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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.

Mouse NEAT1 is required for paraspeckle formation in a subset of cells but is not essential for animal health and viability.

Nuclei of higher organisms are well structured and have multiple, distinct nuclear compartments or nuclear bodies. Paraspeckles are recently identified mammal-specific nuclear bodies ubiquitously found in most cells cultured in vitro. To investigate the physiological role of paraspeckles, we examined the in vivo expression patterns of two long noncoding RNAs, NEAT1_1 and NEAT1_2, which are essential for the architectural integrity of nuclear bodies. Unexpectedly, these genes were only strongly expressed in a particular subpopulation of cells in adult mouse tissues, and prominent paraspeckle formation was observed only in the cells highly expressing NEAT1_2. To further investigate the cellular functions of paraspeckles, we created an animal model lacking NEAT1 by gene targeting. These knockout mice were viable and fertile under laboratory growth conditions, showing no apparent phenotypes except for the disappearance of paraspeckles. We propose that paraspeckles are nonessential, subpopulation-specific nuclear bodies formed secondary to particular environmental triggers.

Paraspeckles are a relatively new class of subnuclear bodies found in the interchromatin space of mammalian cells. They are RNA-protein structures formed by the interaction between a long nonprotein-coding RNA species, NEAT1/Men ε/β, and members of the DBHS (Drosophila Behavior Human Splicing) family of proteins: P54NRB/NONO, PSPC1, and PSF/SFPQ. Paraspeckles are critical to the control of gene expression through the nuclear retention of RNA containing double-stranded RNA regions that have been subject to adenosine-to-inosine editing. Through this mechanism paraspeckles and their components may ultimately have a role in controlling gene expression during many cellular processes including differentiation, viral infection, and stress responses.

Nuclear structures formed by a long noncoding RNA and DBHS proteins are thought to control gene expression by retaining mRNAs that have undergone adenosine-to-inosine editing in the nucleus.

Summary

NEAT1 RNA, a highly abundant 4 kb ncRNA, is retained in nuclei in ~10–20 large foci that we show is completely coincident with paraspeckles, nuclear domains implicated in mRNA nuclear retention. Depletion of NEAT1 RNA via RNAi eradicates paraspeckles, suggesting it controls sequestration of the paraspeckle proteins, PSP1 and p54, factors linked to A-I editing. Unlike over-expression of PSP1, NEAT1 over-expression increases paraspeckle number, and paraspeckles emanate exclusively from the NEAT1 transcription site. The PSP-1 RNA binding domain is required for its co-localization with NEAT1 RNA in paraspeckles, and biochemical analyses supports that NEAT1 RNA binds with paraspeckle proteins. Unlike other nuclear retained RNAs, NEAT1 RNA is not A-I edited, consistent with a structural role in paraspeckles. Collectively results demonstrate that NEAT1 functions as an essential structural determinant of paraspeckles, providing a precedent for a ncRNA as the foundation of a nuclear domain.

In many cells, mRNAs containing inverted repeats (Alu repeats in humans) in their 3′-untranslated regions (3′-UTRs) are inefficiently exported to the cytoplasm. Nuclear retention correlates with adenosine-to-inosine editing and is in paraspeckle-associated complexes containing the proteins p54nrb, PSF and PSP1α. We report that robust editing activity in human embryonic stem cells (hESCs), does not lead to nuclear retention. p54nrb, PSF and PSP1α are all expressed in hESCs, but paraspeckles are absent and only appear upon differentiation. Paraspeckle assembly and function depends on expression of a long nuclear-retained noncoding RNA, hNEAT1. This RNA is not expressed in hESCs, but is induced upon differentiation. Knockdown of hNEAT1 in HeLa cells results both in loss of paraspeckles and enhanced nucleocytoplasmic export of mRNAs containing inverted Alu repeats. Taken together, these results assign a biological function to a large noncoding nuclear RNA in the regulation of mRNA export.

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.

The nucleus of higher eukaryotes, such as humans and mice, is compartmentalized into multiple nuclear bodies, an organization that allows for the regulation of complex gene expression pathways that are characteristic of these organisms. Paraspeckles are recently discovered, mammalian-specific nuclear bodies built on a long, non-protein-coding RNA, NEAT1 (nuclear-enriched abundant transcript 1), which assembles various protein components including RNA-binding proteins of the DBHS (Drosophila behavior and human splicing) family. Paraspeckles have been proposed to control several biological processes, such as stress responses and cellular differentiation, but their function at the whole animal level remains unclear. In this review, we summarize a series of studies on paraspeckles that have been carried out in the decade since their discovery and discuss their physiological function and molecular mechanism.

