Over the last decade, it has been increasingly demonstrated that the genomes of many species are pervasively transcribed, resulting in the production of numerous long noncoding RNAs (lncRNAs). At the ...same time, it is now appreciated that many types of DNA regulatory elements, such as enhancers and promoters, regularly initiate bi-directional transcription. Thus, discerning functional noncoding transcripts from a vast transcriptome is a paramount priority, and challenge, for the lncRNA field. In this review, we aim to provide a conceptual and experimental framework for classifying and elucidating lncRNA function. We categorize lncRNA loci into those that regulate gene expression in cis versus those that perform functions in trans and propose an experimental approach to dissect lncRNA activity based on these classifications. These strategies to further understand lncRNAs promise to reveal new and unanticipated biology with great potential to advance our understanding of normal physiology and disease.
Identifying functional noncoding transcripts within a complex transcriptome remains a challenge. This review explores examples of known functional lncRNAs and then presents a framework for unlocking biological insights from new lncRNAs of interest.
Disease is often the result of an aberrant or inadequate response to physiologic and pathophysiologic stress. Studies over the last 10 years have uncovered a recurring paradigm in which microRNAs ...(miRNAs) regulate cellular behavior under these conditions, suggesting an especially significant role for these small RNAs in pathologic settings. Here, we review emerging principles of miRNA regulation of stress signaling pathways and apply these concepts to our understanding of the roles of miRNAs in disease. These discussions further highlight the unique challenges and opportunities associated with the mechanistic dissection of miRNA functions and the development of miRNA-based therapeutics.
Liquid-liquid phase separation is a major mechanism of subcellular compartmentalization
. Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism
, whether and ...how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins
. We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs
suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.
MicroRNAs (miRNAs) encoded by the miR-17-92 cluster and its paralogs are known to act as oncogenes. Expression of these miRNAs promotes cell proliferation, suppresses apoptosis of cancer cells, and ...induces tumor angiogenesis. New work reveals essential functions for these miRNAs not only in tumor formation but also during normal development of the heart, lungs, and immune system.
Antisense oligonucleotides (ASOs) that trigger RNase-H-mediated cleavage are commonly used to knock down transcripts for experimental or therapeutic purposes. In particular, ASOs are frequently used ...to functionally interrogate long noncoding RNAs (lncRNAs) and discriminate lncRNA loci that produce functional RNAs from those whose activity is attributable to the act of transcription. Transcription termination is triggered by cleavage of nascent transcripts, generally during polyadenylation, resulting in degradation of the residual RNA polymerase II (Pol II)-associated RNA by XRN2 and dissociation of elongating Pol II. Here, we show that ASOs act upon nascent transcripts and, consequently, induce premature transcription termination downstream of the cleavage site in an XRN2-dependent manner. Targeting the transcript 3′ end with ASOs, however, allows transcript knockdown while preserving Pol II association with the gene body. These results demonstrate that the effects of ASOs on transcription must be considered for appropriate experimental and therapeutic use of these reagents.
Display omitted
•ASOs induce cleavage of nascent transcripts•ASO-mediated cleavage of nascent RNAs induces premature transcription termination•XRN2 is required for ASO-mediated premature transcription termination•Targeting the transcript 3′ end with ASOs avoids premature transcription termination
Antisense oligonucleotide (ASO)-mediated transcript knockdown is commonly used to interrogate the function of long noncoding RNAs (lncRNAs). Here, Lee and Mendell demonstrate that ASOs induce cleavage of nascent transcripts and thereby trigger premature transcription termination. Thus, these reagents cannot be used to establish an RNA-mediated function for a lncRNA locus.
Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is ...induced after DNA damage, which we termed “noncoding RNA activated by DNA damage”, or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500–1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.
Display omitted
•NORAD is a broadly expressed, highly abundant, and conserved mammalian lncRNA•Inactivation of NORAD in human cells triggers dramatic aneuploidy•NORAD functions as a potent molecular decoy for PUMILIO proteins (PUM1/PUM2)•PUM1/PUM2 repress a program of genes necessary to maintain genomic stability
NORAD, a conserved, abundant, and broadly expressed long noncoding RNA, preserves genome stability by serving as a molecular decoy for PUMILIO proteins. Without NORAD, PUMILIO hyperactivity leads to aneuploidy in otherwise karyotypically normal cells.
MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs ...encounter highly complementary targets, which can trigger a process called target-directed miRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. We identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adaptor ZSWIM8, that mediates TDMD. The ZSWIM8 CRL interacts with AGO proteins, promotes TDMD in a tailing and trimming-independent manner, and regulates miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.
MicroRNAs (miRNAs) post-transcriptionally repress gene expression by guiding Argonaute (AGO) proteins to target mRNAs. While much is known about the regulation of miRNA biogenesis, miRNA degradation ...pathways are comparatively poorly understood. Although miRNAs generally exhibit slow turnover, they can be rapidly degraded through regulated mechanisms that act in a context- or sequence-specific manner. Recent work has revealed a particularly important role for specialized target interactions in controlling rates of miRNA degradation. Engagement of these targets is associated with the addition and removal of nucleotides from the 3′ ends of miRNAs, a process known as tailing and trimming. Here we review these mechanisms of miRNA modification and turnover, highlighting the contexts in which they impact miRNA stability and discussing important questions that remain unanswered.
Degradation of Argonaute (AGO) proteins by the ubiquitin-proteasome system and modification of miRNA 3′ ends by tailing and trimming are mechanisms that can promote global or miRNA-specific turnover.The consequences of tailing and trimming of small RNAs vary depending on the class of small RNA, the identity of the nucleotides that are added to the small RNA 3′ end, and the species in which the modifications occur.Tailing and trimming appears to regulate the stability of plant miRNAs, animal siRNAs, and animal piRNAs, but usually not animal miRNAs.Target-directed miRNA degradation (TDMD), which is triggered by extensive base-pairing of both the miRNA 5′ and 3′ end with a target, appears to be the major mechanism of regulated turnover of animal miRNAs.The ZSWIM8 ubiquitin ligase mediates TDMD by promoting the destruction of AGO:miRNA complexes engaged with highly complementary targets.
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.
PDF
