C. elegans cholinergic motor neurons (MNs), acts indirectly to prevent alternative neuronal identities . Here, we globally identify the direct targets of UNC-3. Unexpectedly, we find that the suite of UNC-3 targets in MNs is modified across different life stages, revealing ‘temporal modularity’ in terminal selector function. In all larval and adult stages examined, UNC-3 is required for continuous expression of various protein classes  critical for MN function. However, only in late larvae and adults, UNC-3 is required to maintain expression of MN-specific TFs. Minimal disruption of UNC-3’s temporal modularity via genome engineering affects locomotion. Another C. elegans terminal selector (UNC-30/Pitx) also exhibits temporal modularity, supporting the potential generality of this mechanism for the control of neuronal identity.Terminal selectors are transcription factors (TFs) that establish during development and maintain throughout life post-mitotic neuronal identity. We previously showed that UNC-3/Ebf, the terminal selector of  Nervous system development is a multi-step process that culminates in the generation of distinct neuron types necessary for animal behavior. Seminal studies in many model systems have begun to elucidate the molecular mechanisms that control the early steps of neuronal development, such as specification of neural progenitors and generation of post-mitotic neurons . HoweverTerminal selectors represent one class of transcription factors (TFs) with continuous expression – from development through adulthood – in specific neuron types . A definHowever, the molecular mechanisms through which terminal selectors establish and maintain neuronal identity are poorly understood, in part due to two major challenges. First, the majority of studies follow a candidate approach focused on a select subset of terminal identity genes . Hence, C. elegans ortholog of the Collier/Olf/Ebf (COE) family of TFs , which were identified via a candidate approach  and report the identification of ~3500 protein-coding genes as putative direct targets of UNC-3. Protein class ontology analysis suggests that UNC-3, besides terminal identity genes, also controls additional biological processes, such as neuronal metabolism and downstream gene regulatory networks comprised of numerous TFs and nucleic acid-binding proteins. These findings help obtain a comprehensive understanding of terminal selector function.unc-3 mutants at embryonic, larval and adult stages, we identified two groups of target genes with distinct temporal requirements for UNC-3 in cholinergic MNs. One group encodes multiple classes of proteins  that require UNC-3 for both embryonic initiation and post-embryonic maintenance of their expression. Contrasting this stable mode of regulation over time, a second group of targets consists exclusively of TFs  that do not require UNC-3 for initiation, but depend on UNC-3 activity for maintenance. Hence, the suite of UNC-3 targets in cholinergic MNs is modified across different life stages, a phenomenon we term ‘temporal modularity’ in terminal selector function. To provide mechanistic insights, we focused on the second group of targets and identified a molecular mechanism for their unc-3-independent initiation that relies on Hox proteins. Importantly, preventing UNC-3’s ability to selectively maintain expression of a single TF (cfi-1/Arid3a) from the second target group led to locomotion defects, indicating minimal disruption of temporal modularity affects animal behavior. Lastly, we provide evidence for temporal modularity in the function of UNC-30/PITX, the terminal selector of GABAergic MN identity in C. elegans  is unaffected in unc-3::gfp animals  because all unc-3 expressing neurons have been generated by this stage.To identify in an unbiased manner putative UNC-3 target genes, we employed chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq). We used a reporter strain with in-frame GFP sequences inserted immediately upstream of the stop codon of the endogenous c-3 gene . Expresse . Previous studies identified 10 terminal identity genes as putative direct UNC-3 targets and 43 terminal identity genes whose expression is affected by genetic removal of unc-3   . The majof unc-3 . In the of unc-3 . Moreoveneurons) . In concOur bioinformatic analysis of the UNC-3 binding peaks revealed 3502 protein-coding genes as putative UNC-3 targets . To classify these new targets, we performed gene ontology (GO) analysis focused on protein classes using PANTHER . This anC. elegans head and tail , a significant portion of the UNC-3-bound genes may be expressed in MNs. To test this, we used available single-cell RNA-Seq data (CeNGEN project: www.cengen.org) that identified 576 transcripts specifically enriched in cholinergic MNs   with expression in cholinergic MNs. Some of those are also expressed in the unc-3-negative GABAergic MNs of the nerve cord (nhr-49) is exclusively expressed in GABAergic MNs. Expression of five TFs  in ventral cord MNs has not been previously described. The remaining 5 TFs  are known to be expressed in subsets of unc-3-positive MNs  with expression in cholinergic MNs are positively regulated by unc-3; their expression significantly decreased in unc-3 mutants  , Table 1tive MNs , but our mutants . On the  mutants . Togethety genes , these func-3 dependency of terminal identity genes was mostly tested at a single developmental stage, the last larval stage (L4)  are conage (L4) . We theracr-2/AChR, unc-129/TGFbeta, glr-4/GluR, unc-17/VAChT) at larval  and adult (day 1) stages and found that their MN expression, at every stage, critically depends on UNC-3 , whether UNC-3 is continuously required to maintain expression of these genes remained unclear. We therefore employed the auxin-inducible degradation (AID) system to deplete the endogenous UNC-3 protein in cholinergic MNs at late larval and young adult stages  critically depends on UNC-3 at every stage   does not require UNC-3  and TFs  that require UNC-3 for both initiation and maintenance of expression  that depend on UNC-3 for maintenance, but not initiation  , suggestre UNC-3 . Howeverre UNC-3 . Hence, cfi-1/Arid3a) from the ‘maintenance-only’ module, aiming to dissect the molecular mechanisms underlying the temporal modularity of UNC-3 function in cholinergic MNs.Collectively, these findings suggest that, in cholinergic MNs, the suite of UNC-3 target genes can partially change at different life stages. In all larval and adult stages examined, UNC-3 is required for the continuous expression of one set of genes (‘initiation and maintenance’ module). However, only in late larvae and adults, UNC-3 is required to maintain expression of another set of genes (‘maintenance-only’ module). To describe this phenomenon, we use the term ‘temporal modularity in UNC-3 function’ given that the function of a TF in a specific cell type and at a particular life stage is determined by the suite of targets it controls in that cell type and at that stage . In the cis-regulatory analysis suggests that maintenance, but not initiation, of cfi-1 expression depends on UNC-3 (cfi-1 locus (located ~12 kb upstream) demarcates an enhancer element selectively required for maintenance  animals, with each reporter carrying small and contiguous DNA fragments spanning a ~15 kb region  at larval and adult stages  MNs  MNs , which wcfi-1, which also carries an auxin-inducible degron (AID) tag (mNG::AID::cfi-1), enabling inducible depletion of CFI-1 (depletion experiments are described later in Results). Animals carrying the mNG::AID::cfi-1 allele do not show any developmental phenotypes, suggesting that the mNG::AID tag does not detectably alter cfi-1 activity. This reporter showed expression in subsets of unc-3-expressing MNs , GABAergic nerve cord MNs , tail and head neurons, as well as head muscle (cfi-1 expression (cfi-1 reporter (mNG::AID::cfi-1) in MNs to be at the 3-fold embryonic stage, coinciding with the onset of transgenic reporters (#7 and #8) containing the distal enhancer (cfi-1 reporter (Δ enhancer (769 bp))mNG::AID::cfi-1. We found that mNG::AID::cfi-1 expression is selectively eliminated in cholinergic  and GABAergic  nerve cord MNs at all life stages examined   reporter allele for d muscle , a pattepression . We deteenhancer . Next, w1 adult) .cfi-1) is sufficient for cfi-1 expression in nerve cord MNs. Genome editing suggests that a 769 bp sequence within this 2.5 kb enhancer is required for both initiation and maintenance of cfi-1 in nerve cord MNs  by substituting nucleotides known to be critical for DNA binding of UNC-3 orthologs  and adult (day 1) stages, resembling the phenotype of unc-3 null mutants , or in partnership with other TFs . We foun mutants , excludicfi-1 expression in MNs? Previous work showed that two Hox proteins, LIN-39 (Scr/Dfd/HOX4-5) and MAB-5 (Antp/HOX6-8), control cfi-1 expression in MNs , larval  and adult (D1) stages  that carries the distal enhancer is significantly affected in lin-39; mab-5 double mutants at early larval (L2) stages  within the enhancer led to a decrease in reporter gene expression at L2  is mildly affected in lin-39 single mutants, but severely affected in lin-39; mab-5 double mutants  stages . Since oic stage , these rof unc-3 . Moreoveof unc-3  indicateenhancer . Express) stages . These ron at L2 . This de mutants , likely  mutants . We conclin-39::mNG::AID allele  mutants and crossed them to unc-3 null animals. Of note, Hox genes  and unc-3 do not cross-regulate their expression  led to a sustained decrease in reporter gene expression from L2 to adult stages  utilized by UNC-3 to maintain cfi-1  is maintained throughout life by Hox and UNC-3 , a terminal identity gene normally activated by UNC-3 in another MN subtype (SAB), becomes ectopically expressed in DA and DB neurons ,mNG::AID::cfi-1glr-4 expression in these neurons  that selectively affects maintenance, but not initiation, of cfi-1 expression in cholinergic MNs (cfi-1). As controls, we used animals carrying: (a) the endogenous cfi-1 reporter allele (mNG::AID::cfi-1), (b) a putative null cfi-1 allele (ot786) (cfi-1 activity is affected in MNs and other neuron types of the motor circuit (c) a deletion of the distal enhancer (Δenhancer(769 bp)mNG::AID::cfi-1), in which both initiation and maintenance of cfi-1 are abrogated in nerve cord MNs  severely affected in cfi-1 (ot786) putative null animals  display milder, but statistically significant locomotion defects in the adult (Δenhancer (769 bp)mNG::AID::cfi-1  allele, in which both initiation and maintenance of cfi-1 expression is affected. In summary, we specifically disrupted in cholinergic MNs the maintained expression of a single UNC-3 target (cfi-1 from the ‘maintenance-only’ module) by using the 8 COE motifs mutmNG::AID::cfi-1  allele and observed locomotion defects. This analysis suggests that minimal disruption of temporal modularity in UNC-3 function can affect animal behavior.Animals carrying  defects , likely  defects . Genes f defects . Hence, rgic MNs . This alrgic MNs , except  (ot786) , in whic circuit , and (c)cord MNs . We perfcord MNs . We foun animals . Comparehe