SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing.

The survival of motor neurons (SMN) protein is essential for the biogenesis of small nuclear RNA (snRNA)-ribonucleoproteins (snRNPs), the major components of the pre-mRNA splicing machinery. Though it is ubiquitously expressed, SMN deficiency causes the motor neuron degenerative disease spinal muscular atrophy (SMA). We show here that SMN deficiency, similar to that which occurs in severe SMA, has unexpected cell type-specific effects on the repertoire of snRNAs and mRNAs. It alters the stoichiometry of snRNAs and causes widespread pre-mRNA splicing defects in numerous transcripts of diverse genes, preferentially those containing a large number of introns, in SMN-deficient mouse tissues. These findings reveal a key role for the SMN complex in RNA metabolism and in splicing regulation and indicate that SMA is a general splicing disease that is not restricted to motor neurons.

Episodic Hypoxia Promotes Defence Against Cellular Stress.

BACKGROUND/AIMS: Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia. METHODS: In vivo, kidneys of mice challenged daily with CsA for one week were analyzed by microarray analysis, gene ontology analysis, and qPCR. In vitro, renal cells were subjected to hypoxia (1 % O2) which was either episodic (4 h for 6 consecutive days), short-term (4 h), or sustained (24 h). Western blot analysis quantified hypoxia-inducible factor-1α (HIF-1α). 2',7'-dichlorofluorescein diacetate detected intracellular ROS. After re-oxygenation, staurosporine served to induce apoptosis, quantified by active caspase-3. RESULTS: In vivo, HIF target gene expression was suppressed by daily CsA treatment. Yet, we found up-regulation of genes involved in defence against cellular stress, notably against ROS. Renal cells in vitro behaved largely different under episodic and sustained hypoxia, while their response to short-term hypoxia oscillated between the previous two. Episodic hypoxia exhibited the highest total HIF-1α protein level, lowest nucleus-to-cytoplasm ratio, and lowest HIF target gene expression. When compared with normoxia, re-oxygenation after sustained hypoxia increased ROS by 3.04 ± 1.04 fold (p<0.001), and re-oxygenation after episodic hypoxia by 1.26 ± 0.16 fold (p<0.01). Staurosporine-induced active caspase-3 was highest after sustained, and lowest after episodic hypoxia. CONCLUSION: In vitro episodic hypoxia mimics the largely HIF-independent transcriptome observed after repetitive CsA treatment in vivo. In vitro preconditioning with episodic hypoxia protects against stress-induced apoptosis. Despite of its long-term adverse effects, CsA derived episodic hypoxia induces a unique renal hypoxia response that provides adaptation to re-oxygenation mediated ROS damage.

Gemin5 delivers snRNA precursors to the SMN complex for snRNP biogenesis.

The SMN complex assembles Sm cores on snRNAs, a key step in the biogenesis of snRNPs, the spliceosome's major components. Here, using SMN complex inhibitors identified by high-throughput screening and a ribo-proteomic strategy on formaldehyde crosslinked RNPs, we dissected this pathway in cells. We show that protein synthesis inhibition impairs the SMN complex, revealing discrete SMN and Gemin subunits and accumulating an snRNA precursor (pre-snRNA)-Gemin5 intermediate. By high-throughput sequencing of this transient intermediate's RNAs, we discovered the previously undetectable precursors of all the snRNAs and identified their Gemin5-binding sites. We demonstrate that pre-snRNA 3' sequences function to enhance snRNP biogenesis. The SMN complex is also inhibited by oxidation, and we show that it stalls an inventory-complete SMN complex containing pre-snRNAs. We propose a stepwise pathway of SMN complex formation and snRNP biogenesis, highlighting Gemin5's function in delivering pre-snRNAs as substrates for Sm core assembly and processing.

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia.

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, whereas additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pulldown experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease.

Beyond hemoglobin: Critical role of 2,3-bisphosphoglycerate mutase in kidney function and injury.

AIM: 2,3-bisphosphoglycerate mutase (BPGM) is traditionally recognized for its role in modulating oxygen affinity to hemoglobin in erythrocytes. Recent transcriptomic analyses, however, have indicated a significant upregulation of BPGM in acutely injured murine and human kidneys, suggesting a potential renal function for this enzyme. Here we aim to explore the physiological role of BPGM in the kidney. METHODS: A tubular-specific, doxycycline-inducible Bpgm-knockout mouse model was generated. Histological, immunofluorescence, and proteomic analyses were conducted to examine the localization of BPGM expression and the impact of its knockout on kidney structure and function. In vitro studies were performed to investigate the metabolic consequences of Bpgm knockdown under osmotic stress. RESULTS: BPGM expression was localized to the distal nephron and was absent in proximal tubules. Inducible knockout of Bpgm resulted in rapid kidney injury within 4 days, characterized by proximal tubular damage and tubulointerstitial fibrosis. Proteomic analyses revealed involvement of BPGM in key metabolic pathways, including glycolysis, oxidative stress response, and inflammation. In vitro, Bpgm knockdown led to enhanced glycolysis, decreased reactive oxygen species elimination capacity under osmotic stress, and increased apoptosis. Furthermore, interactions between nephron segments and immune cells in the kidney suggested a mechanism for propagating stress signals from distal to proximal tubules. CONCLUSION: BPGM fulfills critical functions beyond the erythrocyte in maintaining glucose metabolism in the distal nephron. Its absence leads to metabolic imbalances, increased oxidative stress, inflammation, and ultimately kidney injury.

