A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin

BACKGROUND: Cajal bodies (CBs) are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus. RESULTS: In this report, we identify a novel protein isoform of EB-1/AIDA-1, termed AIDA-1c, that interacts with the CB marker protein, coilin. Northern and nested PCR experiments reveal that the AIDA-1c isoform is expressed in brain and several cancer cell lines. Competition binding experiments demonstrate that AIDA-1c competes with SmB' for coilin binding sites, but does not bind SMN. When ectopically expressed, AIDA-1c is predominantly nuclear with no obvious accumulations in CBs. Interestingly, another EB-1/AIDA-1 nuclear isoform, AIDA-1a, does not bind coilin in vivo as efficiently as AIDA-1c. Knockdown of EB-1/AIDA-1 isoforms by siRNA altered Cajal body organization and reduced cell viability. CONCLUSION: These data suggest that specific EB-1/AIDA-1 isoforms, such as AIDA-1c, may participate in the regulation of nucleoplasmic coilin protein interactions in neuronal and transformed cells

In Vitro RNase and Nucleic Acid Binding Activities Implicate Coilin in U snRNA Processing

Coilin is known as the marker protein for Cajal bodies (CBs), subnuclear domains important for the biogenesis of small nuclear ribonucleoproteins (snRNPs) which function in pre-mRNA splicing. CBs associate non-randomly with U1 and U2 gene loci, which produce the small nuclear RNA (snRNA) component of the respective snRNP. Despite recognition as the CB marker protein, coilin is primarily nucleoplasmic, and the function of this fraction is not fully characterized. Here we show that coilin binds double stranded DNA and has RNase activity in vitro. U1 and U2 snRNAs undergo a processing event of the primary transcript prior to incorporation in the snRNP. We find that coilin displays RNase activity within the CU region of the U2 snRNA primary transcript in vitro, and that coilin knockdown results in accumulation of the 3′ pre-processed U1 and U2 snRNA. These findings present new characteristics of coilin in vitro, and suggest additional functions of the protein in vivo

Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

BACKGROUND: The Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. RESULTS: In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. CONCLUSION: Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB

Coilin interacts with Ku proteins and inhibits in vitro non-homologous DNA end joining

AbstractCoilin is a nuclear protein that plays a role in Cajal body formation. The function of nucleoplasmic coilin is unknown. Here we report that coilin interacts with Ku70 and Ku80, which are major players in the DNA repair process. Ku proteins compete with SMN and SmB′ proteins for coilin interaction sites. The binding domain on coilin for Ku proteins cannot be localized to one discrete region, and only full-length coilin is capable of inhibiting in vitro non-homologous DNA end joining (NHEJ). Since Ku proteins do not accumulate in CBs, these findings suggest that nucleoplasmic coilin participates in the regulation of DNA repair.Structured summaryMINT-8052983:coilin (uniprotkb:P38432) physically interacts (MI:0915) with SmB′ (uniprotkb:P14678) by pull down (MI:0096)MINT-8052941:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku70 (uniprotkb:P12956) by competition binding (MI:0405)MINT-8052765:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by pull down (MI:0096)MINT-8052971:coilin (uniprotkb:P38432) physically interacts (MI:0915) with SMN (uniprotkb:Q16637) by pull down (MI:0096)MINT-8052957:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by competition binding (MI:0405)MINT-8052894, MINT-8052908:coilin (uniprotkb:P38432) binds (MI:0407) to Ku80 (uniprotkb:P13010) by pull down (MI:0096)MINT-8052804:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku80 (uniprotkb:P13010) by anti bait coimmunoprecipitation (MI:0006)MINT-8052925:coilin (uniprotkb:P38432) binds (MI:0407) to Ku70 (uniprotkb:P12956) by pull down (MI:0096)MINT-8052786:Ku80 (uniprotkb:P13010) physically interacts (MI:0914) with coilin (uniprotkb:P38432) and Ku70 (uniprotkb:P12956) by anti bait coimmunoprecipitation (MI:0006)MINT-8052776:coilin (uniprotkb:P38432) physically interacts (MI:0915) with Ku70 (uniprotkb:P12956) by pull down (MI:0096

Rational selection of small molecules that increase transcription through the GAA repeats found in Friedreich’s ataxia

AbstractFriedreich’s ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA

The relationship of plasma Trans fatty acids with dietary inflammatory index among US adults