adult . As expecis-regulatory analysis suggested that five TFs  require UNC-3 selectively for maintenance  and UNC-3 ensure the continuous expression of cfi-1 in subsets of unc-3-positive MNs  and prevents them from adopting a mixed identity (bnc-1::mNG::AID), we found that LIN-39 and MAB-5 are required for bnc-1 expression at all stages examined , albeit weaker effects were also observed at L2 and L4  expression is affected in GABAergic MNs of unc-30 mutants at all stages examined  , but progressively stronger effects at larval  and adult (day 1) stages  identity in the rve cord . UNC-30/sporter]), but a l[day 1]) , suggest[day 1]) , cfi-1 irgic MNs . Since U) stages , indicatrgic MNs , the funrgic MNs , suggestcis-regulatory region of terminal identity genes   , confirm47/VAGT) . UNC-30 rgic MNs . Howeverrgic MNs . CRISPR-rgic MNs . This fiiotropy’ , in whicC. elegans nerve cord. Through ChIP-Seq, we identify in an unbiased manner the direct targets of UNC-3, uncovering the breadth of biological processes potentially controlled by this terminal selector. Unexpectedly, we find two groups of target genes with distinct temporal requirements for UNC-3 in cholinergic MNs. One group encodes different classes of proteins  that require UNC-3 activation at all life stages examined. However, a second group exclusively encodes MN-specific TFs that selectively require UNC-3 for maintenance of their expression at late larval and adult stages. Hence, the suite of UNC-3 targets in cholinergic MNs is partially modified across different life stages, a phenomenon we term ‘temporal modularity’ in terminal selector function. Minimal disruption of this modularity by selectively removing the ability of UNC-3 to maintain expression of a single target gene (the TF cfi-1/Arid3a from the second group) led to locomotion defects, highlighting the necessity of temporal modularity for animal behavior.Terminal selectors are continuously expressed TFs that determine the identity and function of individual neuron types . HoweverC. elegans ventral nerve cord , specifically expressed in subsets of the six subtypes   are thought to act as transcriptional repressors to prevent the adoption of mixed MN identity  normally expressed in other MNs (cfi-1 (from module #2) during late larval and adult stages. In addition, UNC-3 acts directly to control DA and DB identity by ensuring the continuous expression of unc-129/TGFb, a DA- and DB-specific terminal identity gene (from module #1)  of the rve cord . Howeverrve cord  are exprrve cord , such asrve cord  and thisrve cord  escape iidentity . For exather MNs . Throughdule #1) . Hence, The mechanism of temporal modularity may represent a general paradigm of how terminal selectors establish and maintain subtype identity within a class of functionally related neurons. Indeed, the same terminal selector is often continuously expressed in multiple subtypes of a given neuronal class . In micecis-regulatory region of terminal identity genes, and thereby continuously activate their expression  , suggesting their function can be more dynamic than previously thought. This is based on the finding that UNC-3 is selectively required for maintenance, not initiation, of five conserved TF-encoding genes from module #2 ( nhr-40) . Hence, cfi-1/Arid3a) downstream of UNC-3, we identify a mechanism that potentially enables initiation and maintenance of module #2 genes  (cis-regulatory regions necessary for either initiation or maintenance of cell type-specific gene expression (By honing in on one TF (#2 genes . That isnd UNC-3 . Mechaninhancer) . This sopression .In summary, our findings critically extend the mechanisms underlying UNC-3 function. Previous work demonstrated that, in cholinergic MNs, UNC-3 not only activates expression of terminal identity genes , but alsDuring early neuronal development, Hox proteins are essential for cell survival, neuronal diversity, and circuit assembly . HoweverC. elegans mid-body region  in these neurons  stages  and unc-77/ human NALCN (sodium channel) in cholinergic MNs  stages  and 5G. rgic MNs . Altogetidentity .unc-3 display severe axon guidance defects in cholinergic MNs  can be modified over time, providing evidence for temporal modularity in their function. Given that terminal selectors, as well as other neuron type-specific TFs with continuous expression, have been described in both invertebrate and vertebrate species  plates supplied with d source . A list cis-regulatory analyses and validation of newly identified UNC-3 target genes were made with PCR fusion . Then, mutagenesis PCR was performed, and single clones containing plasmids that carry the desired mutation were isolated. PCR fusion DNA fragments were injected into young adult pha-1(e2123) hermaphrodites at 50 ng/µl together with pha-1 (pBX plasmid) as co-injection marker (50 ng/µl).Reporter gene fusions for R fusion . Genomicunc-3 (ot839 [unc-3::gfp]) worms and N2 worms were cultured on 10 cm plates seeded with OP50 at 20°C overnight. Late L2 worms were cross-linked and resuspended in FA buffer supplemented with protease inhibitors . For each IP experiment, 200 μl worm pellet was collected. The sample was then sonicated using a Covaris S220 at the following settings: 200 W Peak Incident Power, 20% Duty Factor, 200 Cycles per Burst for 1 min. Samples were transferred to centrifuge tubes and spun at the highest speed for 15 min. The supernatant was transferred to a new tube, and 5% of the material was saved as input and stored at −20°C. The remainder was incubated with 2 μl GFP antibody (Abcam Cat.# ab290) at 4°C overnight. Wild-type (N2) worms do not carry the GFP tag and serve as negative control. The unc-3 (ot839 [unc-3::gfp]) CRIPSR generated allele was used in order to immunoprecipitate the endogenous UNC-3 protein. On the next day, 20 μl Dynabeads Protein G (1004D) was added to the immunocomplex, which was then incubated for 2 hr at 4°C. The beads then were washed at 4°C twice with 150 mM NaCl FA buffer (5 min each), and once with 1M NaCl FA buffer (5 min). The beads were transferred to a new centrifuge tube and washed twice with 500 mM NaCl FA buffer (10 min each), once with TEL buffer  for 10 min, and twice with TE buffer (5 min each). The immunocomplex was then eluted in 200 μl elution buffer (1% SDS in TE with 250 mM NaCl) by incubating at 65°C for 20 min. The saved input samples were thawed and treated with the ChIP samples as follows. One (1) μl of 20 mg/ml proteinase K was added to each sample and the samples were incubated at 55°C for 2 hr then 65°C overnight (12–20 hr) to reverse cross-link. The immonuprecipitated DNA was purified with Ampure XP beads (A63881) according to manufacturer’s instructions.ChIP assay was performed as previously described  with theC. elegans genome (ce10) with bowtie2 mNG::AID::cfi-1 was generated by using two guide RNAs flanking the cfi-1 enhancer to guide excision of the genomic region, which was then followed by homology dependent repair (HDR) to create a 769 bp deletion . The UNC-3 binding motif mutation allele 8 COE motifs mutmNG::AID::cfi-1 was generated by creating nucleotide substitutions in the repair template, which carries homology arms complementary to the cfi-1 enhancer region and is then introduced into the genome through HDR.The 3, 100 mM) and mounting them on a 4% agarose pad on glass slides. Images were taken with an automated fluorescence microscope . Images containing several Z-stacks  were taken with Zeiss Axiocam 503 mono using the ZEN software . Representative images are shown following max-projection of 2–5 µm Z-stacks using the maximum intensity projection type. Image reconstruction was performed with Image J (Imaging slides were prepared by anesthetizing worms with sodium azide (NaN Image J .The identification of specific MN subtypes expressing a given UNC-3 target gene was assessed based on the following: (a) co-localization with fluorescent reporters that label specific MN subtypes; (b) Invariant position of neuronal cell bodies along the ventral nerve cord; (c) Birth order of specific MN subtypes (e.g. during embryogenesis or post-embryogenesis); (d) Total cell numbers in each MN subtype.http://cisbp.ccbr.utoronto.ca). To predict and identify LIN-39 binding motifs and UNC-3 binding motifs (identified in this paper) in the cfi-1 enhancer , we utilized tools provided by MEME (Multiple Expectation maximization for Motif Elicitation) bioinformatics suite (http://meme-suite.org/), and performed Find Individual Motif Occurrences (FIMO) motif scanning analysis.Information of the LIN-39 binding motif is curated in the Catalog of Inferred Sequence Binding Preferences database . To induce degradation of proteins , the following alleles were used: lin-39 (kas9 [lin-39::mNG::AID]), cfi-1 (kas16 [mNG::AID::cfi-1]), and unc-3 (ot837 [unc-3::mNG::AID]). Worms at specific developmental stages (see figure legends for details) were grown at 20°C on NGM plates coated with 4 nM auxin (indole-3-acetic acid [IAA] dissolved in ethanol) or ethanol (negative control) for 1 day or 4 days before tested (see figure legends for more details). All plates were shielded from light.Temporally controlled protein degradation was achieved with the AID system . TIR1 exE. coli OP50 as the food source. Worms were bleached and the eggs were allowed to hatch in M9 buffer to arrest as L1 larvae. L1 larvae were refed on OP50 and allowed to grow to day 2 of adulthood. On the day of tracking, five worms were picked from the incubated plates to each of the imaging plates (see below) and allowed to habituate for 30 min before recording for 15 min. Imaging plates are 35 mm plates with 3.5 mL of low-peptone (0.013% Difco Bacto) NGM agar  to limit bacteria growth. Plates are stored at 4°C for at least two days before use. Imaging plates are seeded with 50 μl of a 1:10 dilution of OP50 in M9 the day before tracking and left to dry overnight with the lid on at room temperature.Worms were maintained as mixed stage populations by chunking on NGM plates with All videos were analyzed using Tierpsy Tracker to extract each worm’s position and posture over time . These phttp://www.graphpad.com/quickcalcs/). Differences with p<0.05 were considered significant.For data quantification, graphs show values expressed as mean ± standard error mean (SEM) of animals. The statistical analyses were performed using the unpaired t-test (two-tailed). Calculations were performed using the GraphPad QuickCalcs online software  terminal genes and to other genes, engaging in a broad regulatory networks.