Mammalian cleavage factor I (CF Im) is an essential factor that is required for the first step in pre-mRNA 3′ end processing. Here, we characterize CF Im68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF Im68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF Im68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF Im68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFIm68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF Im68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3′ end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF Im68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus.

Nuclear bodies including nucleoli, Cajal bodies, nuclear speckles, Polycomb bodies, and paraspeckles are membrane-less subnuclear organelles. They are steady-state structures that dynamically respond to basic physiological processes as well as various forms of stress, altered metabolic conditions and alterations in cellular signaling. The formation of specific nuclear bodies has been suggested to follow stochastic and ordered assembly models. In addition, a seeding mechanism has been proposed to assemble, maintain, and regulate particular nuclear bodies. In coordination with noncoding RNAs, chromatin modifiers and other machineries, various nuclear bodies have been shown to sequester and modify proteins, process RNAs and assemble ribonucleoprotein complexes, as well as epigenetically regulate gene expression. Understanding the functional relationships between the three-dimensional organization of the genome and nuclear bodies is essential to fully uncover the regulation of gene expression and its implications in human diseases.

Summary

Although eukaryotic nuclei contain distinct architectural structures associated with noncoding RNAs (ncRNAs), their potential relationship to regulated transcriptional programs remains poorly understood. Here, we report that methylation/demethylation of Polycomb 2 protein (Pc2) controls relocation of growth control genes between Polycomb bodies (PcGs) and interchromatin granules (ICGs) in response to growth signals. This movement is the consequence of binding of methylated and unmethylated Pc2 to the ncRNAs, TUG1 and MALAT1/NEAT2, located in PcGs and ICGs, respectively. These ncRNAs mediate assembly of multiple co-repressors/co-activators, and can alter the histone marks read by Pc2 in vitro. Additionally, binding of NEAT2 to unmethylated Pc2 promotes E2F1 SUMOylation, leading to activation of the growth control gene program. These observations delineate a molecular pathway linking the actions of subnuclear structure-specific ncRNAs and non-histone protein methylation to relocation of transcription units in the three-dimensional space of the nucleus, thus achieving coordinated gene expression programs.

Background

Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment.

Results

This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles.

Conclusion

Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species.

Noncoding RNAs have recently been identified as essential components of the enigmatic nuclear suborganelles called paraspeckles.

Noncoding RNAs have recently been identified as essential components of the nuclear suborganelles called paraspeckles. This finding will facilitate our understanding of the molecular dynamics and physiological role of these enigmatic macromolecular structures.

The phage shock protein (Psp) system is a conserved extracytoplasmic stress response in bacteria that is essential for virulence of the human pathogen Yersinia enterocolitica. This article summarizes some recent findings about Y. enterocolitica Psp system function. Increased psp gene expression requires the transcription factor PspF, but under non-inducing conditions PspF is inhibited by an interaction with another protein, PspA, in the cytoplasm. A Psp-inducing stimulus causes PspA to relocate to the cytoplasmic membrane, freeing PspF to induce psp gene expression. This PspA relocation requires the integral cytoplasmic membrane proteins, PspB and PspC, which might sense an inducing trigger and sequester PspA by direct interaction. The subsequent induction of psp gene expression increases the PspA concentration, which also allows it to contact the membrane directly, perhaps for its physiological function. Mutational analysis of the PspB and PspC proteins has revealed that they both positively and negatively regulate psp gene expression and has also identified PspC domains associated with each function. We also compare the contrasting physiological roles of the Psp system in the virulence of Y. enterocolitica and Salmonella enterica sv. Typhimurium (S. Typhimurium). In S. Typhimurium, PspA maintains the proton motive force, which provides the energy needed to drive ion importers required for survival within macrophages. In contrast, in the extracellular pathogen Y. enterocolitica, PspB and PspC, but not PspA, are the Psp components needed for virulence. PspBC protect Y. enterocolitica from damage caused by the secretin component of its type 3 secretion system, an essential virulence factor.

Expression of the pspABCDE operon of Escherichia coli is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor σ54 and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical pspD gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the psp operon is mediated via protein-protein interactions.

The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ54-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF–A–C–B–ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein–protein interactions, resulting in the release of the PspA–PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.

Background

Long non-protein-coding RNAs (ncRNAs) are emerging as important regulators of cellular differentiation and are widely expressed in the brain.

