Archa Fox

Genome-wide analysis of long noncoding RNA stability.

Publisher: Cold Spring Harbor Laboratory

Date: 09-03-2012

DOI: 10.1101/GR.131037.111

Abstract: Transcriptomic analyses have identified tens of thousands of intergenic, intronic, and cis -antisense long noncoding RNAs (lncRNAs) that are expressed from mammalian genomes. Despite progress in functional characterization, little is known about the post-transcriptional regulation of lncRNAs and their half-lives. Although many are easily detectable by a variety of techniques, it has been assumed that lncRNAs are generally unstable, but this has not been examined genome-wide. Utilizing a custom noncoding RNA array, we determined the half-lives of ∼800 lncRNAs and ∼12,000 mRNAs in the mouse Neuro-2a cell line. We find only a minority of lncRNAs are unstable. LncRNA half-lives vary over a wide range, comparable to, although on average less than, that of mRNAs, suggestive of complex metabolism and widespread functionality. Combining half-lives with comprehensive lncRNA annotations identified hundreds of unstable (half-life 2 h) intergenic, cis -antisense, and intronic lncRNAs, as well as lncRNAs showing extreme stability (half-life 16 h). Analysis of lncRNA features revealed that intergenic and cis -antisense RNAs are more stable than those derived from introns, as are spliced lncRNAs compared to unspliced (single exon) transcripts. Subcellular localization of lncRNAs indicated widespread trafficking to different cellular locations, with nuclear-localized lncRNAs more likely to be unstable. Surprisingly, one of the least stable lncRNAs is the well-characterized paraspeckle RNA Neat1 , suggesting Neat1 instability contributes to the dynamic nature of this subnuclear domain. We have created an online interactive resource ( stability.matticklab.com ) that allows easy navigation of lncRNA and mRNA stability profiles and provides a comprehensive annotation of ∼7200 mouse lncRNAs.

Nuclear matrix binding is critical for progesterone receptor movement into nuclear foci

Publisher: Wiley

Date: 17-10-2008

DOI: 10.1096/FJ.08-113639

Abstract: The ovarian hormone progesterone is essential for normal breast development, and progesterone analogues are implicated in increasing breast cancer risk. The progesterone receptor (PR) is a transcription factor that, when ligand activated, moves rapidly into nuclear foci associated with transcriptional activity. However, the role of intranuclear trafficking signals in the focal location of PR is unknown. We have identified a mutation in PR that ablates its binding to the nuclear matrix and prevents PR movement into nuclear foci. Nuclear matrix binding mutants lack transcriptional activity and inhibit dimerization, demonstrating the critical role of matrix binding for PR dynamics and activity. DNA binding of PR is required for fidelity of location in foci, as DNA binding domain (DBD) mutants form aberrant foci with reduced mobility and altered tethering to the nucleus. Mutations in either the nuclear matrix targeting sequence or DBD domains were dominant in preventing wild-type receptor from moving to appropriate nuclear locations, demonstrating that both partner proteins in a PR dimer must have intact intranuclear trafficking signals for correct receptor positioning within the nucleus. This study has demonstrated that positioning of PR in foci within the nucleus is critically regulated by intranuclear trafficking signals, which play a key role in transcriptional activity and are relevant to its action in normal and malignant breast cells.

Functional dissection of NEAT1 using genome editing reveals substantial localization of the NEAT1_1 isoform outside paraspeckles

Publisher: Cold Spring Harbor Laboratory

Date: 21-03-2017

DOI: 10.1261/RNA.059477.116

Abstract: Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed “microspeckles,” which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.

SOLUTION STRUCTURE OF THE FIRST ZINC FINGER FROM THE DROSOPHILA U-SHAPED TRANSCRIPTION FACTOR

Publisher: Worldwide Protein Data Bank

Date: 04-10-2000

DOI: 10.2210/PDB1FV5/PDB

Confined environments induce polarized paraspeckle condensates

Publisher: Springer Science and Business Media LLC

Date: 03-02-2023

DOI: 10.1038/S42003-023-04528-4

Abstract: Cancer cells experience confinement as they navigate the tumour microenvironment during metastasis. Recent studies have revealed that the nucleus can function as a ‘ruler’ for measuring physical confinement via membrane tension, allowing for compression-sensitive changes in migration. Cell nuclei contain many nuclear bodies that form when their components phase separate and condense within permissive local regions within the nucleus. However, how sub-nuclear organisation and phase separation changes with cell confinement and compression is largely unknown. Here we focus on paraspeckles, stress-responsive subnuclear bodies that form by phase separation around the long non-coding RNA NEAT1. As cells entered moderate confinement, a significant increase in paraspeckle number and size was observed compared to unconfined cells. Paraspeckle polarization bias towards the leading edge was also observed in confinement, correlating with regions of euchromatin. Increasing paraspeckle abundance resulted in increases in confined migration likelihood, speed, and directionality, as well as an enhancement of paraspeckle polarization towards the leading edge. This polarization of paraspeckle condensates may play a key role in regulating confined migration and invasion in cancer cells, and illustrates the utility of microchannel-based assays for identifying phenomena not observed on 2D or 3D bulk substrates.

A class of zinc fingers involved in protein-protein interactions

Publisher: Wiley

Date: 02-2000

DOI: 10.1046/J.1432-1327.2000.01095.X

Abstract: Zinc fingers (ZnFs) are extremely common protein domains. Several classes of ZnFs are distinguished by the nature and spacing of their zinc-coordinating residues. While the structure and function of some ZnFs are well characterized, many others have been identified only through their amino acid sequence. A number of proteins contain a conserved C-X2-C-X12-H-X1-5-C sequence, which is similar to the spacing observed for the 'classic' CCHH ZnFs. Although these domains have been implicated in protein-protein (and not protein-nucleic acid) interactions, nothing is known about their structure or function at a molecular level. Here, we address this problem through the expression and biophysical characterization of several CCHC-type zinc fingers from the erythroid transcription factor FOG and the related Drosophila protein U-shaped. Each of these domains does indeed fold in a zinc-dependent fashion, coordinating the metal in a tetrahedral manner through the sidechains of one histidine and three cysteine residues, and forming extremely thermostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG-F1 in which the final cysteine residue is replaced by histidine remains capable of folding. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GATA-1 binding activity, demonstrating that CCHC and CCHH topologies are not interchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of protein-protein interactions has implications for the prediction of protein function from nucleotide sequences.

Structural basis of dimerization and nucleic acid binding of human DBHS proteins NONO and PSPC1

Publisher: Oxford University Press (OUP)

Date: 14-12-2021

DOI: 10.1093/NAR/GKAB1216

Abstract: The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified ‘β-clasp’ structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets.

Structure of the heterodimer of human NONO and paraspeckle protein component 1 and analysis of its role in subnuclear body formation

Publisher: Proceedings of the National Academy of Sciences

Date: 13-03-2012

DOI: 10.1073/PNAS.1120792109

Abstract: Proteins of the Drosophila behavior/human splicing (DBHS) family include mammalian SFPQ (PSF), NONO (p54nrb), PSPC1, and invertebrate NONA and Hrp65. DBHS proteins are predominately nuclear, and are involved in transcriptional and posttranscriptional gene regulatory functions as well as DNA repair. DBHS proteins influence a wide gamut of biological processes, including the regulation of circadian rhythm, carcinogenesis, and progression of cancer. Additionally, mammalian DBHS proteins associate with the architectural long noncoding RNA NEAT1 (Men ε/β) to form paraspeckles, subnuclear bodies that alter gene expression via the nuclear retention of RNA. Here we describe the crystal structure of the heterodimer of the multidomain conserved region of the DBHS proteins, PSPC1 and NONO. These proteins form an extensively intertwined dimer, consistent with the observation that the different DBHS proteins are typically copurified from mammalian cells, and suggesting that they act as obligate heterodimers. The PSPC1/NONO heterodimer has a right-handed antiparallel coiled-coil that positions two of four RNA recognition motif domains in an unprecedented arrangement on either side of a 20-Å channel. This configuration is supported by a protein:protein interaction involving the NONA araspeckle domain, which is characteristic of the DBHS family. By examining various mutants and truncations in cell culture, we find that DBHS proteins require an additional antiparallel coiled-coil emanating from either end of the dimer for paraspeckle subnuclear body formation. These results suggest that paraspeckles may potentially form through self-association of DBHS dimers into higher-order structures.

A crystallographic study of human NONO (p54nrb): overcoming pathological problems with purification, data collection and noncrystallographic symmetry

Publisher: International Union of Crystallography (IUCr)

Date: 25-05-2016

DOI: 10.1107/S2059798316005830

Abstract: Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54 nrb ) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible s le aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P 2 1 with a unique axis ( b ) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P 1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P 1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L -test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased s le-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.

Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins

Publisher: Elsevier BV

Date: 04-2018

DOI: 10.1074/JBC.RA117.001451

NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma

Publisher: Springer Science and Business Media LLC

Date: 10-09-2020

DOI: 10.1007/S00018-020-03632-6

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

Publisher: Rockefeller University Press

Date: 17-08-2015

DOI: 10.1083/JCB.201504117

Abstract: Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration.

Directed Proteomic Analysis of the Human Nucleolus

Publisher: Elsevier BV

Date: 2002

DOI: 10.1016/S0960-9822(01)00650-9

Abstract: The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

Paraspeckles

Publisher: Cold Spring Harbor Laboratory

Date: 23-06-2010

DOI: 10.1101/CSHPERSPECT.A000687

Key residues characteristic of GATA N-fingers are recognized by FOG

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1074/JBC.273.50.33595

Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing

Publisher: Cold Spring Harbor Laboratory

Date: 18-10-2018

DOI: 10.1101/446773

Abstract: Oligonucleotides and nucleic acid analogues that alter gene expression are showing therapeutic promise for selected human diseases. The modification of synthetic nucleic acids to protect against nuclease degradation and to influence drug function is common practice, however, such modifications may also confer unexpected physicochemical and biological properties. Here we report backbone-specific effects of modified oligonucleotides on subnuclear organelles, altered distribution of nuclear proteins, the appearance of novel structured nuclear inclusions, and modification of RNA processing in cultured cells transfected with antisense oligonucleotides on a phosphorothioate backbone. Phosphodiester and phosphorodiamidate morpholino oligomers elicited no such consequences. Disruption of subnuclear structures and proteins elicit severe phenotypic disturbances, revealed by transcriptomic analysis of fibroblasts exhibiting such disruption. These data suggest that the toxic effects and adverse events reported after clinical evaluation of phosphorothioate nucleic acid drugs may be mediated, at least in part, by non-specific interaction of nuclear components with the phosphorothioate backbone.

The oestrogen receptor alpha-regulated lncRNA NEAT1 is a critical modulator of prostate cancer

Publisher: Springer Science and Business Media LLC

Date: 21-11-2014

DOI: 10.1038/NCOMMS6383

Abstract: The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα-specific non-coding transcriptome signature. Among putatively ERα-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.

PolyA-modulating antisense oligonucleotides reveal opposing functions for long non-coding RNA NEAT1 isoforms in neuroblastoma

Publisher: Cold Spring Harbor Laboratory

Date: 05-2020

DOI: 10.1101/2020.05.01.071696

Abstract: Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One ex le is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2 araspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell-proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.

Distinct Roles of DBHS Family Members in the Circadian Transcriptional Feedback Loop

Publisher: Informa UK Limited

Date: 11-2012

DOI: 10.1128/MCB.00334-12

Paraspeckles: Where Long Noncoding RNA Meets Phase Separation

Publisher: Elsevier BV

Date: 02-2018

DOI: 10.1016/J.TIBS.2017.12.001

Abstract: Long noncoding RNA (lncRNA) molecules are some of the newest and least understood players in gene regulation. Hence, we need good model systems with well-defined RNA and protein components. One such system is paraspeckles - protein-rich nuclear organelles built around a specific lncRNA scaffold. New discoveries show how paraspeckles are formed through multiple RNA-protein and protein-protein interactions, some of which involve extensive polymerization, and others with multivalent interactions driving phase separation. Once formed, paraspeckles influence gene regulation through sequestration of component proteins and RNAs, with subsequent depletion in other compartments. Here we focus on the dual aspects of paraspeckle structure and function, revealing an emerging role for these dynamic bodies in a multitude of cellular settings.

