Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

Chromatin meets its organizers.

Chromatin organization and gene-gene interactions are critical components of carrying out developmental programs. Phillips-Cremins et al. identify a series of unexpected architectural proteins that work in a combinatorial manner to functionally organize chromatin in a cell-type-specific manner at the submegabase-length scale.

POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer.

Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells. Using chromatin- and RNA-profiling experiments, we provide evidence that POU2F3 is a master regulator of tuft cell identity in a variant form of SCLC. Moreover, we show that most SCLC tumors can be classified into one of three lineages based on the expression of POU2F3, ASCL1, or NEUROD1. Our CRISPR screens exposed other unique dependencies in POU2F3-expressing SCLC lines, including the lineage TFs SOX9 and ASCL2 and the receptor tyrosine kinase IGF1R (insulin-like growth factor 1 receptor). These data reveal POU2F3 as a cell identity determinant and a dependency in a tuft cell-like variant of SCLC, which may reflect a previously unrecognized cell of origin or a trans-differentiation event in this disease.

National trends in utilization and safety of gastric bypass, sleeve gastrectomy and conversion surgery in patients with GERD.

BACKGROUND: While some studies have reported improvement in gastro-esophageal reflux disease (GERD) symptoms after sleeve gastrectomy (SG), others have reported higher incidence of de-novo GERD, worsening of prior GERD symptoms and erosive esophagitis post SG. Furthermore, GERD unresponsive to medical management is one of the most common indications for conversion of SG to Roux-en-Y gastric bypass (RYGB). Real-world data on safety of primary SG, primary RYGB and SG to RYGB conversion for obese patients with GERD would be helpful for informing surgeons and patient procedure selection. We sought to evaluate the trends in utilization and safety of primary RYGB and primary SG for patients with GERD requiring medications, and compare the peri-operative outcomes between primary RYGB and conversion surgery from SG to RYGB for GERD using the MBSAQIP database. METHODS: A comparative analysis of post-operative outcomes within 30 days was performed for primary RYGB and primary SG after 1:1 nearest neighbor propensity score matching for patient demographics and preoperative comorbidities using the Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) registry from 2015 to 2021. This was followed by comparison of peri-operative outcomes between conversion surgery from SG to RYGB for GERD and primary RYGB using MBSAQIP 2020-2021 data. RESULTS: Utilization of primary RYGB increased from 38% in 2015 to 45% in 2021, while primary SG decreased from 62% in 2015 to 55% in 2021 for bariatric patients with GERD. Post-operative outcomes including reoperation, reintervention, readmission, major complications, and death within 30 days were significantly higher for patients undergoing primary RYGB compared to primary SG. Increased readmissions and ED visits were seen with conversion surgery. However, there was no difference in rates of reoperation, reintervention, major complications, or death between primary RYGB and SG conversion to RYGB cohorts. CONCLUSIONS: This data suggests that a strategy of performing a primary SG and subsequent SG-RYGB conversion for those with recalcitrant GERD symptoms is not riskier than a primary RYGB. Thus, it may be reasonable to perform SG in patients who are well informed of the risk of worsening GERD requiring additional surgical interventions. However, the impact of such staged approach (SG followed by conversion to RYGB) on long-term outcomes remains unknown.

Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss.

Genome-wide analyses have identified thousands of long noncoding RNAs (lncRNAs). Malat1 (metastasis-associated lung adenocarcinoma transcript 1) is among the most abundant lncRNAs whose expression is altered in numerous cancers. Here we report that genetic loss or systemic knockdown of Malat1 using antisense oligonucleotides (ASOs) in the MMTV (mouse mammary tumor virus)-PyMT mouse mammary carcinoma model results in slower tumor growth accompanied by significant differentiation into cystic tumors and a reduction in metastasis. Furthermore, Malat1 loss results in a reduction of branching morphogenesis in MMTV-PyMT- and Her2/neu-amplified tumor organoids, increased cell adhesion, and loss of migration. At the molecular level, Malat1 knockdown results in alterations in gene expression and changes in splicing patterns of genes involved in differentiation and protumorigenic signaling pathways. Together, these data demonstrate for the first time a functional role of Malat1 in regulating critical processes in mammary cancer pathogenesis. Thus, Malat1 represents an exciting therapeutic target, and Malat1 ASOs represent a potential therapy for inhibiting breast cancer progression.

Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing.

Improved identification of structural variants (SVs) in cancer can lead to more targeted and effective treatment options as well as advance our basic understanding of the disease and its progression. We performed whole-genome sequencing of the SKBR3 breast cancer cell line and patient-derived tumor and normal organoids from two breast cancer patients using Illumina/10x Genomics, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT) sequencing. We then inferred SVs and large-scale allele-specific copy number variants (CNVs) using an ensemble of methods. Our findings show that long-read sequencing allows for substantially more accurate and sensitive SV detection, with between 90% and 95% of variants supported by each long-read technology also supported by the other. We also report high accuracy for long reads even at relatively low coverage (25×-30×). Furthermore, we integrated SV and CNV data into a unifying karyotype-graph structure to present a more accurate representation of the mutated cancer genomes. We find hundreds of variants within known cancer-related genes detectable only through long-read sequencing. These findings highlight the need for long-read sequencing of cancer genomes for the precise analysis of their genetic instability.

Chromatin dynamics and gene positioning.

The mammalian cell nucleus provides a landscape where genes are regulated through their organization and association with freely diffusing proteins and nuclear domains. In many cases, specific genes are highly dynamic, and the principles governing their movements and interchromosomal interactions are currently under intensive study. Recent investigations have implicated actin and myosin in chromatin dynamics and gene expression. Here, we discuss our current understanding of the dynamics of the interphase genome and how it impacts nuclear organization and gene activity.

3' end processing of a long nuclear-retained noncoding RNA yields a tRNA-like cytoplasmic RNA.

MALAT1 is a long noncoding RNA known to be misregulated in many human cancers. We have identified a highly conserved small RNA of 61 nucleotides originating from the MALAT1 locus that is broadly expressed in human tissues. Although the long MALAT1 transcript localizes to nuclear speckles, the small RNA is found exclusively in the cytoplasm. RNase P cleaves the nascent MALAT1 transcript downstream of a genomically encoded poly(A)-rich tract to simultaneously generate the 3' end of the mature MALAT1 transcript and the 5' end of the small RNA. Enzymes involved in tRNA biogenesis then further process the small RNA, consistent with its adoption of a tRNA-like structure. Our findings reveal a 3' end processing mechanism by which a single gene locus can yield both a stable nuclear-retained noncoding RNA with a short poly(A) tail-like moiety and a small tRNA-like cytoplasmic RNA.

Impact of the COVID-19 Pandemic on Inpatient and Outpatient Utilization of Bariatric Surgery.

BACKGROUND: During the COVID-19 pandemic, deferral of inpatient elective surgical procedures served as a primary mechanism to increase surge inpatient capacity. Given the benefit of bariatric surgery on treating obesity and associated comorbidities, decreased access to bariatric surgery may have long-term public health consequences. Understanding the extent of the disruption of the COVID-19 pandemic to bariatric surgery will help health systems plan for appropriate access. MATERIALS AND METHODS: This is an observational cohort study using the PINC AI Healthcare Database from 1/1/2019-6/31/2021. A Poisson regression model with patient characteristics and hospital-fixed effects was used to assess the relative monthly within-hospital reduction in surgical encounters, variations by race and ethnicity, and shift from inpatient to outpatient procedures. A multivariate linear probability model was used to assess the change in 30-day readmissions from 2020 and 2021 compared to 2019. RESULTS: Among 309 hospitals, there were 46,539 bariatric procedures conducted in 2019 with a 14.8% reduction in volume to 39,641 procedures in 2020. There were 22,642 bariatric procedures observed from January to June of 2021. The most pronounced decrease in volume occurred in April with an 89.7% relative reduction from 2019. Black and Hispanic patients were more likely to receive bariatric surgery after the height of the pandemic compared to white patients. A clinically significant shift from inpatient to outpatient bariatric surgical procedures was not observed. Relative to 2019, there were no significant differences in bariatric surgical readmission rates. CONCLUSION: During the pandemic there was a sizable decrease in bariatric surgical volume. There did not appear to be disparities in access to bariatric surgery for minority patients. We did not observe a meaningful shift toward outpatient bariatric surgical procedures. Post-pandemic, monitoring is needed to assess if hospitals have been able to meet the demand for bariatric surgical procedures.

