Approaches for Understanding the Mechanisms of Long Noncoding RNA Regulation of Gene Expression

Mammalian genomes encode tens of thousands of long noncoding RNAs (lncRNAs) that have been implicated in a diverse array of biological processes and human diseases. In recent years, the development of new tools for studying lncRNAs has enabled important progress in defining the mechanisms by which Xist and other lncRNAs function. This collective work provides a framework for how to define the mechanisms by which lncRNAs act. This includes defining lncRNA function, identifying and characterizing lncRNA–protein interactions, and lncRNA localization in the cell. In this review, we discuss various experimental approaches for deciphering lncRNA mechanisms and discuss issues and limitations in interpreting these results. We explore what these data can reveal about lncRNA function and mechanism as well as emerging insights into lncRNA biology that have been derived from these studies

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription

HIV-1 unmasks the plasticity of innate lymphoid cells [preprint]

Pharmaceuticals that suppress HIV-1 viremia preserve CD4+ T cells and prevent AIDS. Nonetheless, HIV-1 infected people taking these drugs have chronic inflammation attributable to persistent disruption of intestinal barrier function with increased rates of cardiovascular mortality. To better understand the etiology of this inflammation we examined the effect of HIV-1 infection on innate lymphoid cells (ILCs). These innate immune counterparts of T cells lack clonotypic antigen receptors, classify according to signature transcription factors and cytokines, and maintain homeostasis in inflamed tissues. ILCs have been defined, in part, by the IL-7Rα, CD127. Here we report that the vast majority of type 1 and 3 ILCs in human adult and placental cord blood are in fact CD127-, as are colon lamina propria ILC1s and many ILC3s. Among ILCs, CD127-ILC1s were the major producer of inflammatory cytokines. In contrast to CD127+ILC3s, CD127-ILC3s did not produce IL-22, a cytokine that maintains epithelial barrier function. In HIV-1+ people taking antivirals that preserve CD4+ T cells, CD127-ILC1s and all homeostatic cytokine-producing CD127+ILCs were decreased in blood and colon. Common γ-chain cytokines that are reported to be elevated in response to HIV-1 infection caused JAK3-dependent downregulation of CD127 and converted CD127-ILC1s into NK cells with heightened cytolytic activity. Consistent with the recent report that human blood CD117+ILCs give rise to both ILC1s and NK cells, pseudotemporal clustering of transcriptomes from thousands of individual cells identified a developmental trajectory from CD127-ILC1s to memory NK cells that was defined by WNT-transcription factor TCF7. WNT inhibition prevented the cytokine-induced transition of CD127-ILC1 cells into memory NK cells. In HIV-1+ people, effector NK cells and TCF7+ memory NK cells were elevated, concomitant with reduction in CD127-ILC1s. These studies describe previously overlooked human ILC subsets that are significant in number and function, identify profound abnormalities in homeostatic ILCs that likely contribute to ongoing inflammation in HIV-1 infection despite control of viremia, provide explanation for increased memory NK cells in HIV-1 infection, and reveal functional plasticity of ILCs

Neighborhood regulation by lncRNA promoters, transcription, and splicing

Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that 'crosstalk' among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs

RNA-RNA Interactions Enable Specific Targeting of Noncoding RNAs to Nascent Pre-mRNAs and Chromatin Sites

Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5′ splice sites and 5′ splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs

Single-cell analysis of upper airway cells reveals host-viral dynamics in influenza infected adults [preprint]

Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection

Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing

The Xist long noncoding RNA orchestrates X chromosome inactivation, a process that entails chromosome-wide silencing and remodeling of the three-dimensional (3D) structure of the X chromosome. Yet, it remains unclear whether these changes in nuclear structure are mediated by Xist and whether they are required for silencing. Here, we show that Xist directly interacts with the Lamin B receptor, an integral component of the nuclear lamina, and that this interaction is required for Xist-mediated silencing by recruiting the inactive X to the nuclear lamina and by doing so enables Xist to spread to actively transcribed genes across the X. Our results demonstrate that lamina recruitment changes the 3D structure of DNA, enabling Xist and its silencing proteins to spread across the X to silence transcription

The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3

Many long non-coding RNAs (lncRNAs) affect gene expression, but the mechanisms by which they act are still largely unknown. One of the best-studied lncRNAs is Xist, which is required for transcriptional silencing of one X chromosome during development in female mammals. Despite extensive efforts to define the mechanism of Xist-mediated transcriptional silencing, we still do not know any proteins required for this role. The main challenge is that there are currently no methods to comprehensively define the proteins that directly interact with a lncRNA in the cell. Here we develop a method to purify a lncRNA from cells and identify proteins interacting with it directly using quantitative mass spectrometry. We identify ten proteins that specifically associate with Xist, three of these proteins—SHARP, SAF-A and LBR—are required for Xist-mediated transcriptional silencing. We show that SHARP, which interacts with the SMRT co-repressor that activates HDAC3, is not only essential for silencing, but is also required for the exclusion of RNA polymerase II (Pol II) from the inactive X. Both SMRT and HDAC3 are also required for silencing and Pol II exclusion. In addition to silencing transcription, SHARP and HDAC3 are required for Xist-mediated recruitment of the polycomb repressive complex 2 (PRC2) across the X chromosome. Our results suggest that Xist silences transcription by directly interacting with SHARP, recruiting SMRT, activating HDAC3, and deacetylating histones to exclude Pol II across the X chromosome

Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus