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Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions., (© 2019 Kuan et al.)

Proper dendritic elaboration of neurons is critical for the formation of functional circuits during brain development. Defects in dendrite morphogenesis are associated with neuropsychiatric disorders, and microRNAs are emerging as regulators of aspects of neuronal maturation such as axonal and dendritic growth, spine formation, and synaptogenesis. Here, we show that miR-214 plays a pivotal role in the regulation of dendritic development. Overexpression of miR-214 increased dendrite size and complexity, whereas blocking of endogenous miR-214-3p, a mature form of miR-214, inhibited dendritic morphogenesis. We also found that miR-214-3p targets quaking (Qki), which is implicated in psychiatric diseases such as schizophrenia, through conserved target sites located in the 3'-untranslated region of Qki mRNA, thereby down-regulating Qki protein levels. Overexpression and knockdown of Qki impaired and enhanced dendritic formation, respectively. Moreover, overexpression of Qki abolished the dendritic growth induced by miR-214 overexpression. Taken together, our findings reveal a crucial role for the miR-214-Qki pathway in the regulation of neuronal dendritic development., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Synapse disruption takes place in many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the mechanistic understanding of this process is still limited. We set out to study a possible role for dynein in synapse integrity. Cytoplasmic dynein is a multisubunit intracellular molecule responsible for diverse cellular functions, including long-distance transport of vesicles, organelles, and signaling factors toward the cell center. A less well-characterized role dynein may play is the spatial clustering and anchoring of various factors including mRNAs in distinct cellular domains such as the neuronal synapse. Here, in order to gain insight into dynein functions in synapse integrity and disruption, we performed a screen for novel dynein interactors at the synapse. Dynein immunoprecipitation from synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, followed by mass spectrometry analysis on synaptic fractions of the ALS model mSOD1(G93A) and wild-type controls, was performed. Using advanced network analysis, we identified Staufen1, an RNA-binding protein required for the transport and localization of neuronal RNAs, as a major mediator of dynein interactions via its interaction with protein phosphatase 1-beta (PP1B). Both in vitro and in vivo validation assays demonstrate the interactions of Staufen1 and PP1B with dynein, and their colocalization with synaptic markers was altered as a result of two separate ALS-linked mutations: mSOD1(G93A) and TDP43(A315T). Taken together, we suggest a model in which dynein's interaction with Staufen1 regulates mRNA localization along the axon and the synapses, and alterations in this process may correlate with synapse disruption and ALS toxicity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Signal sequence-encoding mRNAs undergo translation-dependent localization to the endoplasmic reticulum (ER) and at the ER are anchored via translation on Sec61-bound ribosomes. Recent investigations into the composition and membrane association characteristics of ER-associated mRNAs have, however, revealed both ribosome-dependent (indirect) and ribosome-independent (direct) modes of mRNA association with the ER. These findings raise important questions regarding our understanding of how mRNAs are selected, localized, and anchored to the ER. Using semi-intact tissue culture cells, we performed a polysome solubilization screen and identified conditions that distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. To gain insight into the molecular basis of direct mRNA anchoring to the ER, we performed RNA-protein UV photocross-linking studies in rough microsomes and demonstrate that numerous ER integral membrane proteins display RNA binding activity. Quantitative proteomic analyses of HeLa cytosolic and ER-bound polysome fractions identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins, whereas translocon accessory proteins, such as ribophorin I, were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocross-linked ER protein-RNA adducts identified Sec61α,β and ribophorin I as ER-poly(A) mRNA-binding proteins, suggesting unexpected roles for the protein translocation and modification machinery in mRNA anchoring to the ER. In summary, we propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable an mRNA transcriptome-wide function for the ER in protein synthesis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Initial steps in protein synthesis are highly regulated processes as they define the reading frame of the translation machinery. Eukaryotic translation initiation is a process facilitated by numerous factors (eIFs), aimed to form a "scanning" mechanism toward the initiation codon. Translation initiation of the main open reading frame (ORF) in an mRNA transcript has been reported to be regulated by upstream open reading frames (uORFs) in a manner of re-initiation. This mode of regulation is governed by the phosphorylation status of eIF2α and controlled by cellular stresses. Another mode of translational initiation regulation is leaky scanning, and this regulatory process has not been extensively studied. We have identified arsenite- inducible regulatory particle-associated protein (AIRAP) transcript to be translationally induced during arsenite stress conditions. AIRAP transcript contains a single uORF in a poor-kozak context. AIRAP translation induction is governed by means of leaky scanning and not re-initiation. This induction of AIRAP is solely dependent on eIF1 and the uORF kozak context. We show that eIF1 is phosphorylated under specific conditions that induce protein misfolding and have biochemically characterized this site of phosphorylation. Our data indicate that leaky scanning like re-initiation is responsive to stress conditions and that leaky scanning can induce ORF translation by bypassing poor kozak context of a single uORF transcript.