Caenorhabditis elegansNONO‐1: Insights into DBHS protein structure, architecture, and function

Publisher: Wiley

Date: 29-10-2015

DOI: 10.1002/PRO.2816

Abstract: Members of the Drosophila behavior/human splicing (DBHS) protein family have been characterized in the vertebrates Homo sapiens and Mus musculus , and the invertebrates Drosophila melanogaster and Chironomus tentans . Collectively, both vertebrate and invertebrate DBHS proteins function throughout gene regulation, largely but not always, within the nucleus. In this study, we report a structural and bioinformatic analysis of the DBHS protein family to guide future studies into DBHS protein function. To explore the structural plasticity of the family, we describe the 2.4 Å crystal structure of Caenorhabditis elegans non‐POU domain‐containing octamer‐binding protein 1 (NONO‐1). The structure is dimeric, with a domain arrangement consistent with mammalian DBHS proteins. Comparison with the DBHS structures available from H. sapiens reveals that there is inherent domain flexibility within the homologous DBHS region. Mapping amino acid similarity within the family to the NONO‐1 dimer highlights the dimer interface, coiled‐coil oligomerization motif, and putative RNA binding surfaces. Surprisingly, the interior surface of RNA recognition motif 2 (RRM2) that faces a large internal void is highly variable, but the external β2–β3 loops of RRM2 show remarkable preservation. Overall, the DBHS region is under strong purifying selection, whereas the sequences N‐ and C‐terminal to the DBHS region are less constrained. The findings described in this study provide a molecular basis for further investigation into the mechanistic function of the DBHS protein family in biology.

SOLUTION STRUCTURE OF THE NINTH ZINC-FINGER DOMAIN OF THE U-SHAPED TRANSCRIPTION FACTOR

Publisher: Worldwide Protein Data Bank

Date: 04-10-2000

DOI: 10.2210/PDB1FU9/PDB

NEAT1 long noncoding RNA regulates transcription via protein sequestration within subnuclear bodies

Publisher: American Society for Cell Biology (ASCB)

Date: 2014

DOI: 10.1091/MBC.E13-09-0558

Abstract: Paraspeckles are subnuclear structures formed around nuclear paraspeckle assembly transcript 1 (NEAT1)/MENε/β long noncoding RNA (lncRNA). Here we show that paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded paraspeckle proteins. Of interest, however, using immuno–electron microscopy, we find that key paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1 −/− fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into paraspeckles.

P54nrb Forms a Heterodimer with PSP1 That Localizes to Paraspeckles in an RNA-dependent Manner

Publisher: American Society for Cell Biology (ASCB)

Date: 11-2005

DOI: 10.1091/MBC.E05-06-0587

Abstract: P54nrb is a protein implicated in multiple nuclear processes whose specific functions may correlate with its presence at different nuclear locations. Here we characterize paraspeckles, a subnuclear domain containing p54nrb and other RNA-binding proteins including PSP1, a protein with sequence similarity to p54nrb that acts as a marker for paraspeckles. We show that PSP1 interacts in vivo with a subset of the total cellular pool of p54nrb. We map the domain within PSP1 that is mediating this interaction and show it is required for the correct localization of PSP1 to paraspeckles. This interaction is necessary but not sufficient for paraspeckle targeting by PSP1, which also requires an RRM capable of RNA binding. Blocking the reinitiation of RNA Pol II transcription at the end of mitosis with DRB prevents paraspeckle formation, which recommences after removal of DRB, indicating that paraspeckle formation is dependent on RNA Polymerase II transcription. Thus paraspeckles are the sites where a subset of the total cellular pool of p54nrb is targeted in a RNA Polymerase II-dependent manner.