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Chiou-Fen Chuang (Reviewer #2).Thank you for submitting your article "Establishment and maintenance of motor neuron identity via temporal modularity in terminal selector function" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Your paper addresses the important point of how transcription factors maintain the identity of the tissue they specify. In this case, how does UNC-3 maintain motor neuron identity: Does it retain the same targets though time or is are they dynamic?Your use of a ChIP time series allowed you to identify the gene targets in very few neurons, showing that UNC-3 binds to terminal genes and other genes, engaging in broad regulatory networks. You also showed, however, that some terminal genes might not always require direct UNC-3 input.It is important that you clarify the difference between target genes for which UNC-3 works as a terminal selector versus those where it only initiates activation and is not required for maintenance.The reviewers would therefore like you to examine whether UNC-3 is dispensable for reporter gene maintenance in some of the reporters. This should be a fairly easy experiment as you have UNC-3-AID worms where UNC-3 can be degraded after induction of gene expression. This point should also be discussed in detail in the paper.eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife, either of which would be linked to the original paper.Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in Reviewer #1:C. elegans model to investigate how UNC-3 maintains motor neuron identity. Specifically, the authors aim to address if UNC-3 maintains its targets though time or its binding and targets are dynamic.Understating how neurons keep their identity is an important question in developmental biology and neuroscience. Li and co-workers use a previously we-all established The authors use an appropriate method, a ChIP time series. It is technically challenging to obtain a time series for a gene expressed in a few neurons, and thus by itself is an achievement. UNC-3 binds to "classic" terminal genes but also other regulatory genes. This data suggest UNC-3 is, and perhaps all terminal selectors, engage in broader regulatory networks. The UNC-3 binding dynamics also revealed that some terminal genes might not require direct UNC-3 input at some developmental stages. Moreover, the authors find a possible "initiation module" for the motor neuron program that required UNC-3 for maintenance.cfi-1. Moreover, "it is unclear whether the expression of UNC-3 targets from the "maintenance-only" module is critical for locomotion". Following the authors' logic, it is not clear UNC-3 is a terminal selector because it might not be required to maintain cell fate. I believe it is good to challenge hypotheses, but the experiments must be designed to do so.I am less convinced the presented experiments support "Disruption of temporal modularity in UNC-3 function leads to locomotion defects", as the subtitle claims. The experiments are designed around the eLife publication. Thus, this reviewer believes it is appropriate as a Research Advance.The results are not itself conceptually novel as other temporal TF series have been described, even for motor neurons. However, the work is a significant addition to their previous Reviewer #2:Establishment and maintenance of motor neuron identity via temporal modularity in terminal selector function.C. elegans cholinergic motor neurons, using ChIP-seq. They found that UNC-3 is required for the initiation and maintenance of expression of one group of target genes, but UNC-3 is only required to maintain expression of motor neuron-specific transcription factors such as CFI-1. The authors also found that the Hox proteins LIN-39 and MAB-5 act synergistically to control initiation and maintenance of cfi-1 in cholinergic motor neurons through the same distal enhancer used by UNC-3 to maintain cfi-1 expression. The authors used the auxin-inducible protein degradation system to show that cfi-1 is required post-embryonically to maintain motor neuron subtype identity. In addition, a cfi-1 null mutation and deletion of the cfi-1 enhancer region cause locomotion defects. Furthermore, UNC-30, the terminal selector of GABAergic motor neurons, is required for the maintenance, but not initiation, of cfi-1 expression in GABAergic neurons. In summary, this study reveals temporal modularity in terminal selector function for motor neuron identity.In this paper, the authors identified direct targets of UNC-3/Ebf, the terminal selector of The paper is well written and figures are clearly organized.Reviewer #3:cfi-1. They identify an enhancer that recapitulates to large extent normal cfi-1 expression and provide evidence that the expression is initiated by Hox factors LIN-39 and MAB-5, while maintenance of the expression is controlled by UNC-3, possibly in conjunction with the Hox factors. Failure to maintain expression of CFI-1 in adult motor neurons has detrimental effects on motor neuron gene expression and worm motor behavior.The manuscript by Li et al. describes in depth analysis of UNC-3 target genes. The genes can be divided based on the UNC-3 role as initiation and maintenance genes and maintenance only. The authors focus on the regulation of one target transcription factor – The findings are supported by extensive experimental evidence and in depth genetic and biochemical analysis of the enhancer. Overall this is an interesting in depth analysis of regulatory elements that sheds new light on dynamic changes in transcriptional complexes controlling target gene expression. Below I list several points that the authors should consider during the revision of the manuscript.unc-3 early to turn the reporters on, followed by UNC-3 degradation. This raises the possibility that the first category can be subdivided into another subgroup – initiation only genes. Such possibility should be included in the Discussion.1) The authors place a lot of emphasis on division of target genes into two groups: initiation/ maintenance and maintenance only. However, the maintenance role in the first category has not been rigorously tested. That would require transient expression of 2) The complexity of expression patterns observed with the transgenic reporters , demonstrates that the studied enhancers are regulated by multiple additional transcription factors besides UNC-3 (e.g. MAB-5/LIN-39 shown later in the manuscript). Thus, the conclusion that "UNC-3 can also act directly to either activate or repress expression of multiple TF-encoding genes" needs to be reconsidered, as a likely scenario is that UNC-3 binding to the regulatory element facilitates recruitment of another transcription factor that functions as a strong repressor.3) Describing the results in Figure 4 the authors state: "Instead, it identified a 2.5 kb distal element (reporter #7) demarcated by the UNC-3 binding peak ". Isn't this the same fragment as the one already tested in Figure 3? Please clarify.cfi-1 reporter. Moreover, it seems that even the full 2.5kb fragment is not sufficient to completely recapitulate the kinetics of cfi-1 expression as the transgenic reporter is expressed in ~12 motor neurons  in L2 while cfi-1 endogenous reporter is on in ~25 neurons .4) The authors claim that 769bp sequence is necessary and sufficient for expression of reporter gene, however I do not see data testing the sufficiency of this particular fragment. Sufficiency is tested for construct #8 (552bp) that recapitulates only partially expression of cfi-1 enhancer in the absence of MAB-5 and LIN-39 or do these factors have to "pioneer" the enhancer for UNC-3 recruitment? Would replacement of the enhancer with a synthetic element that carries only multimeric UNC-3 binding sites suffice to induce expression of cfi-1 in cholinergic motor neurons?5) Does UNC-3 bind to the unc-3 mutants? And how about UNC-3 expression in lin-39/mab-5 mutants? The authors should consider the fate switch of motor neurons in unc-3 mutants that could negatively affect maintenance of many motor neuron genes indirectly and independent of the UNC-3 binding sites identified in their promoters.6) What is the status of MAB-5 and LIN-39 expression in glr-4 in adult motor neurons.7) The 8 COE motifs mut animals should be examined for derepression of  It is important that you clarify the difference between target genes for which UNC-3 works as a terminal selector versus those where it only initiates activation and is not required for maintenance.The reviewers would therefore like you to examine whether UNC-3 is dispensable for reporter gene maintenance in some of the reporters. This should be a fairly easy experiment as you have UNC-3-AID worms where UNC-3 can be degraded after induction of gene expression. This point should also be discussed in detail in the paper.We thank the reviewers for their constructive comments. Through additional experiments and text modifications, we now clarify in the revised manuscript how UNC-3 controls over time these two distinct sets of target genes . All new data are included and discussed in detail in the revised manuscript (as described below).Reviewer #1:[…]I am less convinced the presented experiments support "Disruption of temporal modularity in UNC-3 function leads to locomotion defects", as the subtitle claims. The experiments are designed around the cfi-1.Minimal disruption of temporal modularity in UNC-3 function leads to locomotion defects". As the reviewer correctly points out for this behavioral experiment, we only interfered with the ability of UNC-3 to maintain a single target gene (cfi-1) by using a CRISPR-engineered allele that bears mutations in the UNC-3 binding sites located at the distal enhancer of cfi-1 . Apart from these specific mutations that selectively affect cfi-1 maintenance , animals carrying this allele do not bear any other mutations in UNC-3 targets either from module #1 (targets that require UNC-3 for initiation and maintenance) or module #2 (targets that require UNC-3 for maintenance only). Hence, the disruption of temporal modularity in UNC-3 function is minimal because UNC-3 can control all those targets, except one. In the revised manuscript, we modified the text in the Results  to explain better our findings. Also, the phrase “minimal disruption…” appears in Abstract, Introduction, Results, and Discussion.We totally agree with the reviewer and modified the subtitle in the Results to "Moreover, "it is unclear whether the expression of UNC-3 targets from the "maintenance-only" module is critical for locomotion".Following the reviewer’s suggestion, we modified this sentence to:in cholinergic MNs, the maintained expression of any of the UNC-3 targets from the “maintenance-only” module is critical for locomotion”.