Cell-type specific and differential expression of LINC-RSAS long noncoding RNA declines in the testes during ageing of the rat.

Long noncoding RNAs (lncRNAs) have emerged as major regulators of gene expression, chromatin structure, epigenetic changes, post-transcriptional processing of RNAs, translation of mRNAs into proteins as well as contributing to the process of ageing. Ageing is a universal, slow, progressive change in almost all physiological processes of organisms after attaining reproductive maturity and often associated with age-related diseases. Mammalian testes contain various cell-types, vast reservoir of transcriptome complexity, produce haploid male gametes for reproduction and testosterone for development and maintenance of male sexual characters as well as contribute genetic variation to the species. We report age-related decline in expression and cellular localization of Long intergenic noncoding repeat-rich sense-antisense (LINC-RSAS) RNA in the testes and its major cell-types such as primary spermatocytes, Leydig cells and Sertoli cells during ageing of the rat. LINC-RSAS expression in testes increased from immature (4-weeks) to adult (16- and 44-weeks) and declined from adult (44-weeks) to nearly-old (70-weeks) rats. Genomic DNA methylation in the testes showed a similar pattern. Cell-type specific higher expression of LINC-RSAS was observed in primary spermatocytes (pachytene cells), Leydig cells and Sertoli cells of testes of adult rats. Over-expression of LINC-RSAS in cultured human cell lines revealed its possible role in cell-cycle control and apoptosis. We propose that LINC-RSAS expression is involved in molecular physiology of primary spermatocytes, Leydig cells and Sertoli cells of adult testes and its decline is associated with diminishing function of testes during ageing of the rat.

The long noncoding RNA (LINC-RBE) expression in testicular cells is associated with aging of the rat.

Long noncoding RNAs (lncRNAs) are important regulatory biomolecules responsible for many cellular processes. The aging of mammals is manifested by a slow and gradual decline of physiological functions after adulthood, progressively resulting in age-related diseases. Testis comprises different cell-types with defined functions for producing haploid gametes and androgens in males, contributing gene-pool to the next generation with genetic variations to species for evolutionary advantage. The LINC-RBE (long intergenic noncoding-rat brain expressed) RNA showed highest expression in the Leydig cells, responsible for steroidogenesis and production of testosterone; higher expression in primary spermatocytes (pachytene cells), responsible for generation of haploid gametes and high expression in Sertoli cells, the nursing cells of the testes. Testes of immature (4-weeks), adult (16- and 44-weeks), and nearly-old (70-weeks) rats showed low, high, and again low levels of expression, respectively. This along with the nuclear-cytoplasmic localization of LINC-RBE RNA showed age-related expression and function. Thus, expression of LINC-RBE is involved in the molecular physiology of testes, especially Leydig cells, primary spermatocytes, and Sertoli cells. The decline in its expression correlates with diminishing reproductive function of the testes during aging of the rat.

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

BACKGROUND: Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. METHODS: We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). RESULTS: Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1 was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. CONCLUSIONS: Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

Expression of interferon regulatory factors (IRF-1 and IRF-2) during radiation-induced damage and regeneration of bone marrow by transplantation in mouse.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors of IRF-family that regulate expression of genes for cytokines, chemokines and growth factors in mammalian cells. IRF-1 and IRF-2 play crucial roles in the differentiation of bone marrow cells for immune response. Bone marrow (BM) is the soft lymphoid organ that contains many types of stem cells and produces different types of cells of the blood and immune system. Genetic alterations and damage of the bone marrow cells can lead to different types of blood and immune system-related diseases including anemia and cancer. We have studied the expression of IRF-1 and IRF-2 during radiation-induced damage and regeneration of bone marrow cells after transplantation of freshly isolated bone marrow cells in the mouse. Cell cycle analysis, colony forming unit-fibroblast (CFU-F) assay and bone marrow histology showed that after radiation-induced damage, the bone marrow transplantation resulted in regeneration of the bone marrow up to 24-35% recovery. Real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the mRNA expression showed that IRF-1 and IRF-2 were expressed at higher levels in the bone marrow cells of the irradiated (4.34× fold for IRF-1, and 3.87× fold for IRF-2) compared to control and transplanted (1.13× fold for IRF-1, and 1.12× fold IRF-2) mice and immuno-fluorescence analysis for the protein expression showed that IRF-1 and IRF-2 were expressed at higher levels in the bone marrow cells of the irradiated (2.12× fold for IRF-1 and 1.71× fold for IRF-2) compared to control and transplanted (1.73× fold for IRF-1 and 1.21× fold for IRF-2) mice. Thus, IRF-1 and IRF-2 are sensitive and responsive to radiation-induced damage in the bone marrow cells and may also be involved in the bone marrow regeneration process.

Molecular cloning, expression and cellular localization of two long noncoding RNAs (mLINC-RBE and mLINC-RSAS) in the mouse testis.

Long noncoding RNAs (lncRNAs) are transcribed in complex, overlapping, sense- and antisense orientations from intronic and intergenic regions of mammalian genomes. Transcription of genome in mammalian testis is more widespread compared to other organs. LncRNAs are involved in gene expression, chromatin regulation, mRNA stability and translation of proteins during diverse cellular functions. We report molecular cloning of two novel lncRNAs (mLINC-RBE and mLINC-RSAS) and their expression by RT-PCR as well as cellular localization by RNA in-situ hybridization in the mouse testes. mLINC-RBE is an intergenic lncRNA from chromosome 4, with 16.96 % repeat sequences, expressed as a sense transcript with piRNA sequences and its expression is localized into primary spermatocytes. mLINC-RSAS is an intergenic lncRNA from chromosome 2, with 49.7 % repeat sequences, expressed as both sense- and antisense transcripts with miRNA sequences and its expression is localized into different cell types, such as Sertoli cells, primary spermatocytes and round spermatids. The lncRNAs also contain sequences for some short peptides (micropeptides). This suggests that these two repeat sequence containing, intergenic genomic sense- and antisense transcripts expressed as lncRNAs with piRNAs, miRNAs, and showing cell-type specific, differential expression may regulate important functions in mammalian testes. Such functions may be regulated by RNA structures, RNA processing and RNA-protein complexes.

Human buccal epithelial cells as a model system for molecular analysis of DNA, RNA and protein.

We report use of human buccal epithelial cells as an easy model system for isolation and molecular analysis of genomic DNA, RNA, and protein to study any gene of interest by Polymerase Chain Reaction (PCR), RNA expression by Reverse Transcription-PCR (RT-PCR), protein-profiling, and expression by western blot as well as DNA-methylation by Msp I/Hpa II-restriction digestion. We used simple methods to isolate genomic DNA, RNA and protein from human buccal cells collected by oral swab and cultured them in-vitro. The microscopic observation of haematoxylin and eosin (EA-50) stained cells, genomic PCR of house-keeping genes (GAPDH and ß-actin), RT-PCR of GAPDH and ß-actin mRNAs and whole cell protein-profiling by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) were carried out. Expression of ß-actin protein in supernatant and pellet fractions of the cells was determined by western blot analysis. MTT-assay was carried out to assess the cell viability and cell growth. Green Fluorescence Protein (GFP)-DNA was expressed in these cells by transient transfection. DNA-methylation in the genome was analyzed by Msp I/ Hpa II restriction digestion and agarose gel electrophoresis. Thus these methods can be used for molecular analysis of DNA, RNA and protein from the human buccal epithelial cells for studying and monitoring health, disease, population genetics/genomics and epidemiology under different conditions.

Long interspersed nuclear elements (LINEs) show tissue-specific, mosaic genome and methylation-unrestricted, widespread expression of noncoding RNAs in somatic tissues of the rat.

We report strong somatic and germ line expression of LINE RNAs in eight different tissues of rat by using a novel ~2.8 kb genomic PstI-LINE DNA (P1-LINE) isolated from the rat brain. P1-LINE is present in a 93 kb LINE-SINE-cluster in sub-telomeric region of chromosome 12 (12p12) and as multiple truncated copies interspersed in all rat chromosomes. P1-LINEs occur as inverted repeats at multiple genomic loci in tissue-specific and mosaic patterns. P1-LINE RNAs are strongly expressed in brain, liver, lungs, heart, kidney, testes, spleen and thymus into large to small heterogeneous RNAs (~5.0 to 0.2 kb) in tissue-specific and dynamic patterns in individual rats. P1-LINE DNA is strongly methylated at CpG-dinucleotides in most genomic copies in all the tissues and weakly hypomethylated in few copies in some tissues. Small (700-75 nt) P1-LINE RNAs expressed in all tissues may be possible precursors for small regulatory RNAs (PIWI-interacting/piRNAs) bioinformatically derived from P1-LINE. The strong and dynamic expression of LINE RNAs from multiple chromosomal loci and the putative piRNAs in somatic tissues of rat under normal physiological conditions may define functional chromosomal domains marked by LINE RNAs as long noncoding RNAs (lncRNAs) unrestricted by DNA methylation. The tissue-specific, dynamic RNA expression and mosaic genomic distribution of LINEs representing a steady-state genomic flux of retrotransposon RNAs suggest for biological role of LINE RNAs as long ncRNAs and small piRNAs in mammalian tissues independent of their cellular fate for translation, reverse-transcription and retrotransposition. This may provide evolutionary advantages to LINEs and mammalian genomes.

Mouse interferon regulatory factor-2: expression, purification and DNA binding activity.

Interferon regulatory factor-2 (IRF-2) is a mammalian transcription factor for Interferon and Interferon inducible genes; biologically, plays an important role in cell growth regulation and has been shown to be a potential oncogene. We have expressed, purified recombinant Murine IRF-2 (349 amino acid) as a GST (Glutathione-S-Transferase)-IRF-2 soluble fusion protein in E. coli XL-1 blue cells. Recombinant GST-IRF-2 was biologically active in terms of its DNA binding activity with IRF-E oligo (GAAAGT)4. GST-alone expressed from the vector did not bind to it. We observed five different molecular mass complexes of GST-IRF-2/DNA (1-5) with IRF-E, which were competed out by 100×-fold molar excess of IRF-E, suggesting that the complexes were specific for IRF-2. Such GAAANN (N=any nucleotide) hexa nucleotides occur in the promoters of many virus and interferon-inducible mammalian genes. Multimeric GAAAGT/C sequences are inducible by virus, IFN, dsRNA and IRF-1/2. Multiple GST-IRF-2/DNA complexes may be helpful to understand the mechanism of DNA binding activity of IRF-2.

Cell cycle arrest and apoptosis by expression of a novel TPIP (TPIP-C2) cDNA encoding a C2-domain in HEK-293 cells.

The human TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belongs to the PTEN (Phosphatase and TENsin homologue deleted on chromosome 10) family of dual-specific phosphatases and is expressed from the human chromosome 13 as multiple splice-variants, e.g., TPIPα, ß, γ mRNAs. PTEN is a well characterized tumor suppressor, which controls survival, adhesion, motility and migration of mammalian cells, its C2-domain plays crucial role in controlling these functions. However, role of isolated C2-domain protein in regulation of cell proliferation and apoptosis is not reported. We report sequence analysis and function of a novel human TPIP (TPIP-C2) cDNA encoding a 193 amino acid C2-domain in cell proliferation and apoptosis regulation. In silico analysis and homology modelling revealed that the C2-domain of TPIP-C2 is similar to that of PTEN but with short disorder sequences overlapping or adjacent to the post-translational modification sites. Overexpression of TPIP-C2 cDNA in human embryonic kidney (HEK-293) cells caused cell cycle arrest, inhibition of cell proliferation and induced apoptosis in an activated caspase 3 and PARP-dependent manner in comparison to overexpression of the full length human PTEN cDNA. TPIP-C2 overexpressed cells also showed S-phase cell cycle arrest. We suggest that C2-domain of TPIP-C2 may act as a dominant negative effector, which may bind to and arrest the cell proliferation signalling complex and isolated TPIP-C2-domain-like proteins expressed in mammalian cells/tissues may play important role in regulation of cell proliferation and apoptosis. The TPIP-C2 cDNA may be exploited for inducing cell cycle-inhibition and apoptosis in human cancer cells and tissues.

Age-Related Expression of a Repeat-Rich Intergenic Long Noncoding RNA in the Rat Brain.

Genome-wide transcriptome analysis has shown that ∼90 % of the mammalian genome undergoes pervasive transcription into various small and long noncoding RNAs with diverse biological functions and only ∼1.5 % is protein coding. Recent literature suggests that various structurally diverse sense and antisense long noncoding RNAs (lncRNAs) (>200 nt) are expressed from the intronic, intergenic and repeat-rich regions in the mammalian central nervous system (CNS). Till date, many of them have been found to be regulated in developmental, spatio-temporal and cell type-specific manners and are involved in various neurological processes. However, still much is left to be understood regarding their functional relevance in mammalian brain development, maturation and ageing. Furthermore, various signalling factors and metabolites such as all-trans retinoic acid (atRA) have been known to regulate brain functions during development, though their role in adult brain function is much less known. Here, we report differential and age-related expression of a novel repeat sequence-rich, long intergenic nonprotein coding RNA (lincRNA), named as LINC-RSAS (repeat-rich sense-antisense transcript) in different neuroanatomical regions of the rat brain. The LINC-RSAS was found to be moderately conserved and contained regulatory elements of various cell growth- and development-specific transcriptional factors in its up/downstream flanking sequences in the genome. Through RNA expression by reverse transcription polymerase chain reaction (RT-PCR) and localization by in situ RNA hybridization, we found that both sense and antisense transcripts of LINC-RSAS were expressed in the cortex, hippocampus and cerebellum regions of the rat brain in cell type-specific and age-related manner. Furthermore, both the expression level and subcellular localization of the antisense LINC-RSAS transcript were significantly induced in the cultured primary hippocampal neurons after treatment with atRA. Overall, our study provides insights into the possible involvement of an atRA-inducible, intergenic lncRNA in different functional regions of mammalian brain and its association with brain maturation and ageing.

Microgrooved-surface topography enhances cellular division and proliferation of mouse bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells' (MSCs) fate is largely determined by the various topographical features and a range of extracellular matrix (ECM) components present in their niches. Apart from maintaining structural stability, they regulate cell morphology, division, proliferation, migration and differentiation among others. Traditional MSC cultures, which are mainly based on two-dimensional smooth surfaces of culture dishes and plates, do not provide topographical cues similar to in vivo three-dimensional niches, impacting various cellular processes. Therefore, we culture the mouse bone marrow-derived MSCs on microgrooved bearing surface, partially mimicking in vivo reticulated niche, to study its effect on morphology, pluripotency factor-associated stemness, cell division and rate of proliferation. Following culture, morphological features, and MSC-specific marker gene expression, such as CD29, CD44, Sca-1 along with HSC (Haematopoietic stem cell)-specific markers like CD34, CD45, CD11b were evaluated by microscopy and immunophenotyping, respectively. HSC is another type of bone marrow stem cell population, which concertedly interacts with MSC during various functions, including haematopoiesis. In addition, mesenchymal stem cells were further analyzed for gene expression of pluripotency-associated transcription factors such as Oct3/4, Sox-2, Nanog and Myc, as well as differentiated into adipocytes, osteocytes and chondrocytes. Our results show that microgrooved surface-cultured mesenchymal stem cells (MMSCs) expressed higher levels of expected cell surface and pluripotency-associated markers and proliferated more rapidly (2-3×fold) with higher percentage of cells in S/G2-M-phase, consequently giving rise to higher cell yield compared to standard culture flask-grown cells (MSCs), taken as control. Furthermore, both MSCs and MMSCs showed considerable accumulation of intracellular lipid-droplets, higher alkaline phosphatase activity and secretion of extracellular matrix that are characteristics of adipogenesis, osteogenesis and chondrogenesis, respectively.

Relationship between constitutive nuclear factor-kappaB (NF-kappaB) and inhibitor kappaB-alpha (IkappaB-alpha) in an interferon-alpha-sensitive human Burkitt lymphoma cell line.