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.

The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.

Epithelial-mesenchymal transition (EMT), a tightly regulated process that is critical for development, is frequently re-activated during cancer metastasis and recurrence. We reported previously that CD44 isoform switching is critical for EMT and showed that the splicing factor ESRP1 inhibits CD44 isoform switching during EMT. However, the mechanism by which ESRP1 is regulated during EMT has not been fully understood. Here we show that the transcription repressor Snail binds to E-boxes in the ESRP1 promoter, causing repression of the ESRP1 gene. Biochemically, we define the mechanism by which ESRP1 regulates CD44 alternative splicing: ESRP1 binds to the intronic region flanking a CD44 variable exon and causes increased variable exon inclusion. We further show that ectopically expressing ESRP1 inhibits Snail-induced EMT, suggesting that down-regulation of ESRP1 is required for function by Snail in EMT. Together, these data reveal how the transcription factor Snail mediates EMT through regulation of a splicing factor.

The mRNA of hif-1α is considered as being constitutively and ubiquitously expressed, regardless of the level of oxygen tension. However many recent reports have showed that hif-1α mRNA could be regulated by natural antisense transcripts, potential microRNAs, and low O(2). In this study, it was found that a deficiency of JNK2 expression reduced HIF-1α protein induction in response to nickel treatment resulting from the impaired expression of hif-1α mRNA. Both the promoter luciferase assay and mRNA degradation assay clearly showed that depletion of JNK2 affected stability of hif-1α mRNA, rather than regulated its transcription. In addition, nucleolin, a classic histone chaperone, was demonstrated to physically bind to hif-1α mRNA and maintain its stability. Further investigation indicated that JNK2 regulated nucleolin expression and might in turn stabilize hif-1α mRNA. Collectively, we provided one more piece of evidence for the oncogenic role of JNK2 and nucleolin in regulating the cancer microenvironments by controlling HIF-1α expression.

The translation of non-stop mRNA (which lack in-frame stop codons) represents a significant quality control problem for all organisms. In eubacteria, the transfer-messenger RNA (tmRNA) system facilitates recycling of stalled ribosomes from non-stop mRNA in a process termed trans-translation or ribosome rescue. During rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which promotes polypeptide degradation after release from the stalled ribosome. Escherichia coli possesses an additional ribosome rescue pathway mediated by the ArfA peptide. The E. coli arfA message contains a hairpin structure that is cleaved by RNase III to produce a non-stop transcript. Therefore, ArfA levels are controlled by tmRNA through ssrA-peptide tagging and proteolysis. Here, we examine whether ArfA homologues from other bacteria are also regulated by RNase III and tmRNA. We searched 431 arfA coding sequences for mRNA secondary structures and found that 82.8% of the transcripts contain predicted hairpins in their 3'-coding regions. The arfA hairpins from Haemophilus influenzae, Proteus mirabilis, Vibrio fischeri, and Pasteurella multocida are all cleaved by RNase III as predicted, whereas the hairpin from Neisseria gonorrhoeae functions as an intrinsic transcription terminator to generate non-stop mRNA. Each ArfA homologue is ssrA-tagged and degraded when expressed in wild-type E. coli cells, but accumulates in mutants lacking tmRNA. Together, these findings show that ArfA synthesis from non-stop mRNA is a conserved mechanism to regulate the alternative ribosome rescue pathway. This strategy ensures that ArfA homologues are only deployed when the tmRNA system is incapacitated or overwhelmed by stalled ribosomes.

Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy. In myotonic dystrophy, the lack of properly localized MBNL1 leads to missplicing of many pre-mRNAs. One of these events is the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3'-end of intron 4 is non-canonical in that it contains a predicted branch point that is 141 nucleotides from the 3'-splice site and an AAG 3'-splice site. Using a minigene that includes exon 4, intron 4, exon 5, intron 5, and exon 6 of MBNL1, we showed that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branch point confirmed that branching occurs primarily at the predicted distant branch point. Structure probing and footprinting revealed that the highly conserved region between the branch point and 3'-splice site is primarily unstructured and that MBNL1 binds within this region of the pre-mRNA. Deletion of the MBNL1 response element eliminated MBNL1 splicing regulation and led to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.

Post-transcriptional mechanisms play an important role in the control of inflammatory gene expression. The heterogeneous nuclear ribonucleoprotein K homology (KH)-type splicing regulatory protein (KSRP) triggers rapid degradation of mRNAs for various cytokines, chemokines, and other inflammation-related proteins by interacting with AU-rich elements (AREs) in the 3'-untranslated mRNA regions. In addition to destabilizing mRNAs, AU-rich elements can restrict their translation. Evidence that KSRP also participates in translational silencing was obtained in a screen comparing the polysome profiles of cells with siRNA-mediated depletion of KSRP with that of control cells. Among the group of mRNAs showing increased polysome association upon KSRP depletion are those of interleukin (IL)-6 and IL-1α as well as other ARE-containing transcripts. Redistribution of IL-6 mRNA to polysomes was associated with increased IL-6 protein secretion by the KSRP-depleted cells. Silencing of IL-6 and IL-1α mRNAs depended on their 3'-untranslated regions. The sequence essential for translational control of IL-6 mRNA and its interaction with KSRP was located to an ARE. KSRP-dependent silencing was reversed by IL-1, a strong inducer of IL-6 mRNA and protein expression. The results identify KSRP as a protein involved in ARE-mediated translational silencing. They suggest that KSRP restricts inflammatory gene expression not only by enhancing degradation of mRNAs but also by inhibiting translation, both functions that are counteracted by the proinflammatory cytokine IL-1.

According to the "free radical theory" of aging, premature senescence induced by oxidative stress contributes to organismal aging. Polymerase I and transcript release factor (PTRF)/cavin-1 is a structural protein component of caveolae, invaginations of the plasma membrane involved in signal transduction. We show that oxidative stress up-regulates PTRF/cavin-1 protein expression and promotes the interaction between PTRF/cavin-1 and caveolin-1, another structural protein component of caveolae. Consistent with these data, the number of caveolae is dramatically increased in cells subjected to oxidative stress. We demonstrate that down-regulation of PTRF/cavin-1 by shRNA significantly inhibits oxidative stress-induced premature senescence. Mechanistically, we found that PTRF/cavin-1 expression is necessary for the oxidant-induced sequestration of Mdm2, a negative regulator of p53, into caveolar membranes, away from p53, and activation of the p53/p21(Waf1/Cip1) pathway. Expression of a mutant form of PTRF/cavin-1, which fails to localize to caveolar membranes after oxidative stress, inhibits oxidative stress-induced activation of p53 and induction of premature senescence. Thus, PTRF/cavin-1 is a novel regulator of oxidative stress-induced premature senescence by acting as a link between free radicals and activation of the p53/p21(Waf1/Cip1) pathway.

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

Heat shock factor-1 (HSF1) is the central regulator of heat-induced transcriptional responses leading to rapid expression of molecular chaperones that protect mammalian cells against proteotoxic stress. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat shock genes encoding a variety of heat shock proteins, including HSP70, HSP90, HSP27, and other proteins of the network. Herein we report that the zinc finger AN1-type domain-2a gene, also known as AIRAP, behaves as a canonical heat shock gene, whose expression is temperature-dependent and strictly controlled by HSF1. Transcription is triggered at temperatures above 40 degrees C in different types of human cancer and primary cells, including peripheral blood monocytes. As shown by ChIP analysis, HSF1 is recruited to the AIRAP promoter rapidly after heat treatment, with a kinetics that parallels HSP70 promoter HSF1-recruitment. In transfection experiments HSF1-silencing abolished heat-induced AIRAP promoter-driven transcription, which could be rescued by exogenous Flag-HSF1 expression. The HSF1 binding HSE sequence in the AIRAP promoter critical for heat-induced transcription was identified. Because its expression is induced at febrile temperatures in human cells, AIRAP may represent a new potential component of the protective response during fever in humans.

Musashi1 (Msi1) is a highly conserved RNA-binding protein with pivotal functions in stem cell maintenance, nervous system development, and tumorigenesis. Despite its importance, only three direct mRNA targets have been characterized so far: m-numb, CDKN1A, and c-mos. Msi1 has been shown to affect their translation by binding to short elements located in the 3'-untranslated region. To better understand Msi1 functions, we initially performed an RIP-Chip analysis in HEK293T cells; this method consists of isolation of specific RNA-protein complexes followed by identification of the RNA component via microarrays. A group of 64 mRNAs was found to be enriched in the Msi1-associated population compared with controls. These genes belong to two main functional categories pertinent to tumorigenesis: 1) cell cycle, cell proliferation, cell differentiation, and apoptosis and 2) protein modification (including ubiquitination and ubiquitin cycle). To corroborate our findings, we examined the impact of Msi1 expression on both mRNA (transcriptomic) and protein (proteomic) expression levels. Genes whose mRNA levels were affected by Msi1 expression have a Gene Ontology distribution similar to RIP-Chip results, reinforcing Msi1 participation in cancer-related processes. The proteomics study revealed that Msi1 can have either positive or negative effects on gene expression of its direct targets. In summary, our results indicate that Msi1 affects a network of genes and could function as a master regulator during development and tumor formation.

The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse alpha-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe(2)GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe(2)GlyDH, shedding new insights into the protection of heart from hypoxic damages.

Alternative splicing of pre-mRNAs plays an important role in generating biological and functional diversity. Neuro-oncological ventral antigen 1 (Nova1) is a neuron-specific splicing factor that controls the alternative processing of a wide array of mRNAs important for synaptic activity. It is essential for the proper development of the mammalian motor system and for the survival of motoneurons. Because Nova1 gene contains putative regulatory AU-rich elements (ARE) in its highly conserved 3'-untranslated region, we investigated whether its expression is regulated by post-transcriptional mechanisms mediated by ARE-binding proteins. Among these, the neuronal ELAV (nELAV) factors are interesting candidates, because their RNA binding activity is necessary for neuronal differentiation and maintenance. By analysis of ribonucleoprotein complexes in vivo and in vitro we demonstrated that the Nova1 mRNA is a novel target of the nELAV proteins. We defined the nELAV binding site by functional experiments with luciferase reporter gene and Nova1 3'-untranslated region deletion sequences. Gene silencing and overexpression of the nELAV member HuD in motoneuronal NSC34 cells indicate that Nova1 mRNA stability and translation are positively and strongly controlled by the nELAV proteins. In addition, nELAV phosphorylation by a PKC-dependent pathway induces the recruitment of Nova1 mRNA to polysomes. Noteworthy, we found that nELAV proteins are also able to modulate Nova1 splicing activity on its target genes. Our data indicate nELAV proteins as the first factors affecting the expression and activity of the neuronal splicing regulator Nova1 and, consequently, as major candidates for the physiological modulation of Nova1-dependent processing of pre-mRNAs in neurons.

TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression.

mRNAs in eukaryotic cells are presumed to always associate with a set of proteins to form mRNPs. In Xenopus oocytes, a large pool of maternal mRNAs is masked from the translational apparatus as storage mRNPs. Here we identified Xenopus RAP55 (xRAP55) as a component of RNPs that associate with FRGY2, the principal component of maternal mRNPs. RAP55 is a member of the Scd6 or Lsm14 family. RAP55 localized to cytoplasmic foci in Xenopus oocytes and the processing bodies (P-bodies) in cultured human cells: in the latter cells, RAP55 is an essential constituent of the P-bodies. We isolated xRAP55-containing complexes from Xenopus oocytes and identified xRAP55-associated proteins, including a DEAD-box protein, Xp54, and a protein arginine methyltransferase, PRMT1. Recombinant xRAP55 repressed translation, together with Xp54, in an in vitro translation system. In addition, xRAP55 repressed translation in oocytes when tethered with a reporter mRNA. Domain analyses revealed that the N-terminal region of RAP55, including the Lsm domain, is important for the localization to P-bodies and translational repression. Taken together, our results suggest that xRAP55 is involved in translational repression of mRNA as a component of storage mRNPs.