“However, it is unclear whether, Following the authors' logic, it is not clear UNC-3 is a terminal selector because it might not be required to maintain cell fate. I believe it is good to challenge hypotheses, but the experiments must be designed to do so.unc-17/ VAChT, acr-2/AChR, glr-4/GluR) . In previous work , we showed that UNC-3 is also required to maintain expression of another terminal identity gene (unc-129/ TGFb) in cholinergic MNs. In the current manuscript, we further found – by using a null allele – that UNC-3 is required to establish the early expression of all these 4 terminal identity genes . Since the terminal selector definition entails that a TF is required to establish and maintain the expression of key effector molecules  in a specific neuron type, our findings support the idea that UNC-3 operates as a terminal selector in cholinergic MNs. These new data are discussed in the Results  and Discussion .We thank the reviewer for this comment, which enabled us to improve the manuscript in two ways. First, we used the auxin-inducible degradation (AID) system to selectively deplete UNC-3 in MNs at late larval/early adult stages. We found that UNC-3 is required to maintain the expression of 3 terminal identity genes  by explaining that our findings do support the traditional view of how terminal selectors work. We find that module #1 targets do require UNC-3 both for initiation and maintenance, in accordance with the terminal selector concept. At the same time, our findings expand our understanding of terminal selectors because we suggest that terminal selector function can be more dynamic than previously thought. This is based on the data on module #2 targets that selectively require UNC-3 for maintenance (not initiation), suggesting terminal selectors can gain additional targets at later life stages.The results are not itself conceptually novel as other temporal TF series have been described, even for motor neurons. However, the work is a significant addition to their previous eLife publication. Thus, this reviewer believes it is appropriate as a Research Advance.To help the reader, we clarify in the revised manuscript the potential significance of temporal modularity in terminal selector function and how it expands our understanding of how terminal selectors control neuronal identity .Reviewer #3:[…] Below I list several points that the authors should consider during the revision of the manuscript.1) The authors place a lot of emphasis on division of target genes into two groups: initiation/ maintenance and maintenance only. However, the maintenance role in the first category has not been rigorously tested. That would require transient expression of unc-3 early to turn the reporters on, followed by UNC-3 degradation. This raises the possibility that the first category can be subdivided into another subgroup – initiation only genes. Such possibility should be included in the Discussion.unc-17/ VAChT, acr-2/AChR, glr-4/GluR) . In previous work , we showed that UNC-3 is also required to maintain expression of another terminal identity gene (unc-129/ TGFb) in cholinergic MNs. In the current manuscript, we further show – by using a null allele – that UNC-3 is required to establish the early expression of all these 4 genes . Since the terminal selector definition entails that a TF is required to establish and maintain the expression of key effector molecules  in a specific neuron type, our findings support the idea that UNC-3 operates as a terminal selector in cholinergic MNs. These new data are discussed in the Results  and Discussion .We thank the reviewer for this important point. We performed new experiments using the auxin-inducible degradation (AID) system to selectively deplete UNC-3 in MNs at late larval/early adult stages. We found that UNC-3 is required to maintain the expression of 3 terminal identity genes (cfi-1 expression) expand our understanding of how terminal selectors control neuronal identity over time . As the reviewer correctly points out, UNC-3 could also be controlling additional targets in a different manner, for example, by only inducing but not maintaining their expression. This possibility is now mentioned in the Discussion (subsection “Limitations of this study”).Moreover, the identification of UNC-3 targets from module #2  The complexity of expression patterns observed with the transgenic reporters , demonstrates that the studied enhancers are regulated by multiple additional transcription factors besides UNC-3 (e.g. MAB-5/LIN-39 shown later in the manuscript). Thus, the conclusion that "UNC-3 can also act directly to either activate or repress expression of multiple TF-encoding genes" needs to be reconsidered, as a likely scenario is that UNC-3 binding to the regulatory element facilitates recruitment of another transcription factor that functions as a strong repressor.Cis-regulatory analysis reveals novel TFs as direct UNC-3 targets in motor neurons”). In addition, the section entitled “Hox proteins collaborate with stage-specific TFs to establish and maintain MN terminal identity” in the Discussion mentions that additional, yet-to-be-identified factors are involved to control expression of UNC-3 targets.We totally agree and have included this possibility in the Results (subsection “3) Describing the results in Figure 4 the authors state: "Instead, it identified a 2.5 kb distal element (reporter #7) demarcated by the UNC-3 binding peak ". Isn't this the same fragment as the one already tested in Figure 3? Please clarify.unc-3-expressing cholinergic MNs  at larval and adult stages , is also sufficient for embryonic (3-fold stage) MN expression .”Yes, it is the same fragment. We changed this confusing sentence to the following: “Instead, it showed that the same 2.5 kb distal element (reporter #7), that drives RFP expression in subsets of 4) The authors claim that 769bp sequence is necessary and sufficient for expression of reporter gene, however I do not see data testing the sufficiency of this particular fragment. Sufficiency is tested for construct #8 (552bp) that recapitulates only partially expression of cfi-1 reporter. Moreover, it seems that even the full 2.5kb fragment is not sufficient to completely recapitulate the kinetics of cfi-1 expression as the transgenic reporter is expressed in ~12 motor neurons  in L2 while cfi-1 endogenous reporter is on in ~25 neurons .cfi-1) is sufficient for cfi-1 expression in nerve cord MNs. Genome editing suggests that a 769 bp sequence within this 2.5kb enhancer is required for both initiation and maintenance of cfi-1 in nerve cord MNs ”.We apologize for the confusion. The revised manuscript now states: “We conclude that a distal 2.5kb enhancer (located ~12 kb upstream of 5) Does UNC-3 bind to the cfi-1 enhancer in the absence of MAB-5 and LIN-39 or do these factors have to "pioneer" the enhancer for UNC-3 recruitment? Would replacement of the enhancer with a synthetic element that carries only multimeric UNC-3 binding sites suffice to induce expression of cfi-1 in cholinergic motor neurons?lin-39 (-); mab-5 (-) double mutant animals at different developmental stages (embryos and larvae). Getting the required amount of animals for this experiment is extremely difficult, judging from our lab’s experience with ChIP-seq. We also attempted to address this question genetically by increasing the expression levels of UNC-3 in cholinergic motor neurons of lin-39 (-); mab-5 (-) double mutants. However, we were unable to generate the required transgenic animals for this experiment, despite repeated attempts.These are both excellent questions. The first one is technically very challenging as it would require us to perform an UNC-3 ChIP experiment on cfi-1 findings: we propose that the Hox proteins (LIN-39 and MAB-5) cooperate with UNC-3 to control cfi-1 expression in cholinergic motor neurons .To address the second question, we generated transgenic animals carrying 5 copies of the UNC-3 binding site fused to GFP. These animals failed to show GFP expression in cholinergic motor neurons at every life stage examined , suggesting that other factors are needed to cooperate with UNC-3 and activate its target genes. This scenario is in agreement with our 6) What is the status of MAB-5 and LIN-39 expression in unc-3 mutants? And how about UNC-3 expression in lin-39/mab-5 mutants?cfi-1 expression in cholinergic MNs through the same distal enhancer).In a previous study , we showed that the Hox proteins  and UNC-3 do not cross-regulate each other’s expression in cholinergic motor neurons. We added this important information in the Results (subsection “LIN-39 (Scr/Dfd/Hox4-5) and MAB-5 (Antp/Hox6-8) control The authors should consider the fate switch of motor neurons in unc-3 mutants that could negatively affect maintenance of many motor neuron genes indirectly and independent of the UNC-3 binding sites identified in their promoters.We are now mentioning this possibility in the Discussion (subsection “Limitations of this study”).7) The 8 COE motifs mut animals should be examined for derepression of glr-4 in adult motor neurons.glr-4/GluR expression in a subset of cholinergic MNs (~12 cells) that correspond to DA and DB subtypes. Animals carrying 8 COE motifs (UNC-3 sites) mutated in the context of the endogenous cfi-1 reporter allele (8 COE motifs mutmNG::AID::cfi-1) show a reduction (from ~30 to ~15 cfi-1+ cells), but not complete elimination, of cfi-1 expression in adult (Day 1) cholinergic MNs . This partial elimination is likely due to the presence of cryptic UNC-3 sites that we did not identify and therefore did not mutate in the 8 COE motifs mutmNG::AID::cfi-1 allele. Supporting this possibility, cfi-1 expression at Day 1 in unc-3 (-) null animals is more severely affected compared to 8 COE motifs mutmNG::AID::cfi-1 animals . Hence, the remaining expression of cfi-1 in ~ 15 MNs of 8 COE motifs mutmNG::AID::cfi-1 animals is likely the reason that we did not observe glr-4/GluR ectopic expression in MNs of these animals . We mention this reason in the figure legend.As mentioned in the Results, CFI-1 represses Δ enhancer (769 bp)mNG::AID::cfi-1 allele and completely lack cfi-1 expression in cholinergic motor neurons at all stages  do show glr-4/GluR expression in MNs to similar levels observed in cfi-1 (ot786) putative null animals . We mention this data in the Results .However, animals that carry the