The human Burkitt lymphoma Daudi cell line expresses constitutively active nuclear factor kappaB (NF-kappaB) in the nucleus in spite of high levels of inhibitor kappaB-alpha (IkappaB-alpha) in the cytoplasm. The antiproliferative response of these cells to interferon-alpha (IFN-alpha) correlated with the inhibition of the constitutive NF-kappaB activity by the cytokine. The present study shows that IFN-alpha caused an increase in p53 level, inhibited cell proliferation by [(3)H]thymidine incorporation, and stimulated cytotoxicity and apoptosis by PARP-cleavage in the Daudi cells. In order to study the relationship between the constitutively active NF-kappaB and IkappaB-alpha, a dominant negative mutant IkappaB-alpha (IkappaB-alphaDN), lacking the N-terminal 36 amino acids required for the activation of NF-kappaB by tumor necrosis factor-alpha (TNF-alpha), was expressed in the Daudi cells. The expression of IkappaB-alphaDN protein did not inhibit the constitutive NF-kappaB activity, but it inhibited cell proliferation, antiproliferative response to IFN-alpha, and phosphorylated mitogen activated protein kinase (p-MAPK) level. Thus, our results suggest that constitutive NF-kappaB activity in the human Burkitt lymphoma Daudi cells is maintained by a mechanism independent of IkappaB-alpha degradation, and that the IkappaB-alpha is involved in the proliferation of these cells, possibly through the MAP kinase pathway. Therefore, in addition to IFN-alpha treatment, both NF-kappaB and IkappaB-alpha may be used as drug targets for inhibiting cell proliferation in the lymphomas.

All-Trans Retinoic Acid Induces Expression of a Novel Intergenic Long Noncoding RNA in Adult rat Primary Hippocampal Neurons.

Around 90% of the mammalian genome undergoes pervasive transcription into various types of small and long regulatory noncoding RNAs, whereas only ∼ 1.5% codes for proteins. Long noncoding RNAs (lncRNAs) constitute diverse classes of sense- and antisense transcripts that are abundantly expressed in the mammalian central nervous system (CNS) in cell type- and developmental stage-specific manners. They are implicated in brain development, differentiation, neuronal plasticity, and other cognitive functions. Mammalian brain requires the vitamin A metabolite all-trans retinoic acid (atRA) for its normal development, differentiation, and cell-fate determination. However, its role in adult brain function is less understood. Here, we report atRA-mediated transcriptional upregulation of endogenous expression of a novel long intergenic noncoding RNA-rat brain expressed (LINC-RBE) in cultured primary hippocampal neurons from adult rat. We have previously reported LINC-RBE as an intergenic, simple repeat sequence containing lncRNA highly expressed in the rat brain. This is a first-time report of involvement of atRA in transcriptional upregulation of lncRNA expression in rat hippocampal neurons. Therefore, it may be involved in regulation of brain function and disease.

Long noncoding RNAs in aging and age-related diseases.

Aging is the universal, intrinsic, genetically-controlled, evolutionarily-conserved and time-dependent intricate biological process characterised by the cumulative decline in the physiological functions and their coordination in an organism after the attainment of adulthood resulting in the imbalance of neurological, immunological and metabolic functions of the body. Various biological processes and mechanisms along with altered levels of mRNAs and proteins have been reported to be involved in the progression of aging. It is one of the major risk factors in the patho-physiology of various diseases and disorders. Recently, the discovery of pervasive transcription of a vast pool of heterogeneous regulatory noncoding RNAs (ncRNAs), including small ncRNAs (sncRNAs) and long ncRNAs (lncRNAs), in the mammalian genome have provided an alternative way to study and explore the missing links in the aging process, its mechanism(s) and related diseases in a whole new dimension. The involvement of small noncoding RNAs in aging and age-related diseases have been extensively studied and recently reviewed. However, lncRNAs, whose function is far less explored in relation to aging, have emerged as a class of major regulators of genomic functions. Here, we have described some examples of known as well as novel lncRNAs that have been implicated in the progression of the aging process and age-related diseases. This may further stimulate research on noncoding RNAs and the aging process.

Correlation between alterations in nucleosomal organization of LINEs in the promoter of cytochrome P450 2B1/2 gene and induction of CYP 2B1/2B2 mRNA expression by phenobarbitone in rat liver.

