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The long non-coding RNA NEAT1 locus is transcribed into two overlapping isoforms, NEAT1_1 and NEAT1_2, of which the latter is essential for the assembly of nuclear paraspeckles. NEAT1 is abnormally expressed in a wide variety of human cancers. Emerging evidence suggests that the two isoforms have distinct functions in gene expression regulation, and recently it was shown that NEAT1_2, but not NEAT1_1, expression predicts poor clinical outcome in cancer. Here, we report that NEAT1_2 expression correlates with HER2-positive breast cancers and high-grade disease. We provide evidence that NEAT1_1 and NEAT1_2 have distinct expression pattern among different intrinsic breast cancer subtypes. Finally, we show that NEAT1_2 expression and paraspeckle formation increase upon lactation in humans, confirming what has previously been demonstrated in mice. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

The long noncoding RNA (lncRNA) NEAT1 (nuclear enriched abundant transcript 1) is the architectural component of nuclear paraspeckles, and it has recently gained considerable attention as it is abnormally expressed in pathological conditions such as cancer and neurodegenerative diseases. NEAT1 and paraspeckle formation are increased in cells upon exposure to a variety of environmental stressors and believed to play an important role in cell survival. The present study was undertaken to further investigate the role of NEAT1 in cellular stress response pathways. We show that NEAT1 is a novel target gene of heat shock transcription factor 1 (HSF1) and is up-regulated when the heat shock response pathway is activated by sulforaphane (SFN) or elevated temperature. HSF1 binds specifically to a newly identified conserved heat shock element in the NEAT1 promoter. In line with this, SFN induced the formation of NEAT1-containing paraspeckles via an HSF1-dependent mechanism. HSF1 plays a key role in the cellular response to proteotoxic stress by promoting the expression of a series of genes, including those encoding molecular chaperones. We have found that the expression of HSP70, HSP90, and HSP27 is amplified and sustained during heat shock in NEAT1-depleted cells compared with control cells, indicating that NEAT1 feeds back via an unknown mechanism to regulate HSF1 activity. This interrelationship is potentially significant in human diseases such as cancer and neurodegenerative disorders.

Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

Deimination, a posttranslational modification of arginine to citrulline carried out by peptidylarginine deiminases, may compromise tolerance of self-antigens. Patients with connective tissue autoimmunity, particularly rheumatoid arthritis and systemic lupus erythematosus (SLE) or Felty’s syndrome, present with autoantibodies to deiminated histones, which thus form a category of anti-citrullinated protein antibodies (ACPA). In general, ACPA are a sensitive diagnostic for rheumatoid arthritis and may form in response to the release of nuclear chromatin (DNA plus deiminated histones) from granulocytes, usually referred to as neutrophil extracellular traps (NETs). The aim of this study was to examine spontaneously autoimmune mice for autoantibodies and T cell responses to deiminated histones. We compared IgG binding to deiminated and non-deiminated histones by ELISA and Western blotting in spontaneously autoimmune strains of (NZB x NZW) F1 and NZM2410 together with their derivative congenic strains, C57BL/6.Sle1 and C57BL/6.Sle1.Sle3, which display profound auto reactivity against nuclear self-antigens. The splenocyte proliferation against the two antigens was determined in the spontaneously autoimmune (NZB x NZW) F1 strain from which other autoimmune strains used in the study were derived. Immunizations with deiminated and non-deiminated histones were attempted in BALB/c mice to assess their splenocyte response. Splenocytes from BALB/c mice and from autoimmune mice at the time of conversion to autoimmunity proliferated strongly in response to deiminated histones, yet serum IgG from autoimmune (NZB x NZW) F1, NZM2410, and C57BL/6.Sle1.Sle3 mice displayed a remarkable bias against binding to deiminated histones. At the time of seroconversion, the antibodies already exhibited preference for non-deiminated histones and only non-deiminated histones were recovered from circulating immune complexes. Analysis of histone deimination showed constitutive deimination in thymic extracts from C57BL/6 and C57BL/6.Sle1.Sle2.Sle3 triply congenic mice and in spleens of autoimmune triply congenic mice. Our study demonstrates that tolerance mechanisms against deiminated histones are intact in BALB/c and C57BL/6 mice and continue to be effective in mice with overt autoimmunity to non-deiminated histones. We conclude that, in contrast to human RA and SLE patients, where we frequently observe autoantibodies against deiminated histones, autoimmune mice maintain strong tolerance mechanisms to prevent the development of autoantibodies to deiminated histones.

Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB x NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice.

Group I introns are a distinct class of RNA self-splicing introns with an ancient origin. All known group I introns present in eukaryote nuclei interrupt functional ribosomal RNA genes located in ribosomal DNA loci. The discovery of the Tetrahymena intron more than 30 years ago has been essential to our understanding of group I intron catalysis, higher-order RNA structure, and RNA folding, but other intron models have provided information about the biological role. Nuclear group I introns appear widespread among eukaryotic microorganisms, and the plasmodial slime molds (myxomycetes) contain an abundance of self-splicing introns. Here, we summarize the main conclusions from previous work on the Tetrahymena intron on RNA self-splicing catalysis as well as more recent work on myxomycete intron biology. Group I introns in myxomycetes that represent different evolutionary stages, biological roles, and functional settings are discussed.