We demonstrate that the abundance of POP-seq peaks increases with an increase in expression of lncRNAs, suggesting that highly expressed lncRNA are likely to act as sponges for RBPs, contributing to the rewiring of protein–RNA interaction network in cancer cells. Overall, our data supports POP-seq as a robust and cost-effective method that could be applied to primary tissues for mapping global protein occupancies.Interaction between proteins and RNA is critical for post-transcriptional regulatory processes. Existing high throughput methods based on crosslinking of the protein–RNA complexes and poly-A pull down are reported to contribute to biases and are not readily amenable for identifying interaction sites on non poly-A RNAs. We present Protein Occupancy Profile-Sequencing (POP-seq), a phase separation based method in three versions, one of which does not require crosslinking, thus providing unbiased protein occupancy profiles on whole cell transcriptome without the requirement of poly-A pulldown. Our study demonstrates that ~ 68% of the total POP-seq peaks exhibited an overlap with publicly available protein–RNA interaction profiles of 97 RNA binding proteins (RBPs) in K562 cells. We show that POP-seq variants consistently capture protein–RNA interaction sites across a broad range of genes including on transcripts encoding for transcription factors (TFs), RNA-Binding Proteins (RBPs) and long non-coding RNAs (lncRNAs). POP-seq identified peaks exhibited a significant enrichment ( Despite the increasingly appreciated role of protein–RNA interactions, the global occupancy profiles of proteins in a cellular environment is not fully elucidated. For instance dysregulated expression of RNA binding proteins (RBPs) has been associated with a broad spectrum of human pathologies including cancers, neurological and hereditary diseases6. Therefore, it is critical to investigate the diverse protein occupancy sites and their functional impact on physiology and diseases.Interaction of proteins with RNA is crucial for post-transcriptional gene regulation such as capping, splicing, polyadenylation and localization which is indispensable for cellular homeostasis and survival8. CLIP based methods exploit the stability of crosslinked protein–RNA complexes by ultraviolet (UV) irradiation followed by immunoprecipitation and sequencing of the co-purified RNA11. However, these methods are reported to contribute to biases in the interaction profiles due to the inherent nature of UV crosslinking14. Other methods that employ antibody pulldown of protein–RNA complexes such as RIP-seq and DO-RIP-seq17 are difficult to scale up for detecting hundreds of RNA interacting proteins at the same time. Methods like POPPI-seq18 and others have enabled the capture of protein–RNA interaction sites by incorporation of photoreactive nucleosides in UV irradiated cells followed by poly-A pull down and sequencing of the bound RNA. Although, the use of photoreactive nucleosides is known to induce cellular stress that may result in non-physiological protein–RNA interactions and thus limits their application to only in-vitro cultures8. In addition, poly-A pulldown requirement in these methods excludes their application to non-polyadenylated RNAs such as lncRNAs, miRNAs and histone mRNAs19. Several methods also employ formaldehyde crosslinking to capture protein–RNA complexes, however formaldehyde is also known to introduce biases by capturing non-specific interactions22. Therefore, there is a need to develop unbiased and cost-effective method that can map the global occupancy profiles of protein bound sites in a transcriptome wide manner with its application to diverse species of RNA.Experimental approaches such as crosslinking followed by immunoprecipitation (CLIP) have been widely used to identify the binding pockets of specific RBPs across the transcriptome25. Trizol extraction is deployed as a prevalent method to purify total RNA from the cell lysates. This involves solubilization of the biological material by phenol and guanidium isothiocyanate followed by chloroform induced phase separation. After the phase separation, proteins migrate to the organic phase, RNA migrates to the aqueous phase and the DNA/RNA–protein adducts are trapped in the interphase.Trizol based phase separation strategy has emerged as a robust technology that has expanded the identification of protein binding sites independent of the poly-A captureIn this study, we propose a method called POP-seq (Protein Occupancy Profile-sequencing) that incorporates a multi-step phase separation strategy using trizol, followed by high-throughput sequencing of small RNAs, without the requirement of poly-A pulldown. Current study reports three versions of POP-seq; NPOP-seq (no-crosslinking), FPOP-seq  and UPOP-seq (UV crosslinking), among which NPOP-seq can efficiently capture the interactions without crosslinking mediated biases under physiological conditions. Computational analysis of POP-seq data revealed a significant enrichment of clinically relevant somatic variants in the protein–RNA interaction sites. Further, this study also demonstrates that highly expressed lncRNAs act as sponges to titrate the abundance of RBPs thereby altering the protein–RNA regulatory networks. Overall, POP-seq is a robust and cost-effective method that can be utilized by researchers to capture the protein occupied sites on all RNA types.24. However, identification of precise protein occupied pockets across transcriptome remains obscure.We aimed to generate an unbiased transcriptome wide protein–RNA occupancy profiles using a trizol based phase separation method, POP-seq (Protein Occupancy Profile-sequencing) in K562 cells. Recent studies have demonstrated that phase separation using trizol yields abundance of RNA–protein interactions at the interphasePOP-seq is employed in three different versions: NPOP-seq (no crosslinking), FPOP-seq  and UPOP-seq (UV crosslinking) in K562 cells Fig. A. POP-se26 ‘plot correlation’ command. Our data showed a correlation of ~ 64%  between the replicates as shown in Figure 27 profiles of 97 RBPs from the ENCODE project28, with an end to end 50% peak overlap, revealed support for 68.2%, 67.3% and 66.4% in NPOP, FPOP and UPOP-seq respectively. Additionally, we observed that ~ 64% of the genes exhibited by POP-seq peaks are common across the three protocols  were sequenced to generate ~ 20 million reads each. We implemented our NGS pipeline to facilitate the analysis of the POP-seq data which includes quality control and read alignment followed by peak calling, resulting in the identification of 319,657, 288,129, and 320,310 unique peaks in the respective protocols  while a relatively lower proportion were mapped to intronic regions (~ 19%) and 3′UTR’s (~ 16%)  mapped to protein coding followed by ~ 28.4% lncRNA and ~ 18.6% snoRNA with respect to the total genes annotated in the human reference genome (hg38)  of POP-seq peaks with CLIP-seq peaks compared to ChIP-seq peaks as shown in Figure p value < 2.2e−26, Fishers Exact test) of POP-seq signals overlapping with the CLIP-seq profile than the ChIP-seq profile of these 18 proteins  To estimate the ribosomal protein interactions captured by POP-seq, we compared the POP-seq peaks with publicly available ribo-seq data in K562 cells. Our analysis showed that ~ 20% of the total POP-seq peaks (with peak length ≤ 50 bp) exhibited 50% end-to-end overlap with the ribo-seq peaks indicating that some ribosomal protein–RNA interactions are captured by POP-seq  To estimate non-RBP interactions, we compared the POP-seq peaks with ChIP-seq data of 67 proteins and CLIP-seq data of 79 proteins available for K562 cells (from ENCODE project). We observed a significantly higher overlap  DGCR8 , which is involved in microRNA processing and is implicated in the pathogenesis of cancer39 and (b) IGF2BP1 (insulin like growth factor 2 mRNA binding protein 1) which is a critical post-transcriptional regulator of various mRNA involved in cancer progression40. First, we identified the POP-seq peaks from the individual protocol that showed > 50% base-to-base overlap with eCLIP profile of respective RBP (obtained from ENCODE28). Next, we extracted the expression levels of exons ‘proximal’ (< 1000 bp) to overlapped peaks from CRISPR knock out data set . We observed that the cumulative expression level of ‘proximal’ exons was significantly dysregulated with respect to the non-targeting control. We observed that there was a significant reduction in the expression of ‘proximal’ exons in DGCR8 KO and a significant increase in IGF2BP1 KO with respect to their non-targeting CRISPR control 42 and a transcription factor, Jun43. ENO1 is a crucial glycolytic enzyme involved in cell growth and is also reported as an oncogene that promotes metastasis by facilitating cell proliferation in multiple cancers including colorectal, lung, and prostate cancer48. We observed the abundance of protein bound pockets in the genomic loci of ENO1 across the POP-seq protocols ols Fig. A. We als60. We found the occurrence of POP-seq peaks in Jun’s genomic boundary across all the three protocols  are reported as the most common form of somatic variations and are widely associated with metabolism, cell cycle regulation and DNA mismatch repair67 and Ensembl Variation database68  in POP-seq peaks than expected by chance (i.e. 5 random non-peak profiles) using Fisher’s exact test. For this analysis, random ‘non-peak’ files were generated as described previously. Our results indicate a significant enrichment for each SNP cohort with relatively lesser enrichment for GWAS SNP . Similar test for other genomic variations including PhenVar, SomaticVar and ClinVar indicated relatively higher enrichment  in POP-seq peaks compared to non-peaks  have been widely documented with diverse roles in the transcriptional and post-transcriptional regulation of gene expressionlls Fig. ", suggeslls Fig. A, irresplls Fig. B compare78. These studies proposed that lncRNA sponges can extensively rewire the post-transcriptional gene regulatory networks by altering the protein–RNA interaction landscape in cell-type and phenotype specific manner. Based on this finding we tested the hypothesis that highly expressed lncRNAs could sponge the regulatory RBPs that would further provide an insight into lncRNA mediated regulation of RBPs in disease context. We uncovered RP11 − 301G19.1, a highly expressed lncRNA in leukemia80, to illustrate the ‘sponge effect’ in K562 cells. We found an abundance of this lncRNA in AML patients from TCGA81 compared to its expression level in normal whole blood from GTEx cohort82  < 0.00059) prognostic impact in AML patients  exhibited 50% end-to-end overlap with the ribo-seq peaks and this fraction was significantly reduced with increase in % peak overlap as shown in supplementary Table Since ribosomal protein complexes constitute the basic translation machinery and hence are expected to be highly abundant in cells. To evaluate the prevalence of ribosomal protein occupied sites in our data, we compared POP-seq peaks with publicly available ribo-seq dataInterestingly, POP-seq provides evidence for the ‘sponge effect’ depicted by lncRNA on multiple RBPs thus advancing our understanding of the post-transcriptional regulatory mechanism controlled by lncRNAs interacting with RBPs in cancer. In summary, POP-seq is a cost-effective and robust approach to elucidate the binding sites of proteins in a transcriptome-wide manner. Thus, it should stand as a generic framework for mapping the global protein–RNA interactions, widening the scope and application of this technique to primary tissues for rapid profiling of protein occupancies.2 in a humidified incubator and fresh media was replenished every alternate day until confluent.K562 cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco’s minimal essential medium  supplemented with 10% heat-inactivated fetal bovine serum  along with 1% antibiotics . All cells were maintained at 37 °C and 5% CO2 dosage . Immediately after crosslinking, cells were collected in 15 ml tubes and pelleted at 1500 rpm for 5 min. Supernatant was discarded and cells were lysed in 1 ml trizol reagent (Life Technologies) by pipetting up and down to obtain a homogenous cell lysate.Cells were cultured in T-175 flask until a maximum of 90% confluency was reached. A total of 20 million cells per replicate of each sample were used for UV, formaldehyde, and no-crosslinking approaches. Cells were washed twice with 1X PBS, the supernatant was removed by pipetting and cells were resuspended in 1X PBS. For UV crosslinking, cells in PBS suspension were transferred to 100 mm dishes and UV irradiated at 254 nm with 400 mJ/cmFor Formaldehyde crosslinking, cells were first washed in 1X PBS twice until all the media is removed. Next, cells were resuspended in 30 ml PBS and crosslinked with 0.5% formaldehyde for 10 min by gentle shaking at room temperature. To stop crosslinking, 1 M Glycine was added to the cell suspension for 5 min by gentle shaking at room temperature. Cells were pelleted down at 1500 rpm for 5 min, lysed in 1 ml trizol and homogenized by pipetting up and down. For non-crosslinked samples, cells were pelleted down, washed in 1X PBS and immediately lysed in trizol for phase separation.g for 10 min at 4 °C to obtain three phases: aqueous phase (top), interphase (middle) and organic phase (bottom). The aqueous layer was discarded by pipetting and the organic layer was discarded by passing the tip through the interphase leaving behind up to 100 µl of the organic layer. The interphase was resolubilized in trizol followed by phase separation with chloroform three times. After the third phase separation, the interphase was precipitated by adding 1 ml methanol, spun down to remove supernatant containing methanol.Trizol lysed cells were incubated at room temperature for 5 min to dissociate the weak RNA–protein interactions. Phase separation was achieved by adding 200 µl chloroform and thoroughly mixed by vortexing, followed by incubation at room temperature for 5 min. Samples were then centrifuged at 12,000Following the trizol lysis and phase separation, POP-seq was implemented on the three versions in replicates. Interphase pellet was subjected to RNase A/T1 (Thermo Scientific) degradation in RNase buffer . 