Cellular hypoxia response is regulated at the level of hypoxia-inducible factor (HIF) activity. A number of recently identified oxygen sensors are HIF-modifying enzymes that respond to low oxygen by altering HIF modification and thus lead to its activation. In addition to the HIF proline hydroxylases and asparagine hydroxylases, ARD1 is recently described as a HIF-1alpha acetylase that regulates its stability. We found that ARD1 is down-regulated in a number of cell lines in response to hypoxia and hypoxia mimic compounds. After surveying these lines for erythropoietin production and retroviral transfection efficiency, we chose to use HepG2 cells to study the function of ARD1. ARD1 short hairpin RNA delivered by a retroviral vector caused >80% reduction in ARD1 message. We observed decreases in erythropoietin and vascular endothelial growth factor protein production, whereas there was no change in the HIF-1alpha protein level. A gene chip analysis of HepG2 cells transduced with virus expressing ARD1 short hairpin RNA under normoxia and hypoxia conditions or with virus overexpressing recombinant ARD1 confirmed that inhibition of ARD1 does not cause activation of HIF and downstream target genes. However, this analysis revealed that ARD1 is involved in cell proliferation and in regulating a series of cellular metabolic pathways that are regulated during hypoxia response. The role of ARD1 in cell proliferation is confirmed using fluorescence labeling analysis of cell division. From these studies we conclude that ARD1 is not required to suppress HIF but is required to maintain cell proliferation in mammalian cells.

Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients.

In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of l-tryptophan. Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation. Conversely, the accumulation of uncharged tRNA(Trp) induces synthesis of an anti-TRAP protein (AT), which forms a complex with TRAP and inhibits its activity. In this report, we investigate the structural features of TRAP required for AT recognition. A collection of TRAP mutant proteins was examined that were known to be partially or completely defective in tryptophan binding and/or RNA binding. Analyses of AT interactions with these proteins were performed using in vitro transcription termination assays and cross-linking experiments. We observed that TRAP mutant proteins that had lost the ability to bind RNA were no longer recognized by AT. Our findings suggest that AT acts by competing with messenger RNA for the RNA binding domain of TRAP. B. subtilis AT was also shown to interact with TRAP proteins from Bacillus halodurans and Bacillus stearothermophilus, implying that the structural elements required for AT recognition are conserved in the TRAP proteins of these species. Analyses of AT interaction with B. stearothermophilus TRAP at 60 degrees C demonstrated that AT is active at this elevated temperature.

Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a [4Fe-4S] cluster. Recent evidence implicates oxidants in cluster disassembly. We investigated H(2)O(2)-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae. A signal for [3Fe-4S] c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal. H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D). Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) and were more responsive to changes in media iron status. Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases. Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase. This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation. Our results demonstrate that 3Fe c-aconitase is an intermediate in H(2)O(2)-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast. Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.

Intimin-gamma is an outer membrane protein of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that is required for the organism to adhere tightly to HEp-2 cells and to colonize experimental animals. Another EHEC O157:H7 protein, the Transferred intimin receptor (Tir), is considered the primary receptor for intimin-gamma. Nevertheless, Tir-independent binding of intimin-gamma to HEp-2 cells has been reported. This observation suggests the existence of a eukaryotic receptor(s) for intimin-gamma. In this study, we sought to identify that receptor(s). First, we determined by equilibrium binding titration that the association of purified intimin-gamma with HEp-2 cells was specific and consistent with a single host cell receptor. Second, we isolated a protein from lysates of HEp-2 cells that bound intimin-gamma and subsequently identified this molecule as nucleolin, a protein involved in cell growth regulation that can be cell surface-expressed. Third, we established that purified intimin-gamma and nucleolin were co-localized on the surface of HEp-2 cells and that the site of EHEC O157:H7 attachment was associated with regions of nucleolin expression. Finally, we demonstrated that mouse anti-nucleolin sera significantly decreased the adherence of EHEC O157:H7 to HEp-2 cells. From this, we conclude that nucleolin is the HEp-2 cell receptor for intimin-gamma expressed by EHEC O157:H7.

AU-rich elements found in the 3'-untranslated regions of cytokine and proto-oncogene transcripts regulate mRNA degradation and function as binding sites for the mRNA-stabilizing protein HuA and the mRNA-destabilizing protein tristetraprolin. Experiments were performed to evaluate the expression of HuA and tristetraprolin in purified human T lymphocytes and to evaluate the ability of these proteins to recognize specific AU-rich sequences. HuA is a predominantly nuclear protein that can also be found in the cytoplasm of resting T lymphocytes. Within 1 h after stimulation of T lymphocytes with anti-T cell receptor antibodies or a combination of a phorbol myristate acetate and ionomycin, an increase in cytoplasmic HuA RNA-binding activity was observed. Although absent in resting cells, cytoplasmic tristetraprolin protein was detected 3-6 h following activation. HuA recognized specific AU-rich sequences found in c-jun or c-myc mRNA that were poorly recognized by tristetraprolin. In contrast, tristetraprolin recognized an AU-rich sequence in interleukin-2 mRNA that was poorly recognized by HuA. Both HuA and tristetraprolin, however, recognized AU-rich sequences from c-fos, interleukin-3, tumor necrosis factor-alpha, and granulocyte/macrophage colony-stimulating factor mRNA. HuA may transiently stabilize a subset of AU-rich element-containing transcripts following T lymphocyte activation, and tristetraprolin may subsequently mediate their degradation.