Mammalian genome contains a high proportion of repetitive DNA with a sizeable fraction of Long Interspersed Nuclear Elements (LINEs). LINEs have been functionally implicated in the organization and evolution of mammalian genome. However, functions of LINEs in the promoter region and gene expression are poorly understood. Here, we report two small, conserved LINE sequences (3P and 5P) that occur as multiple copies of inverted repeats in the rat Cytochrome P450 2B1/2B2 (CYP 2B1/2) gene promoter. Using 3P or 5P as a single primer, the CYP 2B1/2 promoter DNA was amplified by PCR from the rat genome. Phenobarbitone (PB), a prototype xenobiotic drug, strongly induced CYP 2B1/2 mRNA expression in the rat liver. 3P and 5P LINE sequences showed an alteration in the nucleosomal organization in the CYP 2B1/2 promoter after 2, 4, and 6 h of PB induction, and the promoter was mostly devoid of nucleosomes during the induction. Reorganization of nucleosomes associated with these LINE sequences were strongly correlated with induction of CYP 2B1/2 mRNA expression by PB in vivo. Our results strongly suggest that these LINE sequences in CYP 2B1/2 gene promoter(s) may facilitate transcriptional activation of the gene(s) by PB through retention and reorganization of nucleosomes. This might be a novel function of LINEs in the mammalian genome that correlates the chromatin structure with gene expression during drug metabolism.

Age-dependent differential expression profile of a novel intergenic long noncoding RNA in rat brain.

Long noncoding RNAs (lncRNAs) are ≥ 200 nt long, abundant class of non-protein coding RNAs that are transcribed in complex, sense- and antisense patterns from the intergenic and intronic regions of mammalian genome. Mammalian central nervous system constitutes the largest repertoire of noncoding transcripts that are known to be expressed in developmentally regulated and cell-type specific manners. Although many lncRNAs, functioning in the brain development and diseases are known, none involved in brain aging has been reported so far. Here, we report involvement of a novel, repeat sequence (simple repeats and SINES)-containing, trans-spliced, long intergenic non-protein coding RNA (lincRNA), named as LINC-RBE (rat brain expressed transcript) involved in maturation and aging of mammalian brain. The LINC-RBE is strongly expressed in the rat brain and the upstream/downstream sequences of its DNA in the chromosome 5 contain binding sites for many cell growth, survival and development-specific transcriptional factors. Through RT-PCR and RNA in situ hybridization, LINC-RBE was found to be expressed in an age-dependent manner with significantly higher level of expression in the brain of adult (16 week) compared to both immature (4 week) and old (70 week) rats. Moreover, the expression pattern of the LINC-RBE showed distinct association with the specific neuro-anatomical regions, cell types and sub-cellular compartments of the rat brain in an age-related manner. Thus, its expression increased from immature stage to adulthood and declined further in old age. This is a first-time report of involvement of an intergenic repeat sequence-containing lncRNA in different regions of the rat brain in an age-dependent manner.

Age-dependent differential expression profile of a novel intergenic long noncoding RNA in rat brain.

Long noncoding RNAs (lncRNAs) are ≥200 nt long, abundant class of non-protein coding RNAs that are transcribed in complex, sense- and antisense patterns from the intergenic and intronic regions of mammalian genome. Mammalian central nervous system constitutes the largest repertoire of noncoding transcripts that are known to be expressed in developmentally regulated and cell-type specific manners. Although many lncRNAs, functioning in the brain development and diseases are known, none involved in brain aging has been reported so far. Here, we report involvement of a novel, repeat sequence (simple repeats and SINES)-containing, trans-spliced, long intergenic non-protein coding RNA (lincRNA), named as LINC-RBE (rat brain expressed transcript) involved in maturation and aging of mammalian brain. The LINC-RBE is strongly expressed in the rat brain and the upstream/downstream sequences of its DNA in the chromosome 5 contain binding sites for many cell growth, survival and development-specific transcriptional factors. Through RT-PCR and RNA in situ hybridization, LINC-RBE was found to be expressed in an age-dependent manner with significantly higher level of expression in the brain of adult (16 weeks) compared to both immature (4 weeks) and old (70 weeks) rats. Moreover, the expression pattern of the LINC-RBE showed distinct association with the specific neuro-anatomical regions, cell types and sub-cellular compartments of the rat brain in an age-related manner. Thus, its expression increased from immature stage to adulthood and declined further in old age. This is a first-time report of involvement of an intergenic repeat sequence-containing lncRNA in different regions of the rat brain in an age-dependent manner.

Induction of the interferon-inducible RNA-degrading enzyme, RNase L, by stress-inducing agents in the human cervical carcinoma cells.