Fellowship training influences learning curves for laparoscopic sleeve gastrectomy.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is the most commonly performed bariatric surgical procedure. Little is known about how surgeon training background influences the learning curve of this procedure. We examined operating times (OT), weight loss outcomes, and 30-day complications between surgeons with and without fellowship training in LSG. We hypothesize that post-residency training specific to LSG influences learning curves. METHODS: Surgeons from a single institution were split into two groups: those who had not completed fellowship training in LSG (NF, n = 3), and those who had completed LSG specific training in fellowship (SGF, n = 3). OTs, BMI changes at 1 year, and 30-day readmissions, reoperations, and complications were extracted for the first 100 LSG cases of each surgeon. Data were analyzed in bins of 20 cases. Comparisons were made between cohorts within a bin and between adjacent bins of the same surgeon cohort. Logistic regression analyses were performed of OT and weight loss outcomes. RESULTS: SGF surgeons showed no difference in OTs over their first 100 cases. NF surgeons had statistically significant increased OTs compared to SGF surgeons during their first 60 cases and progressively shortened OTs during that interval (109 min to 78 min, p < 0.001 for NF surgeons vs. 73 min to 69 min, SGF surgeons). NF surgeons had a significantly steeper slope for improvement in OT over case number. There was no correlation between case number and weight loss outcomes in either group, and no differences in 30-day outcomes between groups. CONCLUSION: Surgeons who trained to perform LSG in fellowship demonstrate faster and consistent OR times on their initial independent LSG cases compared to surgeons who did not, with no correlation between case number and weight loss outcomes or safety profiles for either group. This suggests that learning curves for LSG are achieved during formal case-specific fellowship training.

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation.

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

MALAT1 long non-coding RNA and breast cancer.

Non-coding RNAs are becoming major players in disease pathogenesis such as cancer. Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) is a nuclear enriched long non-coding RNA that is generally overexpressed in patient tumors and metastases. Overexpression of MALAT1 has been shown to be positively correlated with tumor progression and metastasis in a large number of tumor types including breast tumors. Surprisingly, a recent report by Kim et al shows a metastasis suppressive role for Malat1. Here, we discuss these results in the context of a large body of published literature that support a pro-tumorigenic role for MALAT1 in order to gain potential insights into the basis of these observed differences.

Regulation of the ESC transcriptome by nuclear long noncoding RNAs.

Long noncoding (lnc)RNAs have recently emerged as key regulators of gene expression. Here, we performed high-depth poly(A)(+) RNA sequencing across multiple clonal populations of mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs) to comprehensively identify differentially regulated lncRNAs. We establish a biologically robust profile of lncRNA expression in these two cell types and further confirm that the majority of these lncRNAs are enriched in the nucleus. Applying weighted gene coexpression network analysis, we define a group of lncRNAs that are tightly associated with the pluripotent state of ESCs. Among these, we show that acute depletion of Platr14 using antisense oligonucleotides impacts the differentiation- and development-associated gene expression program of ESCs. Furthermore, we demonstrate that Firre, a lncRNA highly enriched in the nucleoplasm and previously reported to mediate chromosomal contacts in ESCs, controls a network of genes related to RNA processing. Together, we provide a comprehensive, up-to-date, and high resolution compilation of lncRNA expression in ESCs and NPCs and show that nuclear lncRNAs are tightly integrated into the regulation of ESC gene expression.

Regulation of the histone H4 monomethylase PR-Set7 by CRL4(Cdt2)-mediated PCNA-dependent degradation during DNA damage.

The histone methyltransferase PR-Set7/Set8 is the sole enzyme that catalyzes monomethylation of histone H4 at K20 (H4K20me1). Previous reports document disparate evidence regarding PR-Set7 expression during the cell cycle, the biological relevance of PR-Set7 interaction with PCNA, and its role in the cell. We find that PR-Set7 is indeed undetectable during S phase and instead is detected during late G2, mitosis, and early G1. PR-Set7 is transiently recruited to laser-induced DNA damage sites through its interaction with PCNA, after which 53BP1 is recruited dependent on PR-Set7 catalytic activity. During the DNA damage response, PR-Set7 interaction with PCNA through a specialized "PIP degron" domain targets it for PCNA-coupled CRL4(Cdt2)-dependent proteolysis. PR-Set7 mutant in its "PIP degron" is now detectable during S phase, during which the mutant protein accumulates. Outside the chromatin context, Skp2 promotes PR-Set7 degradation as well. These findings demonstrate a stringent spatiotemporal control of PR-Set7 that is essential for preserving the genomic integrity of mammalian cells.