2 µg of RNase A/T1 mix was added to the interphase pellet, mixed by pipetting, and incubated at 37 °C for 1 h. Interphase-RNase mixture was resolubilized in trizol reagent to recover the RNA–protein complexes. The aqueous and organic layers were discarded as described previously. Interphase pellet was precipitated in 1 ml methanol. Next, the interphase was mixed with Proteinase K (Ambion) in appropriate buffer  and incubated at 50 °C for 2 h. After proteinase K digestion, free RNA was recovered from the aqueous layer by trizol extraction as described previously.Purified RNA concentration was estimated using Nanodrop and up to 1 µg of RNA was incubated with DNase I, 1U (Thermo Scientific) at 37 °C for 30 min to remove any traces of DNA contamination. 1 µl of 50 mM EDTA was added to the reaction mixture and incubated at 65 °C for 10 min to terminate the reaction. RNA was purified using trizol extraction from the aqueous layer. At this point, r-RNA depletion was performed with 1 µg input RNA using Ribo-cop kit (Lexogen) as per manufacturer instructions. Further, the ends of r-RNA depleted RNA were modified by treating with Calf intestine alkaline phosphatase  and T4 polynucleotide kinase  as per manufacturer protocol. The end modification enabled the library preparation of these RNA fragments.RNA purity and concentration were assessed at each step using Nanodrop, based on the absorbance ratio 260/280 > 2. RNA integrity was evaluated using Agilent 2100 bioanalyzer system. At least 50 ng of r-RNA depleted RNA was used to generate sequencing libraries using the True-seq small RNA library prep kit (Illumina). All libraries were barcoded and sequenced in parallel on a Next-seq platform for 400 million reads to obtain 75 bp single end reads.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and deployed FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) for removal of adapters and low quality read fragments wherever applicable. Next, we aligned the high quality reads onto human reference genome (GRCh38.p12) using HISAT94 followed by post processing using Samtools95. To ensure the reproducibility between the replicates, we compared the aligned reads per 10 kb genome using deepTools ‘plot correlation’ module26. We employed Piranha96 for peak calling with default parameters and obtained the resulting POP-seq peaks in bed format. Source code of the POP-seq data processing pipeline is accessible at GitHub (https://github.com/Janga-Lab/POP-seq). We merged the replicate bed files of respective POP-seq protocols and used several tools such as bedtools97, HOMER29 (annotatePeaks.pl), and R (https://www.r-project.org/) for annotation, statistical testing and other downstream analysis. We also downloaded the publicly available formaldehyde RNA ImmunoPrecipitation (fRIP-seq) data32 for 24 RBPs from GSE67963 and raw ribo-seq data93 from GSE125218, both generated in K562 cells and processed them using the same pipeline and identified the peaks. Next, we computed the fraction of POP-seq peaks overlapping with the identified peaks from both the datasets independently using bedtools97.We implemented NGS data processing pipeline to facilitate the analysis of the POP-seq data as shown in Fig. 98 profiles of 97 RBPs from ENCODE project28 and concatenated the unique coordinates into a bed file. Resulting coordinates of the binding sites of RBPs were compared with the POP-seq peaks using bedtools97. All POP-seq data have been deposited under Gene Expression Omnibus (GEO) accession number GSE142460. We also downloaded the raw FATSQ reads of total RNA (accession no. ENCLB822JYE from ENCODE project) and processed using the standard NGS pipeline as described in below section.Similarly, we downloaded the eCLIP28. We computed the overlap of POP-seq peaks with CLIP-seq peaks and ChIP-seq peaks using bedtools97. This dataset also includes 18 proteins for which both ChIP and CLIP-seq data is available for K562 cells. We generated 5 random peak profiles for each POP-seq protocols using bedtools ‘shuffle’ function. Each random peak profile contains equal but “non-peak” locations (with consistent peak width distribution), within the gene boundary. Then, we compared the POP-seq derived peaks (and the random peaks separately) with the ChIP and CLIP-seq profile of 18 proteins. We employed Fisher’s exact test to estimate the level of significance for the enrichment of POP-seq in protein bound DNA(ChIP-seq)/RNA(CLIP-seq) locations compared to non-peaks.We downloaded the ChIP-seq and CLIP-seq profile of available proteins for K562 cells from ENCODE project28 to facilitate the understanding of downstream biological processes associated to loss of function of the respective RBP. We downloaded the raw RNA-sequencing dataset of CRISPR experiments of DGCR8 (n = 6), IGF2BP1 (n = 2) where gRNAs were used to deplete the functional form of RBPs and their non-specific CRISPR control (n = 8) in K562 cells92. We processed the raw sequencing reads using standard NGS data analysis pipeline (as described previously). Briefly, we filtered the low-quality reads (Phred Score < 30) using FASTQC tool and aligned them onto human reference genome (hg38) using HISAT94. After post processing (using samtools), we used StringTie99 to quantify the expression levels in Transcripts Per Million (TPM) reads for all the genes annotated in human genome (hg38). Thereafter, we used an ad-hoc script to calculate the exon levels from the resulting gtf files of StringTie and converted the resulting files into an exon expression matrix.CRISPR/Cas9 Knock Down (KD) followed by expression profiling on several RBPs have been conducted as part of the ENCODE project100.To further investigate the functional relevance of protein–RNA interactions, we identified the POP-seq peaks from the individual protocols that exhibited at least 50% overlap with an eCLIP profile of the respective RBP (as described previously). Next, we extracted the expression levels of exons, proximal (< 1000 bp) to overlapped peaks, from exon expression matrix of the knock down experiments. We compared the distribution of the expression levels of these proximal exons in non-targeting control to that in respective KD and statistically examined the condition specific expression differences using Wilcoxon test101 i.e. by altering RNA secondary structure or TAM (Transcript associated mutation)102 that further contribute to transcriptome complexity in higher eukaryotes. Therefore, it is imperative to investigate the genomic variations occurring in protein–RNA interaction sites identified by POP-seq protocols. Hence, we downloaded the somatic variants reported in the GWAS catalog67 and Ensembl Variation database68 including phenotype and clinically associated somatic variations (ftp://ftp.ensembl.org/pub/release-97/variation/vcf/homo_sapiens/). In order to detect the somatic variations captured by POP-seq, we tested the enrichment of genomic variations from GWAS catalog67 and Ensembl Variation database68  in POP-seq peaks than expected by chance (i.e. 5 random non-peak profiles) using Fisher’s exact test. For this analysis, random ‘non-peak’ files were generated as described previously. To gain disease specific understanding of the role of SNPs in impacting protein–RNA interactions, we also investigated the POP-seq peaks overlapping with SNPs associated with leukemia (from GWAS) and generated genomic tracks in Integrative Genomics Viewer (IGV)103 for CDC20 and HDLBP genes.Several studies suggest that single nucleotide variations (SNVs) play an important role in gene regulation via riboSNitches’104 were extracted from the resulting matrix and averaged for each lncRNA across the replicates. Further, we binned all the expressed lncRNAs into two groups based on their median TPM value. We compared the number of POP-seq peaks  mapped to the two groups of lncRNAs categorized based on low and high median expression in K562 cells. The difference in normalized peak counts between the two groups was statistically tested using Wilcoxon test100.We mapped the protein–RNA interaction sites captured by POP-seq protocols onto known lncRNAs using bedtools. For each lncRNA, the number of POP-seq peaks normalized by respective gene length was calculated. To obtain the expression levels, we processed the raw RNA sequencing dataset  of K562 cells from ENCODE using a standard NGS data analysis pipeline described earlier and generated a gene expression matrix. TPM values of known lncRNAs105 and 174 AML patient samples from The Cancer Genome Atlas (TCGA)81. We processed the dataset using the NGS data processing pipeline, to generate expression levels for all human genes annotated in the human genome (hg38). We extracted the expression level of lncRNA—RP11 − 301G19.1 (ENSG00000227706) from the two groups; AML and GTEx ‘whole blood’. The difference in expression levels was statistically examined between the two groups using Wilcoxon test. We also calculated the patient’s survival over time using the expression levels of this lncRNA in AML patients using the Kaplan–Meier method implemented in ‘The survival’, an R package83. We generated a genomic track for this lncRNA using IGV and illustrated the regulatory sites identified by POP-seq. We re-investigated the genomic coordinates of this gene in SliceIt36 and added a panel to enable all the possible regulatory sites captured by eCLIP of RBPs in ENCODE project28.Additionally, we downloaded the raw RNA sequencing dataset of ‘whole blood’ cohort from 141 individuals from the GTExSupplementary Legends.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Tables.