Construct optimization for studying protein complexes: obtaining diffraction-quality crystals of the pseudosymmetric PSPC1–NONO heterodimer

Publisher: International Union of Crystallography (IUCr)

Date: 19-10-2011

DOI: 10.1107/S0907444911039606

Different low-complexity regions of SFPQ play distinct roles in the formation of biomolecular condensates

Publisher: Cold Spring Harbor Laboratory

Date: 30-11-2022

DOI: 10.1101/2022.11.30.518278

Abstract: Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism governing the organisation of molecules within the cell. Long disordered low complexity regions (LCRs) are a common feature of proteins that form biomolecular condensates. RNA-binding proteins with prion-like composition have been highlighted as drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. Here, we show that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell. In contrast, we find that, unexpectedly, the longer N-terminal prion-like LCR of SFPQ attenuates condensation, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. Our data add nuance to the emerging understanding of biomolecular condensate formation, by providing the first ex le of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation.

RNA binding is essential for NONO condensates to modulate pre-mRNA processing of super enhancer-associated genes in neuroblastoma

Publisher: Cold Spring Harbor Laboratory

Date: 28-02-2022

DOI: 10.1101/2022.02.28.482217

Abstract: High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA binding protein, NONO, in neuroblastoma is associated with poor patient outcome, however there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biophysical and molecular analysis to reveal complex NONO-dependent regulation of gene expression, finding that NONO forms RNA- and DNA-tethered phase-separated condensates throughout the nucleus. CLIP analyses show that NONO mainly binds to the 5’ end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA binding activity. NONO preferentially regulates super enhancer-associated genes, including HAND2 and GATA2. In the absence of functional NONO-RNA condensates, inefficient pre-mRNA processing at these loci leads to decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA binding activity of NONO may have therapeutic potential in this cancer context.

Paraspeckles: nuclear bodies built on long noncoding RNA

Publisher: Rockefeller University Press

Date: 31-08-2009

DOI: 10.1083/JCB.200906113

Abstract: Paraspeckles are ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei. These structures play a role in regulating the expression of certain genes in differentiated cells by nuclear retention of RNA. The core paraspeckle proteins (PSF/SFPQ, P54NRB/NONO, and PSPC1 [paraspeckle protein 1]) are members of the DBHS (Drosophila melanogaster behavior, human splicing) family. These proteins, together with the long nonprotein-coding RNA NEAT1 (MEN-ε/β), associate to form paraspeckles and maintain their integrity. Given the large numbers of long noncoding transcripts currently being discovered through whole transcriptome analysis, paraspeckles may be a paradigm for a class of subnuclear bodies formed around long noncoding RNA.

Paraspeckle nuclear condensates: Global sensors of cell stress?

Publisher: Wiley

Date: 22-03-2021

DOI: 10.1002/BIES.202000245

SPArking Interest in the Long Noncoding RNA World: A New Class of 5′ SnoRNA-Stabilized LncRNA that Influences Alternative Splicing

Publisher: Elsevier BV

Date: 11-2016

DOI: 10.1016/J.MOLCEL.2016.10.026

Abstract: Delving deeply into the locus deleted in Prader-Willi syndrome, in this issue of Molecular Cell, Wu et al. (2016) identify SPA RNAs, a new class of 5' snoRNA-capped lncRNAs that sequester RNA binding proteins and influence alternative splicing.

Extracting, Enriching, and Identifying Nuclear Body Sub-Complexes Using Label-Based Quantitative Mass Spectrometry

Publisher: Springer New York

Date: 05-12-2014

DOI: 10.1007/978-1-4939-2253-6_13

Abstract: Determining the proteome of a nuclear body is a crucial step toward understanding its function however, it is extremely challenging to obtain pure nuclear body preparations. Moreover, many nuclear proteins dynamically associate with multiple bodies and subnuclear compartments, confounding analysis. We have found that a more practical approach is to carry out affinity purification of nuclear body sub-complexes via the use of tagged nuclear-body-specific marker proteins. Here we describe in detail the method to identify new nuclear body protein sub-complexes through SILAC (stable isotope labeling by amino acids in culture)-based affinity purification followed by quantitative mass spectrometry.

