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.

Cross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program.

Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans.

Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.

Transcriptome profiling is widely used to infer functional states of specific cell types, as well as their responses to stimuli, to define contributions to physiology and pathophysiology. Focusing on microglia, the brain's macrophages, we report here a side-by-side comparison of classical cell-sorting-based transcriptome sequencing and the 'RiboTag' method, which avoids cell retrieval from tissue context and yields translatome sequencing information. Conventional whole-cell microglial transcriptomes were found to be significantly tainted by artifacts introduced by tissue dissociation, cargo contamination and transcripts sequestered from ribosomes. Conversely, our data highlight the added value of RiboTag profiling for assessing the lineage accuracy of Cre recombinase expression in transgenic mice. Collectively, this study indicates method-based biases, reveals observer effects and establishes RiboTag-based translatome profiling as a valuable complement to standard sorting-based profiling strategies.

The status of the human gene catalogue.

Scientists have been trying to identify every gene in the human genome since the initial draft was published in 2001. In the years since, much progress has been made in identifying protein-coding genes, currently estimated to number fewer than 20,000, with an ever-expanding number of distinct protein-coding isoforms. Here we review the status of the human gene catalogue and the efforts to complete it in recent years. Beside the ongoing annotation of protein-coding genes, their isoforms and pseudogenes, the invention of high-throughput RNA sequencing and other technological breakthroughs have led to a rapid growth in the number of reported non-coding RNA genes. For most of these non-coding RNAs, the functional relevance is currently unclear; we look at recent advances that offer paths forward to identifying their functions and towards eventually completing the human gene catalogue. Finally, we examine the need for a universal annotation standard that includes all medically significant genes and maintains their relationships with different reference genomes for the use of the human gene catalogue in clinical settings.

lincRNAs: genomics, evolution, and mechanisms.

Long intervening noncoding RNAs (lincRNAs) are transcribed from thousands of loci in mammalian genomes and might play widespread roles in gene regulation and other cellular processes. This Review outlines the emerging understanding of lincRNAs in vertebrate animals, with emphases on how they are being identified and current conclusions and questions regarding their genomics, evolution and mechanisms of action.

Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing.

To use microRNAs to downregulate mRNA targets, cells must first process these ~22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.

Gene Architecture and Sequence Composition Underpin Selective Dependency of Nuclear Export of Long RNAs on NXF1 and the TREX Complex.

The core components of the nuclear RNA export pathway are thought to be required for export of virtually all polyadenylated RNAs. Here, we depleted different proteins that act in nuclear export in human cells and quantified the transcriptome-wide consequences on RNA localization. Different genes exhibited substantially variable sensitivities, with depletion of NXF1 and TREX components causing some transcripts to become strongly retained in the nucleus while others were not affected. Specifically, NXF1 is preferentially required for export of single- or few-exon transcripts with long exons or high A/U content, whereas depletion of TREX complex components preferentially affects spliced and G/C-rich transcripts. Using massively parallel reporter assays, we identified short sequence elements that render transcripts dependent on NXF1 for their export and identified synergistic effects of splicing and NXF1. These results revise the current model of how nuclear export shapes the distribution of RNA within human cells.

Transcription Dynamics Regulate Poly(A) Tails and Expression of the RNA Degradation Machinery to Balance mRNA Levels.

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.

SARS-CoV-2 uses a multipronged strategy to impede host protein synthesis.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-191. Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response2,3. Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression4-7, a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.