Although mild hypothermia generally reduces protein synthesis in mammalian cells, the expression of a small number of proteins, including Rbm3, is induced under these conditions. In this study, we identify an Rbm3 mRNA with a complex 5' leader sequence containing multiple upstream open reading frames. Although these are potentially inhibitory to translation, monocistronic reporter mRNAs containing this leader were translated relatively efficiently. In addition, when tested in the intercistronic region of dicistronic mRNAs, this leader dramatically enhanced second cistron translation, both in transfected cells and in cell-free lysates, suggesting that the Rbm3 leader mediates cap-independent translation via an internal ribosome entry site (IRES). Inasmuch as Rbm3 mRNA and protein levels are both increased in cells exposed to mild hypothermia, the activity of this IRES was evaluated at a cooler temperature. Compared to 37 degrees C, IRES activity at 33 degrees C was enhanced up to 5-fold depending on the cell line. Moderate enhancements also occurred with constructs containing other viral and cellular IRESes. These effects of mild hypothermia on translation were not caused by decreased cell growth, as similar effects were not observed when cells were serum starved. The results suggest that cap-independent mechanisms may facilitate the translation of particular mRNAs during mild hypothermia.

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding beta-alpha-beta fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the filamentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein.

The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.

Iron regulatory proteins (IRP1 and IRP2) are two cytoplasmic RNA-binding proteins that control iron metabolism in mammalian cells. Both IRPs bind to specific sequences called iron-responsive elements (IREs) located in the 3' or 5' untranslated regions of several mRNAs, in particular mRNA encoding ferritin and transferrin receptor. In this study, we followed in parallel the in vivo regulation of the two IRPs in physiologically stimulated macrophages. We show that stimulation of mouse RAW 264.7 macrophage-like cells increased IRP1 IRE binding activity 4-fold, whereas IRP2 activity decreased 2-fold 8 h after interferon-gamma/lipopolysaccharide treatment. Decrease in IRP2 was not due to nitric oxide (NO) production and did not require de novo protein synthesis. Our data therefore indicate that the two IRPs can be conversely regulated in response to the same stimulus. In addition, the effect of endogenously produced NO on IRP1 was further characterized in an activated macrophage/target cell system. We show that NO acts as an intercellular signal to increase IRP1 activity in adjacent cells. As the effect was detectable within 1 h and did not require de novo protein synthesis, this result supports a direct action of NO on IRP1.

Eukaryotic initiation factor 4E (eIF4E) is the subunit of eIF4F that binds to the cap structure at the 5' end of messenger RNA and is a critical component for the regulation of translation initiation. Using 7-methyl-GTP-Sepharose affinity chromatography, two distinct cap-binding proteins that migrate on SDS-polyacrylamide gel electrophoresis at approximately 35 kDa were purified from Drosophila adults. Peptide microsequence analysis indicated that these two proteins differ at their amino termini. Analysis of a set of cDNA clones encoding eIF4E led to the conclusion that the two different protein isoforms, which we term eIF4EI and eIF4EII, result from three alternatively spliced transcripts from a single eIF4E gene, which maps to region 67A8-B2 on polytene chromosomes. The three eIF4E transcripts also vary greatly in the lengths of their 5'-UTRs, suggesting the possibility of complex translational control of expression of the two eIF4E isoforms.

Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.