Protocadherin 19 (PCDH19) interacts with paraspeckle protein NONO to co-regulate gene expression with estrogen receptor alpha (ERα)

Publisher: Oxford University Press (OUP)

Date: 17-03-2017

DOI: 10.1093/HMG/DDX094

The structure of human SFPQ reveals a coiled-coil mediated polymer essential for functional aggregation in gene regulation

Publisher: Oxford University Press (OUP)

Date: 12-03-2015

DOI: 10.1093/NAR/GKV156

Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1016/J.MOLCEL.2018.05.019

Abstract: A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.

Effects of a Novel Long Noncoding RNA, lncUSMycN, on N-Myc Expression and Neuroblastoma Progression

Publisher: Oxford University Press (OUP)

Date: 06-2014

DOI: 10.1093/JNCI/DJU113

Abstract: Patients with neuroblastoma due to the lification of a 130-kb genomic DNA region containing the MYCN oncogene have poor prognoses. Bioinformatics data were used to discover a novel long noncoding RNA, lncUSMycN, at the 130-kb licon. RNA-protein pull-down assays were used to identify proteins bound to lncUSMycN RNA. Kaplan-Meier survival analysis, multivariable Cox regression, and two-sided log-rank test were used to examine the prognostic value of lncUSMycN and NonO expression in three cohorts of neuroblastoma patients (n = 47, 88, and 476, respectively). Neuroblastoma-bearing mice were treated with antisense oligonucleotides targeting lncUSMycN (n = 12) or mismatch sequence (n = 13), and results were analyzed by multiple comparison two-way analysis of variance. All statistical tests were two-sided. Bioinformatics data predicted lncUSMycN gene and RNA, and reverse-transcription polymerase chain reaction confirmed its three exons and two introns. The lncUSMycN gene was co lified with MYCN in 88 of 341 human neuroblastoma tissues. lncUSMycN RNA bound to the RNA-binding protein NonO, leading to N-Myc RNA upregulation and neuroblastoma cell proliferation. High levels of lncUSMycN and NonO expression in human neuroblastoma tissues independently predicted poor patient prognoses (lncUSMycN: hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.06 to 3.28, P = .03 NonO: HR = 2.48, 95% CI = 1.34 to 4.57, P = .004). Treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice statistically significantly hindered tumor progression (P < .001). Our data demonstrate the important roles of lncUSMycN and NonO in regulating N-Myc expression and neuroblastoma oncogenesis and provide the first evidence that lification of long noncoding RNA genes can contribute to tumorigenesis.

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions

Publisher: Elsevier BV

Date: 11-2000

DOI: 10.1016/S0969-2126(00)00527-X

Abstract: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.

Nuclear bodies: news insights into structure and function

Publisher: Elsevier BV

Date: 06-2017

DOI: 10.1016/J.CEB.2017.05.001

Abstract: The cell nucleus contains a number of different dynamic bodies that are variously composed of proteins and generally, but not always, specific RNA molecules. Recent studies have revealed new understanding about nuclear body formation and function in different aspects of nuclear metabolism. Here, we focus on findings describing the role of nuclear bodies in the biogenesis of specific ribonucleoprotein complexes, processing of key mRNAs, and subnuclear sequestration of protein factors. We highlight how nuclear bodies are involved in stress responses, innate immunity and tumorigenesis. We further review organization of nuclear bodies and principles that govern their assembly, highlighting the pivotal role of scaffolding noncoding RNAs, and liquid-liquid phase separation, which are transforming our picture of nuclear body formation.