Random monoallelic expression: regulating gene expression one allele at a time.

Monoallelic gene expression is a remarkable process in which transcription occurs from only one of two homologous alleles in a diploid cell. Interestingly, between 0.5% and 15% of autosomal genes exhibit random monoallelic gene expression, in which different cells express only one allele independently of the underlying genomic sequence, in a cell type-specific manner. Recently, genome-wide studies have increased our understanding of the cell type-specific incidence of random monoallelic gene expression, and how the imbalance in allelic expression is distinguished within the cell and potentially maintained across cell generations. Monoallelic gene expression is likely generated through stochastic independent regulation of the two alleles upon differentiation, and has varied implications for the cell and organism, in particular with respect to disease.

Long noncoding RNAs: functional surprises from the RNA world.

Most of the eukaryotic genome is transcribed, yielding a complex network of transcripts that includes tens of thousands of long noncoding RNAs with little or no protein-coding capacity. Although the vast majority of long noncoding RNAs have yet to be characterized thoroughly, many of these transcripts are unlikely to represent transcriptional "noise" as a significant number have been shown to exhibit cell type-specific expression, localization to subcellular compartments, and association with human diseases. Here, we highlight recent efforts that have identified a myriad of molecular functions for long noncoding RNAs. In some cases, it appears that simply the act of noncoding RNA transcription is sufficient to positively or negatively affect the expression of nearby genes. However, in many cases, the long noncoding RNAs themselves serve key regulatory roles that were assumed previously to be reserved for proteins, such as regulating the activity or localization of proteins and serving as organizational frameworks of subcellular structures. In addition, many long noncoding RNAs are processed to yield small RNAs or, conversely, modulate how other RNAs are processed. It is thus becoming increasingly clear that long noncoding RNAs can function via numerous paradigms and are key regulatory molecules in the cell.

Quantitative analysis of chromatin interaction changes upon a 4.3 Mb deletion at mouse 4E2.

BACKGROUND: Circular chromosome conformation capture (4C) has provided important insights into three dimensional (3D) genome organization and its critical impact on the regulation of gene expression. We developed a new quantitative framework based on polymer physics for the analysis of paired-end sequencing 4C (PE-4Cseq) data. We applied this strategy to the study of chromatin interaction changes upon a 4.3 Mb DNA deletion in mouse region 4E2. RESULTS: A significant number of differentially interacting regions (DIRs) and chromatin compaction changes were detected in the deletion chromosome compared to a wild-type (WT) control. Selected DIRs were validated by 3D DNA FISH experiments, demonstrating the robustness of our pipeline. Interestingly, significant overlaps of DIRs with CTCF/Smc1 binding sites and differentially expressed genes were observed. CONCLUSIONS: Altogether, our PE-4Cseq analysis pipeline provides a comprehensive characterization of DNA deletion effects on chromatin structure and function.

Identification and initial functional characterization of a human vascular cell-enriched long noncoding RNA.

OBJECTIVE: Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. APPROACH AND RESULTS: RNA sequencing (RNA-seq) of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA (Smooth muscle and Endothelial cell-enriched migration/differentiation-associated long NonCoding RNA [SENCR]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5' end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-seq experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. CONCLUSIONS: SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.

Receptor-mediated delivery of engineered nucleases for genome modification.

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.

Biogenesis and function of nuclear bodies.

Nuclear bodies including nucleoli, Cajal bodies, nuclear speckles, Polycomb bodies, and paraspeckles are membraneless subnuclear organelles. They are present at steady-state and dynamically respond to basic physiological processes as well as to various forms of stress, altered metabolic conditions and alterations in cellular signaling. The formation of a specific nuclear body has been suggested to follow a stochastic or ordered assembly model. In addition, a seeding mechanism has been proposed to assemble, maintain, and regulate particular nuclear bodies. In coordination with noncoding RNAs, chromatin modifiers and other machineries, various nuclear bodies have been shown to sequester and modify proteins, process RNAs and assemble ribonucleoprotein complexes, as well as epigenetically regulate gene expression. Understanding the functional relationships between the 3D organization of the genome and nuclear bodies is essential to fully uncover the regulation of gene expression and its implications for human disease.