Nucleosomes represent a set of major autoantigens in the induction of systemic lupus erythematosus (SLE), and appear to be essential for the development of lupus nephritis. Deposition of nucleosome-containing immune complexes within the mesangial matrix and activation of mesangial cells may be important in the progression of lupus nephritis from a "sleeping" minimal change disease state to a full blown disease state. This study investigated the renal cytokine profile both in vivo during the development of the disease in (NZBxNZW)F1 (B/W) mice, and in vitro in primary B/W mesangial cells stimulated with nucleosomes, nucleosome-IgG complexes, and anti-dsDNA mAb respectively. Nucleosomes alone stimulated primary mesangial cells in a dose dependent manner. Of the chemokines produced by activated mesangial cells, CCL2, CCL7, CCL20, CXCL1, CXCL2 and CXCL5 were highly up-regulated compared to unstimulated cells. These chemokines were also increased in vivo in anti-dsDNA antibody positive and nephritic B/W kidneys, and was accompanied by infiltration of neutrophils, macrophages, T and B cells. This study provides a link between nucleosome-containing immune complexes, activation of mesangial cells, infiltration of effector cells and the development of lupus nephritis. Nucleosomes are pro-inflammatory and trigger innate immune responses, and thus are not only passive targets for autoantibodies but may play an active role in lupus pathogenesis. The removal or increased enzymatic destruction of nucleosomes, and/or the inhibition of mesangial cell activation may be possible treatment strategies in lupus nephritis.

Objective Association of nucleosome–IgG immune complexes with glomerular basement membranes (GBMs) is an important event in the development of lupus nephritis. Preventing this binding and/or increasing nuclease sensitivity of nucleosomes may be viable strategies for the prevention of the disease. Theoretically, heparin may alter nucleosomal structure and increase sensitivity to proteinases and nucleases, and may also inhibit binding of nucleosomes and nucleosome–IgG complexes to basement membrane structures. The aim of this study was to investigate whether and eventually how heparin prevents murine lupus nephritis. Methods Surface plasmon resonance was used to analyze if heparin inhibits binding of nucleosomes to laminin and collagen. The effect of heparin on nuclease- and proteinase-mediated degradation of nucleosomes was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and agarose gel electrophoresis. In vitro results were compared with analyses in vivo in heparin-treated (NZB × NZW)F1 mice. Anti–double-stranded DNA antibody production, deposition of nucleosome–IgG complexes in GBMs, and development of proteinuria were monitored, and circulating chromatin fragments were quantified using quantitative polymerase chain reaction. Results In vitro studies demonstrated that heparin increased enzymatic degradation of nucleosomes and almost completely inhibited binding of nucleosomes to laminin and collagen. (NZB × NZW)F1 mice treated with heparin demonstrated delayed or no antibody production and higher variation of circulating chromatin levels compared with untreated control mice. This effect was accompanied by highly reduced nucleosome–IgG complexes in GBMs and delayed development of nephritis. Conclusion Increasing the degradation of nucleosomes, reducing their immunogenicity, and preventing binding of nucleosome–IgG complexes in glomeruli together provide an alternative basis for the treatment of lupus nephritis.

Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB x NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice.

Murine and human lupus nephritis are characterized by glomerular deposits of electron-dense structures (EDS). Dominant components of EDS are chromatin fragments and IgG antibodies. Whether glomerular EDS predispose for similar deposits in skin is unknown. We analysed (i) whether dermo-epidermal immune complex deposits have similar molecular composition as glomerular deposits, (ii) whether chromatin fragments bind dermo-epidermal structures, and (iii) whether deposits in nephritic glomeruli predispose for accumulation of similar deposits in skin. Paired skin and kidney biopsies from nephritic (NZBxNZW)F1 and MRL-lpr/lpr mice and from five patients with lupus nephritis were analysed by immunofluorescence, immune electron microscopy (IEM) and co-localization TUNEL IEM. Affinity of chromatin fragments for membrane structures was determined by surface plasmon resonance. Results demonstrated (i) presence of EDS containing chromatin fragments and IgG in both organs in nephritic patients, (ii) chromatin fragments possessed high affinity for dermo-epidermal laminins and collagens, (iii) glomerular immune complex deposits did not predict similar interstitial deposits in skin, although such complexes were present in capillary lumina in glomeruli and skin of all nephritic individuals. Thus, chromatin-IgG complexes accounting for lupus nephritis seem to reach skin through circulation, but other undetermined factors are required for these complexes to deposit within skin membranes.

The present review focuses on pathogenic molecular and transcriptional events in patients with lupus nephritis. These factors are renal DNaseI, exposed chromatin fragments and the corresponding chromatin-reactive autoantibodies. Lupus nephritis is the most serious complication in human systemic lupus erythematosus, and is characterised by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes. The latter deposition defines end-stage disease. This event is stringently linked to a renal-restricted shutdown of expression of the DNaseI gene, as determined by loss of DNaseI mRNA level and DNaseI enzyme activity. The major aim of the present review is to generate new therapeutic strategies based on new insight into the disease pathogenesis.