In this review, we will discuss these findings, candidate chromatin architectural long non-coding RNAs and possible mechanisms by which RNA, along with RNA binding proteins might be mediating chromatin organization.RNA plays a well-established architectural role in the formation of membraneless interchromatin nuclear bodies. However, a less well-known role of RNA is in organizing chromatin, whereby specific RNAs have been found to recruit chromatin modifier proteins. Whether or not RNA can act as an architectural molecule for chromatin remains unclear, partly because dissecting the architectural role of RNA from its regulatory role remains challenging. Studies that have addressed RNA's architectural role in chromatin organization rely on  Of the three components of the central dogma , RNA is the most versatile molecule. RNA functions universally as the genetic messenger and as transfer RNAs that deliver amino acids to the site of protein synthesis on the RNA-scaffolded ribosome. In addition, ribozymes catalyze certain specific biochemical reactions, small RNAs (∼20–30 nucleotides) silence gene expression via transcriptional, post-transcriptional as well as chromatin-dependent gene silencing pathways and a functionally diverse class of >200 nucleotides long non-coding RNAs (lncRNAs) play roles in gene expression, genomic imprinting and genome organization . AnotherRNA is an excellent architectural molecule due to its ability to form extensive secondary structures which provide surfaces for various RNA binding proteins (RBPs) thereby forming RNA–RBP scaffolds. These RNA–RBP architectural scaffolds are well known to form nuclear bodies, such as nucleoli, nuclear speckles and nuclear stress bodies, in the interchromatin space where RNA is highly abundant. Removal of RNA leads to collapse of these nuclear bodies providing clear evidence for the architectural role of RNA in formation of these structures . A signiHere, we will discuss the potential role of RNA in maintaining the native structure of chromatin. RNA was shown to be a structural component of the nuclear matrix, a seemingly filamentous structure that spans the interchromatin space in electron microscopy images. However, the existence of the nuclear matrix as a discrete structure is now considered to have been a fixation artifact, as such structures were not visualized in living cells or using high-resolution cryo-electron tomography ,26. In cA significant fraction of RNA, mostly non-coding, has also been found to associate with chromatin ,32,33. Rin situ assays rely on in situ RNA depletion using RNase A , which become inactive after cells have been permeabilized with a detergent. In addition, both cytological visualization and in situ conformation capture assays (discussed below) preserve overall nuclear structures and can be analyzed in presence or absence of RNA.Each of the studies that have addressed the architectural contribution of RNA in chromatin integrity by either cytological visualization or other  RNase A  36–38]..in situ in situ RNase A treatment as compared with the control untreated cells [One of the earliest reports of an architectural role of RNA came from observing RNase A treated Dinoflagellate cells/nuclei . Dinoflaed cells . The foued cells . Bindinged cells . SUV39H1ed cells . These fin situ depletion of RNA by RNase A treatment reduces centromere binding of CENP-C significantly while leaving CENP-A levels unaffected [in vivo and not in situ RNA depletion alters CENP-A binding suggests that α-satellite transcripts might play both architectural and regulatory roles.Possible architectural roles of specific transcripts have also been investigated for the binding of chromatin components at centromeres, DNA loci that mediate chromosome segregation by assembling a multproteinaceous structure called kinetochore, which then binds to spindles ,46. Centaffected . These rin situ chromosome conformation capture technique has been applied to test for a chromatin architectural role for RNA. Hi-C captures 3-D interactions by ligating proximal genomic regions and can be combined with in situ RNase digestion to probe the effect of total RNA depletion on genome organization [Recently, the Hi-C nization . Hi-C ofnization ,53. Hi-Cnization . These rAlthough a small subset of total lncRNAs are known to play essential roles, many of them are involved in chromatin regulation by recruiting chromatin modifying proteins ,55 Tabl. LncRNAsThe best-studied candidate chromatin architectural lncRNA is XIST, which coats the entire inactive X-chromosome (Xi) in therian female mammals . XIST exExact molecular mechanisms whereby lncRNAs might function as chromatin architectural elements remain speculative. Some lncRNAs such as HOTAIR that interact with two different chromatin modifying protein complexes, might act as bridges between two chromatin contact points . RNA–RNAKd < 1 nM) and is known to interact with thousands of transcripts  [LncRNAs also impact the 3-D organization of mammalian genomes by facilitating the function of architectural proteins involved in chromatin looping. The multiple zinc-finger architectural protein CCCTC-binding factor (CTCF) acts as an insulator between facultative heterochromatin and euchromatin and organizes them into spatially disjoint domains . CTCF alnd XITE) ,92. CTCFnd XITE) .in vitro reconstituted H3K9me-containing nucleosome arrays and bridges two H3K9me nucleosomes [in vitro is only modest relative to the massive compaction at pericentric regions seen in vivo, suggesting that additional factors are required to condense H3K9me heterochromatin in the nucleus [Histone H3K9 methyl-marked constitutive heterochromatin is predominantly formed on repetitive DNA and is bound to the heterochromatin protein HP1 . HP1 conleosomes ,97. The  nucleus . Interes nucleus . Drosoph nucleus . In situ nucleus . Further nucleus . Chromoc nucleus . These s nucleus .Components of membraneless RNA-filled nuclear bodies do not mix with the nucleoplasm because they undergo liquid-liquid phase separation (LLPS). Nuclear bodies, the best known examples of LLPS in the nucleus to date, phase-separate from the nucleoplasm due to multivalent weak interactions between RNA and RBPs ,104,105.in vitro [in vitro, and SAF-B knockdown results in a dispersed localization of pericentric heterochromatin suggesting that SAF-B-mediated phase separation may contribute to chromocenter condensates [in vivo heterochromatin LLPS research is at an early stage and it remains to be determined how liquid droplets that form spontaneously from pure recombinant heterochromatin components in vitro, are regulated in vivo where one or more phase separating proteins (e.g. HP1 and SAF-B around pericentric regions) are present in a crowded environment of a given region. A recent investigation of biochemical and biophysical properties of in vivo HP1 foci showed a lack of LLPS behavior attributable to HP1 [in vivo constitutive heterochromatin is much weaker when compared with nuclear bodies, and that the majority of the properties of heterochromatin can be explained by a classic condensed polymer model in which heterochromatin can be percolated with nucleoplasm.Recent studies have attributed LLPS behavior to heterochromatin domains as well . Componein vitro . SAF-B adensates . Unlike e to HP1 . This suin vitro and interact with several RNAs [in vitro and in vivo LLPS behavior of HP1 and SAF-B was shown to be enhanced by pericentric MajSat RNA [To the extent that heterochromatin exhibits a degree of LLPS behavior, it is possible that architectural RNA is involved. HP1 and CBX2, respectively components of constitutive and facultative heterochromatin that compact nucleosome arrays, exhibit LLPS behavior ral RNAs ,117,118.jSat RNA ,119. TheThe ability of RNA to participate in multivalent interactions via its sequence and secondary structures makes it an excellent architectural molecule, as is evident from its scaffolding role for nuclear bodies . Architein situ and that map RNA-chromatin interactions, such as GRID-seq, PIRCh-seq and CUT&RUN [A next step in understanding the roles of architectural RNAs in maintaining chromatin structure is to apply genome-scale approaches that deplete specific RNAs from the intact nucleus  CUT&RUN . CharactUnderstanding the role of RNA mediated compaction in the formation of heterochromatic foci such as chromocenters in the mouse nucleus should shed light on the possible role of LLPS in heterochromatin condensation.Finally, RNA components of heterochromatin assemblies might provide sequence specificity to RBPs in heterochromatin, including CENPs, HP1, SAF-B and PRC1, and understanding the mechanistic basis for these interactions is an important goal of chromatin research.