Seamless Coupling of Chemical Glycan Release and Labeling for an Accelerated Protein N-Glycan Sample Preparation Workflow.

Analytical methods fr direct quantitative N-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled N-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-N-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent N-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS m/z peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.

Release of protein N-glycans by effectors of a Hofmann carboxamide rearrangement.

Background: Chemical methods for glycan release have gained traction because of their cost efficiency, accelerated reaction time and ability to release glycans not amenable to enzymatic cleavage. Oxidative chemical glycan release via hypochlorite treatment has been shown to be a convenient and efficient method that yields N-glycans similar to classical PNGase F digestion. We observed that the initial steps of the suggested mechanism for the oxidative release of glycans from glycoproteins by hypohalites showed similarities to the initiating steps of the classical Hofmann rearrangement of carboxamides. Therefore, we investigated the ability of different stable effectors of a Hofmann-type carboxamide rearrangement to efficiently and selectively release N-glycans from glycoproteins. Methods: Released glycans obtained from different experimental chemical release approaches were analyzed by HILIC-FLD, BHZ-FACE and ESI-MS and evaluated with respect to electrophoretic mobility, retention time and integrated peak area for resolved glycans. Results: We show that the known Hoffmann catalysts 1,3-dichloro-5,5-dimethylhydantoin, the hypervalent organoiodine (III) compound diacetoxy-iodobenzene as well as in-situ hypobromite generation using Oxone® and potassium bromide are all capable of releasing protein-bound N-glycans in good yield. Among the compounds investigated, diacetoxy-iodobenzene was capable of releasing glycans in the absence of alkali. Detailed investigations of the bromide/Oxone® method revealed a dependence of N-glycan release efficiency from the temporal order of bromide addition to the reaction mix as well as from a molar excess of bromide over Oxone®. Conclusions. These findings suggest that the oxidative release of N-glycans occurs via the initiating steps of a Hofmann carboxamide rearrangement. Hypervalent organoiodine compounds hold the promise of releasing glycans in the absence of alkali. The in-situ generation of hypobromite by bromide/Oxone® produces a consistent defined amount of reagent for rapid N-glycan release for both analytical and preparative purposes.

Functional characterization of the re-face loop spanning residues 536-541 and its interactions with the cofactor in the flavin mononucleotide-binding domain of flavocytochrome P450 from Bacillus megaterium.

Flavocytochrome P450BM-3, a bacterial monooxygenase, contains a flavin mononucleotide-binding domain bearing a strong structural homology to the bacterial flavodoxin. The flavin mononucleotide (FMN) serves as the one-electron donor to the heme iron, but in contrast to the electron transfer mechanism of mammalian cytochrome P450 reductase, the FMN semiquinone state is not thermodynamically stable and appears transiently as the anionic rather than the neutral form. A unique loop region comprised of residues (536)Y-N-G-H-P-P(541), which forms a type I' reverse turn and provides several interactions with the FMN isoalloxazine ring, was targeted in this study. Nuclear magnetic resonance studies support the presence of a strong hydrogen bond between the backbone amide of Asn537 and FMN N5, the anionic ionization state of the hydroquinone, and for a change in the hybridization state of the N5 upon reduction. Replacement of Tyr536, which flanks the flavin ring, with a basic residue (histidine or arginine) did not significantly influence the redox properties of the FMN or the accumulation of the anionic semiquinone. The central residues of the type I' turn (Asn-Gly) were replaced with various combinations of glycine and alanine as a means of altering the turn and its interactions. Gly538 was found to be crucial in maintaining the type I' turn conformation of the loop and the strong H-bonding interaction at N5. The functional role of the tandem Pro-Pro sequence which anchors and possible "rigidifies" the loop was investigated through alanine replacements. Despite changes in the stabilities of the oxidized and hydroquinone redox states of the FMN, none of the replacements studied significantly altered the two-electron midpoint potentials. Pro541 does contribute to some degree to the strength of the N5 interaction and the formation of the anionic semiquinone. Unlike that of the flavodoxin, it would appear that the conformation of the FMN rather than the loop changes in response to reduction in this flavoprotein.

PBRM1 and VHL expression correlate in human clear cell renal cell carcinoma with differential association with patient's overall survival.