Paraspeckles

Publisher: Elsevier BV

Date: 2002

DOI: 10.1016/S0960-9822(01)00632-7

Abstract: The cell nucleus contains distinct classes of subnuclear bodies, including nucleoli, splicing speckles, Cajal bodies, gems, and PML bodies. Many nuclear proteins are known to interact dynamically with one or other of these bodies, and disruption of the specific organization of nuclear proteins can result in defects in cell functions and may cause molecular disease. A proteomic study of purified human nucleoli has identified novel proteins, including Paraspeckle Protein 1 (PSP1) (see accompanying article, this issue of Current Biology). Here we show that PSP1 accumulates in a new nucleoplasmic compartment, termed paraspeckles, that also contains at least two other protein components: PSP2 and p54/nrb. A similar pattern of typically 10 to 20 paraspeckles was detected in all human cell types analyzed, including primary and transformed cells. Paraspeckles correspond to discrete bodies in the interchromatin nucleoplasmic space that are often located adjacent to splicing speckles. A stable cell line expressing YFP-PSP1 has been established and used to demonstrate that PSP1 interacts dynamically with nucleoli and paraspeckles in living cells. The three paraspeckle proteins relocalize quantitatively to unique cap structures at the nucleolar periphery when transcription is inhibited. We have identified a novel nuclear compartment, termed paraspeckles, found in both primary and transformed human cells. Paraspeckles contain at least three RNA binding proteins that all interact dynamically with the nucleolus in a transcription-dependent fashion.

hFOG-2, a novel zinc finger protein, binds the co-repressor mCtBP2 and modulates GATA-mediated activation.

Publisher: Elsevier BV

Date: 08-1999

DOI: 10.1074/JBC.274.33.23491

Highly Ordered Spatial Organization of the Structural Long Noncoding NEAT1 RNAs within Paraspeckle Nuclear Bodies

Publisher: American Society for Cell Biology (ASCB)

Date: 15-11-2010

DOI: 10.1091/MBC.E10-08-0690

Abstract: Paraspeckles (PSPs) are nuclear bodies associated with the retention in the nucleus of specific mRNAs. Two isoforms of a long noncoding RNA (NEAT1_v1/Menε and NEAT1_v2/Menβ) are required for the integrity of PSPs. Here, we analyzed the molecular organization of PSPs by immuno- and in situ hybridization electron microscopy. Detection of the paraspeckle markers PSPC1 and P54NRB/NONO confirm the identity between PSPs and the previously described interchromatin granule-associated zones (IGAZs). High-resolution in situ hybridization of NEAT1 transcripts revealed a highly ordered organization of IGAZ/PSPs. Although the 3.7-kb NEAT1_v1 and the identical 5′ end of the 22.7-kb NEAT1_v2 transcripts are confined to the periphery, central sequences of NEAT1_v2 are found within the electron-dense core of the bodies. Moreover, the 3′ end of NEAT1_v2 also localize to the periphery, indicating possible architectures for IGAZ/PSPs. These results further suggest that the organization of NEAT1 transcripts constrains the geometry of these bodies. Accordingly, we observed in HeLa and NIH 3T3 cells that IGAZ/PSPs are elongated structures with a well-defined diameter. Our results provide new insight on the ability of noncoding RNAs to form subcellular structures.

A mitochondria–paraspeckle crosstalk

Publisher: Springer Science and Business Media LLC

Date: 24-09-2018

DOI: 10.1038/S41556-018-0207-Z

Caveolin‐1‐driven membrane remodelling regulates hnRNPK‐mediated exosomal microRNA sorting in cancer

Publisher: Wiley

Date: 04-2021

DOI: 10.1002/CTM2.381

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold

Publisher: Oxford University Press (OUP)

Date: 15-04-2016

DOI: 10.1093/NAR/GKW271

The long and short of non-coding RNAs during post-natal growth and differentiation of skeletal muscles: Focus on lncRNA and miRNAs

