Here, we combine a multi-omics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype associated with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies

Nuclear lncRNA stabilization in the host response to bacterial infection.

Long non-coding RNAs (lncRNAs) play important roles in many cellular pathways, but their contribution to the defense of eukaryotic cells against pathogens remains poorly understood. A new study from Imamura et al in The EMBO Journal reports that Salmonella infection in human cells impacts nuclear RNA decay, which in turn drives the accumulation of otherwise unstable nuclear lncRNAs, some of which may have protective effects against this common bacterial pathogen. These unexpected findings demand more efforts to fully decrypt the molecular functions of lncRNAs in innate and adaptive immunity

Atlas der SARS-CoV-2-RNA-Protein-Interaktionen in infizierten Zellen

Using RNA antisense purification and mass spectrometry, we identified more than 100 human proteins that directly and specifically bind SARS-CoV-2 RNA in infected cells. To gain insights into the functions of selected RNA interactors, we applied genetic perturbation and pharmacological inhibition experiments, and mapped the contact sites on the viral RNA. This led to the identification of host dependency factors and defense strategies, which can guide the design of novel therapeutics against SARS-CoV-2

ProbeSHIFTR - Oligonucleotide probe designer for <b>S</b>elective RNase-<b>H</b>-mediated <b>I</b>nteractome <b>F</b>raming for <b>T</b>arget <b>R</b>NA Regions (SHIFTR)

Software to design a specific set of DNA probes for a target specific RNA sequence. ProbeSHIFTR is a highly versatile computational tool to design, filter and select an optimized probe set for SHIFTR experiments in any species of interest. In brief, Probe-SHIFTR extracts each possible k-mer for a given target RNA and filters sequences containing stretches of polybases, repeat regions, or low complexity regions. The remaining k-mers are filtered based on sequence homology to regions in the target genome or transcriptome. All filtering parameters are fully customizable.See publication for further details:SHIFTR enables the unbiased identification of proteins bound to specific RNA regions in live cells. Jens Aydin, Alexander Gabel, Sebastian Zielinski, Sabina Ganskih, Nora Schmidt, Christina R. Hartigan, Monica Schenone, Steven A. Carr, and Mathias Munschauer.</p

Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer

Despite some well-characterized functions in cancer, the impact of most long non-coding RNAs remains unknown. Here, the authors discover the lncRNA lincNMR which is upregulated in cancer and drives cell proliferation by interacting with YBX1 and controlling nucleotide metabolism