Results

Here we show that many long ncRNAs exhibit dynamic expression patterns during neuronal and oligodendrocyte (OL) lineage specification, neuronal-glial fate transitions, and progressive stages of OL lineage elaboration including myelination. Consideration of the genomic context of these dynamically regulated ncRNAs showed they were part of complex transcriptional loci that encompass key neural developmental protein-coding genes, with which they exhibit concordant expression profiles as indicated by both microarray and in situ hybridization analyses. These included ncRNAs associated with differentiation-specific nuclear subdomains such as Gomafu and Neat1, and ncRNAs associated with developmental enhancers and genes encoding important transcription factors and homeotic proteins. We also observed changes in ncRNA expression profiles in response to treatment with trichostatin A, a histone deacetylase inhibitor that prevents the progression of OL progenitors into post-mitotic OLs by altering lineage-specific gene expression programs.

Conclusion

This is the first report of long ncRNA expression in neuronal and glial cell differentiation and of the modulation of ncRNA expression by modification of chromatin architecture. These observations explicitly link ncRNA dynamics to neural stem cell fate decisions, specification and epigenetic reprogramming and may have important implications for understanding and treating neuropsychiatric diseases.

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the σ54 subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of σ54 AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own expression. The PspA protein is distributed between the cytoplasm and the inner membrane fraction. In addition to its transcriptional inhibitory role, PspA assists maintenance of the proton motive force and protein export. Several lines of in vitro evidence indicate that PspA-PspF interactions inhibit the ATPase activity of PspF, resulting in the inhibition of PspF-dependent gene expression. In this study, we characterize sequences within PspA and PspF crucial for the negative effect of PspA upon PspF. Using a protein fragmentation approach, we show that the integrity of the three putative N-terminal α-helical domains of PspA is crucial for the role of PspA as a negative regulator of PspF. A bacterial two-hybrid system allowed us to provide clear evidence for an interaction in E. coli between PspA and PspF in vivo, which strongly suggests that PspA-directed inhibition of PspF occurs via an inhibitory complex. Finally, we identify a single PspF residue that is a binding determinant for PspA.

The phage shock protein operon (pspABCE) of Escherichia coli is strongly induced by adverse environmental conditions. Expression is controlled principally at the transcriptional level, and transcription is directed by the sigma factor sigma 54. PspB and PspC are required for high-level psp expression during osmotic shock, ethanol treatment and f1 infection, but heat-induced expression is independent of these proteins. We report here that the promoter region contains an upstream activation sequence (UAS) that is required for psp induction and has the enhancer-like ability to activate at a distance. A DNA-binding activity is detected in crude protein extracts that is dependent on the UAS and induced by heat shock. We further show that integration host factor (IHF) binds in vitro to a site between the UAS and sigma 54 recognition sequence. In bacteria lacking IHF, psp expression is substantially reduced in response to high temperature and ethanol. During osmotic shock in contrast, psp expression is only weakly stimulated by IHF, and IHF mutants can strongly induce the operon. The dependence of psp expression on IHF varies with the inducing condition, but does not correlate with dependence on PspB and PspC, indicating distinct, agent-specific activation mechanisms.

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TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP), we demonstrated that TDP-43 preferentially binds long clusters of UG-rich sequences in vivo. Analysis of TDP-43 RNA binding in FTLD-TDP brains revealed the greatest increases in binding to MALAT1 and NEAT1 non-coding RNAs. We also showed that TDP-43 binding on pre-mRNAs influences alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A significant proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or are implicated in neurological diseases, highlighting the importance of TDP-43 for splicing regulation in the brain.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

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The widely conserved phage shock protein (Psp) extracytoplasmic stress response has been studied extensively in Escherichia coli and Yersinia enterocolitica. Both species have the PspF, -A, -B, and -C proteins, which have been linked to robust phenotypes, including Y. enterocolitica virulence. PspB and PspC are cytoplasmic membrane proteins required for stress-dependent induction of psp gene expression and for bacterial survival during the mislocalization of outer membrane secretin proteins. Previously, we reported that Y. enterocolitica PspB functions to positively control the amount of PspC by an uncharacterized posttranscriptional mechanism. In this study, we have discovered that the cytoplasmic membrane protease FtsH is involved in this phenomenon. FtsH destabilizes PspC in Y. enterocolitica, but coproduction of PspC with its binding partner PspB was sufficient to prevent this destabilization. In contrast, FtsH did not affect any other core component of the Psp system. These data suggested that uncomplexed PspC might be particularly deleterious to the bacterial cell and that FtsH acts as an important quality control mechanism to remove it. This was supported by the observation that toxicity caused by PspC production was reduced either by coproduction of PspB or by increased synthesis of FtsH. We also found that the phenomenon of FtsH-dependent PspC destabilization is conserved between Y. enterocolitica and E. coli.