Many intricate pathways contribute to the timely control of gene expression during development. Polycomb repressive complexes (PRC1 and PRC2) and long non-coding RNAs (lncRNAs) are players associated with gene repression in various developmental processes such as X chromosome inactivation (XCI) and genomic imprinting. Historically, lncRNAs were proposed to directly recruit PRC2. However, recent evidence suggests that promiscuous interactions between PRC2 and RNA fine-tune the function of the complex through a multiplicity of mechanisms. A PRC2-recruitment model was definitively overturned in the paradigm of XCI by Xist RNA, being replaced by a novel mechanism which puts PRC1 in the spotlight. This review focuses on these recent advances in understanding the interplay between RNA and Polycomb complexes for gene expression control. Current Opinion in Genetics and Development 2020, 61:53–61Genome architecture and expressionThis review comes from a themed issue on Kerstin Bystricky and Matthias MerkenschlagerEdited by Issue and the EditorialFor a complete overview see the Available online 11th May 2020https://doi.org/10.1016/j.gde.2020.02.023http://creativecommons.org/licenses/by/4.0/).0959-437X/© 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license  which catalyses H2AK119ub1, and Polycomb repressive complex 2 (PRC2) which methylates lysine 27 of histone H3 (H3K27me1/2/3) (reviewed in Ref. .•.+ nucleTaken together, recent evidence argues against the idea that RNA binding contributes to PRC2 recruitment. Instead, RNA binding to PRC2 fine tunes its activity, keeping PRC2 poised and in check as previously proposed ,32. Thisin vivo models and a paradigm for functional associations between Polycomb and lncRNA. After years of debate, there is an emerging consensus of how Xist RNA recruits Polycomb through a mechanism that seems to be common to other lncRNAs.The recruitment of Polycomb complexes and their respective post-translational histone modifications are hallmarks of X chromosome inactivation (XCI) seen to occur rapidly in response to Xist RNA expression. Alongside other integral pathways , 49, 50,As noted above, early models for Xist-mediated Polycomb recruitment invoked a direct interaction of the PRC2 subunit EZH2 with the A-repeat element of Xist RNA , leadingA series of studies, using a variety of models and techniques , have noA landmark study showed a strict requirement for PRC1 upstream of PRC2 redefining the hierarchy of Polycomb complex recruitment in XCI and moreover defined a key role for the non-canonical PCFG3/5-PRC1 complex ••. The nRecent experiments in PRC1 (RING1A/B) knockout fibroblasts, in which XCI is already established, show a reduction but not erasure of H3K27me3 domains •. This oFrom RNA-sequencing it has also become clear that in cellular models of Xist-induced gene repression the functional effect of Polycomb removal is significant but not absolute  46•,57•••,59•,60Ring1A/B, Eed or deletion of Xist B-repeat region [It is well-documented that initial gain of Polycomb modifications correlates with Xist spreading over the Xi ,69, and t region •. These t region ••,72, alin cis by two lncRNAs paternally expressed in trophoblast stem cells, Airn and Kncq1ot1 [The emergence of the new model for recruitment of Polycomb by Xist RNA has led to silencing mechanisms downstream of other lncRNAs previously reported to functionally interact with PRC2 being revisited. One recent example profiled accumulation of Polycomb over Mb-long genomic imprinted regions controlled Kncq1ot1 ••. Of noKncq1ot1 ••, implyIn recent years the model for Polycomb recruitment by RNA has been overturned, with roles for PRC1 and hnRNPK emerging as key determinants. Consideration of this mechanism may be useful for redefining other previously reported functional interactions between lncRNAs and Polycomb. One such example is HOTAIR, where a recent study which tethered HOTAIR to a reporter found that PRC2 deletion had no effect on its ability to mediate transcriptional silencing . HoweverAlthough RNA does not seem to directly recruit PRC2, there is evidence to support a function for RNA in regulating PRC2 activity. Future work will need to establish how these RNA-associated mechanisms are integrated with others, such as H2AK119ub1 and DNA binding, to establish appropriate Polycomb repression during development.Nothing declared.• of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as


While the important functions of long noncoding RNAs (lncRNAs) in nuclear organization are well documented, their orchestrating and architectural roles in the cytoplasmic environment have long been underestimated. However, recently developed fractionation and proximity labelling approaches have shown that a considerable proportion of cellular lncRNAs is exported into the cytoplasm and associates nonrandomly with proteins in the cytosol and organelles. The functions of these lncRNAs range from the control of translation and mitochondrial metabolism to the anchoring of cellular components on the cytoskeleton and regulation of protein degradation at the proteasome. In the present review, we provide an overview of the functions of lncRNAs in cytoplasmic structures and machineries und discuss their emerging roles in the coordination of the dense intracellular milieu. It is becoming apparent that further research into the functions of these lncRNAs will lead to an improved understanding of the spatiotemporal organization of cytoplasmic processes during homeostasis and disease. The RNA pool of mammalian cells primarily consists of ribosomal RNA, followed by other types of RNA, such as messenger RNA (mRNA), transfer RNA (tRNA), small nuclear RNA (snRNA) or microRNA (miRNA). A common feature of these RNA classes is their association with distinct cellular machineries and RNA maturation pathways . Long noIndividual ncRNAs exceeding the 200 nt length threshold were already discovered and characterized as early as in the 1980s and recognized as critical components of diverse cellular machineries. Examples are the signal recognition particle (SRP) RNA, which serves as a scaffold for SRP assembly , the RMRThe relatively loose definition and variety of different molecular functions raises the question of whether subgroups of lncRNAs, which can be mechanistically assigned to specific subcellular machineries or organelles, should be separated from the generic term “lncRNAs” and given their own function-related designations. In support of this view, multiple lncRNA subgroups with characteristic sedimentation rates can be discriminated on density gradients . At presRecent studies have questioned the classic narrative that lncRNAs mostly act in the nucleus. Using subcellular fractionation techniques, a large percentage of lncRNAs was found to be exported to the cytoplasm and, at least in some cell types, the number of predominantly cytoplasmic lncRNAs seems to outnumber the nuclear-enriched lncRNAs ,15,16. TBesides random ribosomal scanning events or active translation of micro-ORFs, the association of lncRNAs with ribosomes may also be attributed to regulatory interactions, serving the adjustment of mRNA translation. LincRNA-p21 for instance, was reported to associate with JUNB and CTNNB1 mRNAs to lower their translation through recruitment of the translational repressor RCK . SimilarTaken together, besides occasional ribosomal scanning or productive translation, several cytoplasmic lncRNAs associate with active or assembling ribosomes  and posiMitochondria are not only essential to cellular energy balance but also participate in cell death programs and in signal transduction in response to environmental changes. Upon detection of pathogens by intracellular innate immune receptors or during removal of damaged mitochondria for instance, proteins such as MAVS or OPTN establish signaling platforms on the mitochondrial outer membrane, which trigger TBK1 kinase dependent type I interferon immune responses and autophagy, respectively . Severalf indicated that all three lncRNAs form RNA duplexes [By systematic transcriptome profiling, three lncRNAs were found to be encoded in the mitochondrial DNA, complementary to the ND5, ND6 and CytB mRNAs. Expression of these mitochondria-retained lncRNAs, termed lncND5, lncND6 and lncCytB, depends on their processing by mitochondrial RNaseP. Resistance to the ssRNA-specific nuclease RNase Iduplexes . lncND5,duplexes , which, duplexes ,49. Howeduplexes . Nuclearduplexes . Mutatioduplexes . Anotherduplexes . Mechaniduplexes . During duplexes . Thus, mduplexes  suggestsWhile several lncRNAs are known to act in mitochondria, little is known about the potential roles of lncRNAs in other organelles, such as the endoplasmic reticulum (ER) or the Golgi apparatus. Several lncRNAs were reported to act in their periphery, e.g., regulating ER-dependent autophagy  or ER-stBesides their association with vesicles and membranes of intracellular organelles, several lncRNAs were shown to interact with phospholipids or adherence-junction proteins at the plasma membrane . To systBesides organelles, separated from the cytosol by intracellular membranes, the role of liquid–liquid phase-separation in compartmentalization of cellular processes is gaining increasing attention. Phase separation is a cellular means of delimiting biochemical reactions in the intracellular milieu in liquid-like droplets, which contribute to subcellular organization . Stress By systematically profiling the sedimentation-rates of lncRNAs on glycerol-gradients, 22 sub-groups of human phagocyte lncRNAs could be discriminated, cosedimenting with protein-components of diverse subcellular machineries. Interestingly, a prominent subgroup, comprising dozens of lncRNAs, was found to comigrate with components of the ubiquitin-proteasome system (UPS) . The UPSNumerous other lncRNAs have been characterized, which interact with a variety of cytoplasmic components and machineries. Several lncRNAs for instance interact with cytoskeletal components. LncRNA Dreh binds to vimentin and regulates cytoskeleton structure in tumor cells . LncRNA In recent years, significant methodological advances have been made enabling the systematic mapping of lncRNA binding substrates and lncRNA subcellular localization patterns. Questioning the classic narrative, multiple studies suggest that lncRNAs do not primarily locate to the nucleus but also appear in the cytoplasm in large quantities ,15,16. TPreliminary work on stress-granules suggests that lncRNAs might have an architectural function during cytoplasmic phase separation. Specifically, lncRNA NORAD is enriched in stress granules and their PUMILIO-protein-dependent formation seems to depend at least partially on RNA. This is reminiscent of the requirement of architectural RNAs, such as NEAT1 or HSATIII for the assembly of phase-separated nuclear bodies ,96. A fuWhile assembly of large protein structures such as the ribosome or proteasome results in a near-equilibrium state, the maintenance of highly dynamic structures, such as the cytoskeleton, requires constant energy input . Therefo