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.DIFF.2016.05.003

Abstract: Post-natal growth of skeletal muscle is a dynamic process involving proliferation and fusion of myoblasts with elongating myofibres (hyperplasia of myonuclei) until 3 weeks post-natally in mice, with ongoing differentiation and further increases in myofibre size mostly by hypertrophy until about 12 weeks of age. The expression of mRNAs that control these events are well described, but little is known about the in vivo roles of non-coding RNAs (ncRNAs), including both microRNAs (miRNAs) and the lesser-studied long non-coding RNAs (lncRNAs). We analysed expression patterns for a broad range of lncRNAs (including Neat1, Malat1, Sra, Meg3, LncMyoD and linc-MD1), miRNAs and mRNAs in muscles of normal male C57Bl/6J mice at 2 days and 2, 4, 6 and 12 weeks after birth. These post-natal patterns were compared with expression of these RNAs during classic C2C12 myogenesis and differentiation in tissue culture. This overview of RNAs during post-natal skeletal muscle growth provides a novel focus on ncRNAs during this often overlooked growth period, with many potential applications to normal muscle growth in humans and livestock, and to childhood muscle disorders.

NONO enhances mRNA processing of super‐enhancer‐associated GATA2 and HAND2 genes in neuroblastoma

Publisher: EMBO

Date: 23-11-2022

DOI: 10.15252/EMBR.202254977

Abstract: High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.

The ins and outs of lncRNA structure: How, why and what comes next?

Publisher: Elsevier BV

Date: 2016

DOI: 10.1016/J.BBAGRM.2015.08.009

Abstract: The field of structural biology has the unique advantage of being able to provide a comprehensive picture of biological mechanisms at the molecular and atomic level. Long noncoding RNAs (lncRNAs) represent the new frontier in the molecular biology of complex organisms yet remain the least characterised of all the classes of RNA. Thousands of new lncRNAs are being reported each year yet very little structural data exists for this rapidly expanding field. The length of lncRNAs ranges from 200 nt to over 100 kb in length and they generally exhibit low cellular abundance. Therefore, obtaining sufficient quantities of lncRNA to use for structural analysis is challenging. However, as technologies develop structures of lncRNAs are starting to emerge providing important information regarding their mechanism of action. Here we review the current methods used to determine the structure of lncRNA and lncRNA:protein complexes and describe the significant contribution structural biology has and will make to the field of lncRNA research. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

An Architectural Role for a Nuclear Noncoding RNA: NEAT1 RNA Is Essential for the Structure of Paraspeckles

Publisher: Elsevier BV

Date: 03-2009

DOI: 10.1016/J.MOLCEL.2009.01.026

The noncoding RNA revolution

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BIOCEL.2014.07.021

Structural, super-resolution microscopy analysis of paraspeckle nuclear body organization

Publisher: Rockefeller University Press

Date: 19-09-2016

DOI: 10.1083/JCB.201601071

Abstract: Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the paraspeckle spheroids. We propose that paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.

Tadpole-like conformations of huntingtin exon 1 are characterized by conformational heterogeneity that persists regardless of polyglutamine length.

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1016/J.JMB.2018.03.031

Crystallization of a paraspeckle protein PSPC1–NONO heterodimer

Publisher: International Union of Crystallography (IUCr)

Date: 29-09-2011

DOI: 10.1107/S1744309111026212

NONO Detects the Nuclear HIV Capsid to Promote cGAS-Mediated Innate Immune Activation

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.CELL.2018.08.062

Abstract: Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.

Transcriptional cofactors of the FOG family interact with GATA proteins by means of multiple zinc fingers.

Publisher: Wiley

Date: 11-02-1970

DOI: 10.1093/EMBOJ/18.10.2812

Involvement of the N-finger in the self-association of GATA-1

Publisher: Elsevier BV

Date: 11-1998

DOI: 10.1074/JBC.273.46.30560

University Of Dundee

Location: United Kingdom of Great Britain and Northern Ireland

View Organisation

University Of Sydney

Location: Australia

View Organisation

University Of Western Australia

Location: Australia

View Organisation

Discovery Projects - Grant ID: DP160102435

Start Date: 2016

End Date: 12-2018

Amount: $408,800.00

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT180100204

Start Date: 2019

End Date: 12-2023

Amount: $878,125.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180103573

Start Date: 2018

End Date: 12-2021

Amount: $604,543.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100122

Start Date: 08-2020

End Date: 08-2021

Amount: $620,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP220103667

Start Date: 03-2022

End Date: 03-2025

Amount: $650,573.00

Funder: Australian Research Council