Iron is an essential requirement for life for nearly all organisms. The human pathogen Staphylococcus aureus is able to acquire iron from the heme cofactor of hemoglobin (Hb) released from lysed erythrocytes. IsdB, the predominant Hb receptor of S. aureus, is a cell wall-anchored protein that is composed of two NEAT domains. The N-terminal NEAT domain (IsdB-N1) binds Hb, and the C-terminal NEAT domain (IsdB-N2) relays heme to IsdA for transport into the cell. Here we present the 1.45 Å resolution X-ray crystal structure of the IsdB-N2–heme complex. While the structure largely conforms to the eight-strand β-sandwich fold seen in other NEAT domains such as IsdA-N and uses a conserved Tyr residue to coordinate heme-iron, a Met residue is also involved in iron coordination, resulting in a novel Tyr-Met hexacoordinate heme-iron state. The kinetics of the transfer of heme from IsdB-N2 to IsdA-N can be modeled as a two-step process. The rate of transfer of heme between the isolated NEAT domains (82 s–1) was found to be similar to that measured for the full-length proteins. Replacing the iron coordinating Met with Leu did not abrogate high-affinity heme binding but did reduce the heme transfer rate constant by more than half. This unusual Met-Tyr heme coordination may also bestow properties on IsdB that help it to bind heme in different oxidation states or extract heme from hemoglobin.

Human lactoferrin is an iron-binding glycoprotein that is particularly prominent in exocrine secretions and leukocytes and is also found in serum, especially during inflammation. It is able to sequester iron from microbes and has immunomodulatory functions, including inhibition of both complement activation and cytokine production. This study used mutants lacking pneumococcal surface protein A (PspA) and PspC to demonstrate that the binding of human lactoferrin to the surface of Streptococcus pneumoniae was entirely dependent on PspA. Lactoferrin bound both family 1 and family 2 PspAs. Binding of lactoferrin to PspA was shown by surface colocalization with PspA and was verified by the lack of binding to PspA-negative mutants. Lactoferrin was expressed on the body of the cells but was largely absent from the poles. PspC showed exactly the same distribution on the pneumococcal surface as PspA but did not bind lactoferrin. PspA's binding site for lactoferrin was mapped using recombinant fragments of PspA of families 1 and 2. Binding of human lactoferrin was detected primarily in the C-terminal half of the α-helical domain of PspA (amino acids 167 to 288 of PspA/Rx1), with no binding to the N-terminal 115 amino acids in either strain. The interaction was highly specific. As observed previously, bovine lactoferrin bound poorly to PspA. Human transferrin did not bind PspA at all. The binding of lactoferrin to S. pneumoniae might provide a way for the bacteria to interfere with host immune functions or to aid in the acquisition of iron at the site of infection.

ABSTRACT

Most of the human genome is transcribed into protein-noncoding RNAs (ncRNAs), including small ncRNAs and long ncRNAs (lncRNAs). Over the past decade, rapidly emerging evidence has increasingly supported the view that lncRNAs serve key regulatory and functional roles in mammal cells. HIV-1 replication relies on various cell functions. To date, while the involvement of host protein factors and microRNAs (miRNAs) in the HIV-1 life cycle has been extensively studied, the relationship between lncRNAs and HIV-1 remains uncharacterized. Here, we have profiled 83 disease-related lncRNAs in HIV-1-infected T cells. We found NEAT1 to be one of several lncRNAs whose expression is changed by HIV-1 infection, and we have characterized its role in HIV-1 replication. We report here that the knockdown of NEAT1 enhances virus production through increased nucleus-to-cytoplasm export of Rev-dependent instability element (INS)-containing HIV-1 mRNAs.

IMPORTANCE

Long protein-noncoding RNAs (lncRNAs) play roles in regulating gene expression and modulating protein activities. There is emerging evidence that lncRNAs are involved in the replication of viruses. To our knowledge, this report is the first to characterize a role contributed by an lncRNA, NEAT1, to HIV-1 replication. NEAT1 is essential for the integrity of the nuclear paraspeckle substructure. Based on our findings from NEAT1 knockdown, we have identified the nuclear paraspeckle body as another important subcellular organelle for HIV-1 replication.