UV cross-linking was used to identify estrogen-induced hepatocyte proteins that bind to apoII mRNA. Probes spanning the entire message revealed the presence of eight estrogen-induced proteins cross-linked to the 3'-untranslated region (UTR), but not to the coding region or the 5'-UTR. Two estrogen-induced proteins of 132 and 50 kDa were either absent or barely detectable in control animals, whereas six additional proteins of 93, 83, 74, 65, 58, and 45 kDa were clearly present in control animals and increased 2-5-fold by estrogen. A similar profile of estrogen-induced proteins was seen with the 3'-UTRs of the estrogen-regulated mRNAs for apoB and vitellogenin II, but not with the 3'-UTRs of the non-estrogen-regulated mRNAs for apoA-I and glyceraldehyde-phosphate dehydrogenase. These findings indicate that the estrogen-induced proteins discriminate among mRNAs and suggest that they interact selectively with the family of estrogen-regulated mRNAs. The estrogen-induced proteins are found in the cytoplasmic fraction of liver extracts, and a subset of them are also found in adrenal glands, testes, heart, brain, and kidneys, but they are estrogen-induced only in the liver. Deletion analysis defined a 150-nucleotide region of the apoII 3'-UTR that is necessary for maximal binding of the estrogen-induced proteins. An internal deletion of endonucleolytic cleavage sites previously identified within the apoII 3'-UTR selectively reduced the binding of the 58-kDa protein. These findings reveal remarkable complexity in estrogen-stimulated protein-RNA interactions within the 3'-UTRs of estrogen-regulated mRNAs. These proteins may participate in the mRNA degradation process or in other aspects of cytoplasmic mRNA metabolism that accompany estrogen-stimulated vitellogenesis.

A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.

We have examined the effects of illumination, carbon source, and levels of chloroplast protein synthesis on trans-acting proteins that bind to the leaders of five representative chloroplast mRNAs. The accumulation of these five chloroplast mRNAs and the proteins they encode were measured in cells grown under identical conditions. Extracts from all cell types examined contain a minimum set of six chloroplast 5'-untranslated region (UTR)-binding proteins (81, 62, 56, 47, 38, and 15 kDa). Fractionation results suggest that multiple forms of the 81-, 62-, and 47-kDa proteins may exist. A 36-kDa protein was found in all cells except those deficient in chloroplast protein synthesis. Binding of the 81-, 47-, and 38-kDa proteins to the rps12 leader is effectively competed by the atpB or rbcL 5'-UTRs, indicating that the same proteins bind to all three leaders. In contrast, these three proteins do not bind to the nuclear-encoded alpha-1 tubulin leader, which bound novel proteins of 110, 70, and 43 kDa. Cis-acting sequences within the 5'-UTRs of two chloroplast mRNAs (rps7 and atpB) have been identified which are protected from digestion by RNase T1 by extracts enriched for the 81-, 47-, and 38-kDa proteins.

Iron-responsive elements (IREs) are cis-acting mRNA stem-loop structures that specifically bind cytoplasmic iron regulatory proteins (IRPs). IRP-IRE interactions mediate the coordinate post-transcriptional regulation of key proteins in iron metabolism, such as ferritin, transferrin receptor, and erythroid 5-aminolevulinic acid synthase. Depending on whether the IRE is located in the 5'- or 3'-untranslated region (UTR), binding of IRP will inhibit mRNA translation or degradation, respectively. Here we describe a new IRE in the 5'-UTR of succinate dehydrogenase subunit b (SDHb) mRNA of Drosophila melanogaster. The SDHb IRE binds in vitro to vertebrate and insect IRPs with a high affinity equal to that of human ferritin H chain IRE. Under conditions of iron deprivation, SDHb mRNA of Drosophila SL-2 cells shifts to a non-polysome-bound pool. Moreover, translation of a human growth hormone mRNA with the SDHb IRE in its 5'-UTR is iron-dependent in stably transfected L cells. We conclude that the SDHb IRE mediates translational inhibition both in insect and vertebrate cells. This constitutes the first identification of a functional IRE in insects. Furthermore, Drosophila SDHb represents the second example, after porcine mitochondrial aconitase, of an enzyme of the citric acid cycle whose mRNA possesses all necessary features for translational regulation by cellular iron levels.

Astrocytes have a critical role in the neuronal response to ischemia, as their production of neurotrophic mediators can favorably impact on the extreme sensitivity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed two ribonucleoprotein domains and serine/arginine-rich domains, suggestive of their function as RNA splicing factor. Northern analysis displayed striking induction only in cultured astrocytes within 15 min of reoxygenation and reached a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrated expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/reoxygenated astrocytes. Induction of RA301 mRNA was mediated, in large part, by endogenously generated reactive oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase which blocks oxygen-free radical formation by astrocytes. Similarly, increased expression of RA301 in supporting a neurotrophic function of astrocytes was suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expression is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues.

The Xenopus Y-box protein FRGY2 has a role in the translational silencing of masked maternal mRNA. Here, we determine that FRGY2 will recognize specific RNA sequences. The evolutionarily conserved nucleic acid-binding cold shock domain is required for sequence-specific interactions with RNA. However, RNA binding by FRGY2 is facilitated by N- and C-terminal regions flanking the cold shock domain. The hydrophilic C-terminal tail domain of FRGY2 interacts with RNA independent of the cold shock domain but does not determine sequence specificity. Thus, both sequence-specific and nonspecific RNA recognition domains are contained within the FRGY2 protein.