OBJECTIVE: To identify the clinicopathological association of PBRM1 (Polybromo-1 gene) and VHL (von Hippel-Lindau gene) expression at mRNA and protein levels in clear cell renal cell carcinoma (ccRCC) and its role in tumor progression. PATIENTS AND METHODS: Immunohistochemical analysis, Western blotting and qPCR analysis of PBRM1 and VHL were performed on fresh-frozen ccRCC and adjacent normal tissue obtained from 70 patients who underwent radical nephrectomy. In addition, a tissue microarray (TMA) from specimens of 326 ccRCC patients was used to evaluate the effect of loss of PBRM1 and VHL immunohistological expression on clinicopathological features as well as patient survival. RESULTS: In frozen tissue, PBRM1 and VHL mRNA were significantly down-regulated in most ccRCC tumors (77.6%/80.6%). Simultaneous weak PBRM1 and VHL protein expression was observed in 21.4% of frozen tumors. In the TMA samples, weak PBRM1 and VHL immunohistochemical staining was observed in 60.4% of the cases and was correlated (P<0.001). The association of PBRM1 and VHL immunohistochemical expression with clinicopathological parameters depicts a variable picture: predominantly weak PBRM1 and VHL expression were significantly associated with higher Fuhrman grade (P = 0.012 and 0.024, respectively) but only weak VHL expression was associated with a higher pT stage (P = 0.023). PBRM1 expression did not affect the overall survival, whereas weak VHL expression was associated with decreased patient overall survival (P = 0.013). CONCLUSIONS: Our data suggest that reduced expression of PBRM1 and VHL is correlated with an increased tumor aggressiveness. Low VHL expression was identified as a risk factor for worse patient overall survival, independently from PBRM1 expression pattern.

Achaete-Scute Homolog 1 Expression Controls Cellular Differentiation of Neuroblastoma.

Neuroblastoma, the major cause of infant cancer deaths, results from fast proliferation of undifferentiated neuroblasts. Treatment of high-risk neuroblastoma includes differentiation with retinoic acid (RA); however, the resistance of many of these tumors to RA-induced differentiation poses a considerable challenge. Human achaete-scute homolog 1 (hASH1) is a proneural basic helix-loop-helix transcription factor essential for neurogenesis and is often upregulated in neuroblastoma. Here, we identified a novel function for hASH1 in regulating the differentiation phenotype of neuroblastoma cells. Global analysis of 986 human neuroblastoma datasets revealed a negative correlation between hASH1 and neuron differentiation that was independent of the N-myc (MYCN) oncogene. Using RA to induce neuron differentiation in two neuroblastoma cell lines displaying high and low levels of hASH1 expression, we confirmed the link between hASH1 expression and the differentiation defective phenotype, which was reversed by silencing hASH1 or by hypoxic preconditioning. We further show that hASH1 suppresses neuronal differentiation by inhibiting transcription at the RA receptor element. Collectively, our data indicate hASH1 to be key for understanding neuroblastoma resistance to differentiation therapy and pave the way for hASH1-targeted therapies for augmenting the response of neuroblastoma to differentiation therapy.

Multilevel regulation of HIF-1 signaling by TTP.

Hypoxia-inducible factor-1 (HIF-1) is a well-studied transcription factor mediating cellular adaptation to hypoxia. It also plays a crucial role under normoxic conditions, such as in inflammation, where its regulation is less well understood. The 3'-untranslated region (UTR) of HIF-1α mRNA is among the most conserved UTRs in the genome, hinting toward posttranscriptional regulation. To identify potential trans factors, we analyzed a large compilation of expression data. In contrast to its known function of being a negative regulator, we found that tristetraprolin (TTP) positively correlates with HIF-1 target genes. Mathematical modeling predicts that an additional level of posttranslational regulation of TTP can explain the observed positive correlation between TTP and HIF-1 signaling. Mechanistic studies revealed that TTP indeed changes its mode of regulation from destabilizing to stabilizing HIF-1α mRNA upon phosphorylation by p38 mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2. Using a model of monocyte-to-macrophage differentiation, we show that TTP-driven HIF-1α mRNA stabilization is crucial for cell migration. This demonstrates the physiological importance of a hitherto-unknown mechanism for multilevel regulation of HIF-1α in normoxia.

SMN-independent subunits of the SMN complex. Identification of a small nuclear ribonucleoprotein assembly intermediate.

The survival of motor neurons (SMN) complex is essential for the biogenesis of small nuclear ribonucleoprotein (snRNP) complexes in eukaryotic cells. Reduced levels of SMN cause the motor neuron degenerative disease, spinal muscular atrophy. We identify here stable subunits of the SMN complex that do not contain SMN. Sedimentation and immunoprecipitation experiments using cell extracts reveal at least three complexes composed of Gemin3, -4, and -5; Gemin6, -7, and unrip; and SMN with Gemin2, as well as free Gemin5. Complexes containing Gemin3-Gemin4-Gemin5 and Gemin6-Gemin7-unrip persist at similar levels when SMN is reduced. In cells, immunofluorescence microscopy shows differential localization of Gemin5 after cell stress. We further show that the Gemin5-containing subunits bind small nuclear RNA independently of the SMN complex and without a requirement for exogenous ATP. ATP hydrolysis is, however, required for displacement of small nuclear RNAs from the Gemin5-containing subunits and their assembly into snRNPs. These findings demonstrate a modular nature of the SMN complex and identify a new intermediate in the snRNP assembly process.