Probing the function of long noncoding RNAs in the nucleus.

The nucleus is a highly organized and dynamic environment where regulation and coordination of processes such as gene expression and DNA replication are paramount. In recent years, noncoding RNAs have emerged as key participants in the regulation of nuclear processes. There are a multitude of functional roles for long noncoding RNA (lncRNA), mediated through their ability to act as molecular scaffolds bridging interactions with proteins, chromatin, and other RNA molecules within the nuclear environment. In this review, we discuss the diversity of techniques that have been developed to probe the function of nuclear lncRNAs, along with the ways in which those techniques have revealed insights into their mechanisms of action. Foundational observations into lncRNA function have been gleaned from molecular cytology-based, single-cell approaches to illuminate both the localization and abundance of lncRNAs in addition to their potential binding partners. Biochemical, extraction-based approaches have revealed the molecular contacts between lncRNAs and other molecules within the nuclear environment and how those interactions may contribute to nuclear organization and regulation. Using examples of well-studied nuclear lncRNAs, we demonstrate that the emerging functions of individual lncRNAs have been most clearly deduced from combined cytology and biochemical approaches tailored to study specific lncRNAs. As more functional nuclear lncRNAs continue to emerge, the development of additional technologies to study their interactions and mechanisms of action promise to continually expand our understanding of nuclear organization, chromosome architecture, genome regulation, and disease states.

Under-reporting by surgical pathologists in tissue removed during revision surgery for metal-on-metal arthroplasties.

Although adverse local tissue reactions (ALTR) have been reported for metal-on-metal implants (MoM) requiring early revision surgery, no study has looked at the accuracy of surgical pathologists in diagnosing ALTR. This study aims to investigate the accuracy of reporting adverse local tissue reactions in tissue samples following revision surgery from metal-on-metal implants. The authors reviewed histology glass slides as well as the original pathology reports of tissue processed in revision arthroplasties in 23 cases. These samples were microscopically analyzed for tissue necrosis and cystic degeneration, the presence of metal particles, corrosion byproducts, membrane formation, histiocytic cells, lymphocytic cells, and vascular pathology. The authors' findings were then compared to their corresponding original pathology reports. The authors found consistent under-reporting of the tissue findings. Most importantly, 18 samples showed evidence of metal present compared to 2 samples on original pathology reporting. The authors found that 15 samples showed evidence of pathological membranous tissue compared to just 6 on original pathology reporting. While just 3 of the original pathology reports indicated the presence of areas of predominantly lymphocytic inflammatory cells, the authors found 13 examples of such areas. Although ALTR reactions have been described as a sequala of failed MoM, the authors' data suggest that ALTR may occur more frequently than previously described. Under-reported findings of ALTR deprive both the patient and orthopaedic surgeon of important information that can help guide further follow-up.

Dual IKZF2 and CK1α degrader targets acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a hematologic malignancy for which several epigenetic regulators have been identified as therapeutic targets. Here we report the development of cereblon-dependent degraders of IKZF2 and casein kinase 1α (CK1α), termed DEG-35 and DEG-77. We utilized a structure-guided approach to develop DEG-35 as a nanomolar degrader of IKZF2, a hematopoietic-specific transcription factor that contributes to myeloid leukemogenesis. DEG-35 possesses additional substrate specificity for the therapeutically relevant target CK1α, which was identified through unbiased proteomics and a PRISM screen assay. Degradation of IKZF2 and CK1α blocks cell growth and induces myeloid differentiation in AML cells through CK1α-p53- and IKZF2-dependent pathways. Target degradation by DEG-35 or a more soluble analog, DEG-77, delays leukemia progression in murine and human AML mouse models. Overall, we provide a strategy for multitargeted degradation of IKZF2 and CK1α to enhance efficacy against AML that may be expanded to additional targets and indications.

N6-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation.

N6-Methyladenosine (m6A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m6A dictates its diverse molecular and cellular effects in leukemias remains unknown. We found that YTHDC1 is the essential m6A reader in myeloid leukemia from a genome-wide CRISPR screen and that m6A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m6A condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m6A-mRNAs from the PAXT complex and exosome-associated RNA degradation. Collectively, m6A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.

Transcriptional control of CBX5 by the RNA binding proteins RBMX and RBMXL1 maintains chromatin state in myeloid leukemia.

RNA binding proteins (RBPs) are key arbiters of post-transcriptional regulation and are found to be found dysregulated in hematological malignancies. Here, we identify the RBP RBMX and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX/L1 are overexpressed in acute myeloid leukemia (AML) primary patients compared to healthy individuals, and RBMX/L1 loss delayed leukemia development. RBMX/L1 loss lead to significant changes in chromatin accessibility, as well as chromosomal breaks and gaps. We found that RBMX/L1 directly bind to mRNAs, affect transcription of multiple loci, including CBX5 (HP1α), and control the nascent transcription of the CBX5 locus. Forced CBX5 expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through their downstream target CBX5. These findings identify a mechanism for RBPs directly promoting transcription and suggest RBMX/L1, as well as CBX5, as potential therapeutic targets in myeloid malignancies.

Variable levels of drift in tunicate cardiopharyngeal gene regulatory elements.

BACKGROUND: Mutations in gene regulatory networks often lead to genetic divergence without impacting gene expression or developmental patterning. The rules governing this process of developmental systems drift, including the variable impact of selective constraints on different nodes in a gene regulatory network, remain poorly delineated. RESULTS: Here we examine developmental systems drift within the cardiopharyngeal gene regulatory networks of two tunicate species, Corella inflata and Ciona robusta. Cross-species analysis of regulatory elements suggests that trans-regulatory architecture is largely conserved between these highly divergent species. In contrast, cis-regulatory elements within this network exhibit distinct levels of conservation. In particular, while most of the regulatory elements we analyzed showed extensive rearrangements of functional binding sites, the enhancer for the cardiopharyngeal transcription factor FoxF is remarkably well-conserved. Even minor alterations in spacing between binding sites lead to loss of FoxF enhancer function, suggesting that bound trans-factors form position-dependent complexes. CONCLUSIONS: Our findings reveal heterogeneous levels of divergence across cardiopharyngeal cis-regulatory elements. These distinct levels of divergence presumably reflect constraints that are not clearly associated with gene function or position within the regulatory network. Thus, levels of cis-regulatory divergence or drift appear to be governed by distinct structural constraints that will be difficult to predict based on network architecture.