RNA-degradation is one of the fundamental mechanisms of interferon (IFN)-inducible antiviral response in mammalian cells. This is primarily brought about by the IFN-inducible 2',5'-oligoadenylate (2-5A)-cofactor dependent ribonuclease L (RNase L). RNase L also functions as a tumor suppressor gene in case of prostate cancer due to its role in apoptosis. We report that RNase L is induced by stress-inducing agents such as double-stranded RNA [poly(I:C)], chemotherapeutic drugs, hydrogen peroxide (H(2)O(2)), calcium chloride (CaCl(2)) and tumor necrosis factor-alpha (TNF) in the human cervical carcinoma (HeLa) cells. The level of RNase L was not detected in the untreated cells. Induction of RNase L by such stress-inducing agents correlated with degradation of cellular RNA, fragmentation of chromatin-DNA and induction of apoptosis. We checked the stress-inducible transcription factor, nuclear factor kappa B (NF-kappaB), which was persistently activated by cycloheximide but not by other agents after 24 hours indicating no role of NFkappaB in the RNase L-induction. However, as expected, TNF-induced NF-kappaB activity was stimulated within 10-30 minutes through degradation of IkappaB-alpha. Our results strongly suggest that the IFN-inducible RNase L is induced by a broad range of stress-inducing signals such as double-stranded RNA (dsRNA) produced during viral infection, membrane- and osmotic shock caused by CaCl(2) and oxidative stress induced by H(2)O(2), inflammation stimulated by TNF-alpha and chemotherapy. Thus, in addition to its antiviral function, the IFN-inducible RNase L may play an important role during stress-response through RNA-degradation and apoptosis.

Interferon regulatory factor-1 (IRF-1) interacts with regulated in development and DNA damage response 2 (REDD2) in the cytoplasm of mouse bone marrow cells.

IRF-1 is a critical hematopoietic transcription factor, which regulates cell growth, development of immune cells, immune response, tumor suppression, apoptosis and autophagy in mammalian cells. Protein-protein interactions of IRF-1 in mouse bone marrow cells (BMCs) by GST-IRF-1 pull-down followed by mass spectrometry, coimmunoprecipitation, immunoblotting and colocalization show that regulated in development and DNA damage response 2 (REDD2) is an IRF-1-interacting protein. REDD2 is a highly conserved mammalian regulatory protein of the TSC2/mTOR pathway. It is structurally similar to REDD1 but has a distinct loop region. Cellular IRF-1 and REDD2 complex is present in the cytoplasm of BMCs as distinct speckles in punctate pattern. In vitro interaction of recombinant IRF-1 and REDD2 shows their physical interaction. Taken together, our results suggest that IRF-1 physically interacts with REDD2 in the large cytoplasmic protein complex, which may function as cellular signaling proteins for 'cross-talk' of mTOR and cytokine pathways during regulation of cell growth/proliferation, apoptosis and autophagy of mammalian bone marrow cells during health and disease.

Expression of mRNA and protein-protein interaction of the antiviral endoribonuclease RNase L in mouse spleen.

The interferon-inducible, 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L is a unique antiviral RNA-degrading enzyme involved in RNA-metabolism, translational regulation, stress-response besides its anticancer/tumor-suppressor and antibacterial functions. RNase L represents complex cellular RNA-regulations in mammalian cells but diverse functions of RNase L are not completely explained by its 2-5A-regulated endoribonuclease activity. We hypothesized that RNase L has housekeeping function(s) through interaction with cellular proteins. We investigated RNase L mRNA expression in mouse tissues by RT-PCR and its protein-protein interaction in spleen by GST-pulldown and immunoprecipitation assays followed by proteomic analysis. RNase L mRNA is constitutively and differentially expressed in nine different mouse tissues, its level is maximum in immunological tissues (spleen, thymus and lungs), moderate in reproductive tissues (testis and prostate) and low in metabolic tissues (kidney, brain, liver and heart). Cellular proteins from mouse spleen [fibronectin precursor, ß-actin, troponin I, myosin heavy chain 9 (non-muscle), growth-arrest specific protein 11, clathrin light chain B, a putative uncharacterized protein (Ricken cDNA 8030451F13) isoform (CRA_d) and alanyl tRNA synthetase] were identified as cellular RNase L-interacting proteins. Thus our results suggest for more general cellular functions of RNase L through protein-protein interactions in the spleen for immune response in mammals.