BACKGROUND: It has been suggested that trans fatty acids (TFAs) play an important role in cardiovascular diseases. We investigated the association between plasma TFAs and the dietary inflammatory index (DII) ™ in US adults. METHODS: National Health and Nutrition Examination Survey (NHANES) participants with data on plasma TFAs measured from 1999 to 2010 were included. Energy-adjusted-DII ™ (E-DII ™) expressed per 1000 kcal was calculated from 24-h dietary recalls. All statistical analyses accounted for the survey design and sample weights. RESULTS: Of the 5446 eligible participants, 46.8% (n = 2550) were men. The mean age of the population was 47.1 years overall, 47.8 years for men and 46.5 years for women (p = 0.09). After adjustment for C-reactive protein, body-mass-index, smoking, race, age, education, and marital status in linear regressions, trans 9-hexadecenoic acid [β coefficient 0.068 (95% CI: 0.032 to 0.188)], trans 11-octadecenoic acid [β coefficient 0.143 (95% CI: 0.155 to 0.310)], trans 9-octadecenoic acid [β coefficient 0.122 (95% CI: 0.120 to 0.277)], trans 9, and trans 12-octadienoic acid [β coefficient 0.103 (95% CI: 0.090 to 0.247)] were positively associated with the DII (all p < 0.001). CONCLUSION: The association of plasma TFAs with a marker of dietary inflammation suggests an underlying mechanism in the initiation and progression of cardiovascular diseases

Association of Markers of Inflammation with Sleep and Physical Activity among People Living with HIV or AIDS

This study examined associations of sleep and minutes spent in moderate-vigorous physical activity (MVPA) with C-reactive protein (CRP) and interleukin (IL)-6 among persons living with HIV (PLWH). Cross-sectional analyses (n=45) focused on associations of inflammatory outcomes (i.e., CRP and IL-6) with actigraph-derived sleep duration, latency, and efficiency; bedtime; wake time; and wake-after-sleep-onset; as well as MVPA. Least square means for CRP and IL-6 by levels of sleep and MVPA were computed from general linear models. Individuals below the median of sleep duration, above the median for bedtime, and below the median of MVPA minutes had higher CRP or IL-6 levels. Generally, individuals with both low MVPA and poor sleep characteristics had higher inflammation levels than those with more MVPA and better sleep. Understanding the combined impact of multiple lifestyle/behavioral factors on inflammation could inform intervention strategies to reduce inflammation and therefore, chronic disease risk

Identification of Birds through DNA Barcodes

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10× average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens

Coilin levels modulate cell cycle progression and γH2AX levels in etoposide treated U2OS cells

AbstractCoilin is considered the Cajal body (CB) marker protein. In this report, we investigated the role of coilin in the DNA damage response and found that coilin reduction correlated with significantly increased levels of soluble γH2AX in etoposide treated U2OS cells. Additionally, coilin levels influenced the proliferation rate and cell cycle distribution of cells exposed to etoposide. Moreover, coilin overexpression inhibited nucleolar localization of endogenous coilin in etoposide treated U2OS cells. Collectively, these data provide additional evidence for coilin and CBs in the DNA damage response

Testing of a Tool for Prostate Cancer Screening Discussions in Primary Care

BackgroundAs prostate cancer (PCa) screening decisions often occur in outpatient primary care, a brief tool to help the PCa screening conversation in busy clinic settings is needed.MethodsA previously created 9-item tool to aid PCa screening discussions was tested in five diverse primary care clinics. Fifteen providers were recruited to use the tool for 4 weeks, and the tool was revised based upon feedback. The providers then used the tool with a convenience sample of patients during routine clinic visits. Pre- and post-visit surveys were administered to assess patients’ knowledge of the option to be screened for PCa and of specific factors to consider in the decision. McNemar’s and Stuart–Maxwell tests were used to compare pre-and post-survey responses.Results14 of 15 providers completed feedback surveys and had positive responses to the tool. All 15 providers then tested the tool on 95 men aged 40–69 at the five clinics with 2–10 patients each. The proportion of patients who strongly agreed that they had the option to choose to screen for PCa increased from 57 to 72% (p = 0.018) from the pre- to post-survey, that there are factors in the personal or family history that may affect PCa risk from 34 to 47% (p = 0.012), and that their opinions about possible side effects of treatment for PCa should be considered in the decision from 47 to 61% (p = 0.009).ConclusionA brief conversation tool for the PCa screening discussion was well received in busy primary-care settings and improved patients’ knowledge about the screening decision