The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. In a previous report, we showed that recombinant NC protein from HIV-1, a 71-amino-acid protein (NC71), is apparently able to form two types of protein-nucleic acid complexes under low [NaCl], pH 8.3 and 25 degrees C. These appeared to differ in occluded apparent site size, napp, forming n = 8 and n = 14 complexes on poly(A) (Dib-Hajj, F., Khan, R., and Giedroc, D. P. (1993) Protein Sci. 2, 331-243) under conditions of high and low protein-nucleotide ratios, respectively. Here we show that both NC71-poly(A) complexes strongly scatter light under these solution conditions. Examination of the wavelength dependence of the light scattering at lambda < or = 320 nm indicates that each complex is characterized by a different scattering coefficient. Optical density measurements suggest that upon formation of the saturated n = 8 complex, additional polynucleotide is not incorporated into the complex over a period of hours, i.e. the n = 14 complex is not formed via redistribution of the n = 8 complex under low salt conditions, 25 degrees C. In contrast, the n = 14 complex readily incorporates additional protein until that sufficient to form the n = 8 complex is present. The n = 14 complex efficiently precipitates poly(A) and shows spectral characteristics expected for an extensively charge-neutralized nucleic acid complex. At [NC71] in excess of that required to form the n = 8 complex, this n = 14 complex is best described as a kinetic intermediate on the pathway to the n = 8 complex, which forms over a period of hours under low salt conditions, 25 degrees C. This slow kinetics of binding provides a possible explanation for the finding that the previously observed moderate cooperativity of Zn2 NC71 binding to poly(A) (omega = 200) at pH 8.3 and 0.29 M NaCl (Khan, R., and Giedroc, D. P. (1992) J. Biol. Chem. 267, 6689-6695) is shown here to represent a nonequilibrium phenomenon, apparently converting to a low or no cooperativity complex over a period of hours. Proteolytic removal of the COOH-terminal 14 amino acids from NC71, forming a 57-amino-acid protein (denoted NC57), removes this apparent binding site size heterogeneity of NC71 on poly(A). At 20 mM NaCl, NC57 binds with n = 6-7 nucleotides, in a manner which is independent of the protein-poly(A) nucleotide ratio. The implications of these findings on processing of the gag precursor which leads to mature NC in HIV-1 virions is discussed.

Delineating the molecular basis for agonist-induced destabilization of mRNA of G-protein-linked receptors that contributes to receptor down-regulation is fundamental to our understanding of long-term regulation of receptors by agonist. Previously we identified a prominent, M(r) 35,000 cytosolic RNA-binding protein that (i) binds selectively to beta 1 and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind either to alpha 1b-adrenergic receptor mRNA, which does not undergo agonist-induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A),-(C), or -(G) RNA; and (iv) its abundance varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA (Port, J. D., Huang, L.-y., and Malbon (1992) J. Biol. Chem. 267, 24103-24108). We demonstrate here that the binding of beta-adrenergic receptor mRNA by this protein, termed beta-ARB protein, is sensitive to competition by AU-rich domains of the 3'-untranslated regions of c-fos, c-myc, and human granulocyte-macrophage colony-stimulating factor. Using the AU-rich 3'-untranslated regions of wild-type adenovirus IVa2 mRNA and variants with defined mutations in the AUUUApentamer, AU-rich, and U-rich domains, we were able to define sequences critical to the binding of the beta 2-receptor mRNA by the beta-ARB protein. Recognition of beta-ARB protein requires not only an AUUUA destabilization pentamer, but also a flanking U-rich domain(s). Using radiolabeled 3'-untranslated regions of short-lived mRNA, we were able to identify this same M(r) 35,000 cytosolic RNA-binding protein(s), beta-ARB protein, as selective for beta 2-adrenergic receptor mRNA.

In an effort to explore the molecular basis for agonist-induced destabilization of beta-adrenergic receptor mRNA, we investigated the nature of RNA-binding proteins both in untreated and agonist-treated DDT1-MF2 smooth muscle cells. Messenger RNAs for the alpha 1b-, beta 1-, and beta 2-adrenergic receptors as well as for beta-globin were transcribed in vitro, incubated with cytosolic fractions, covalently cross-linked by short-wave UV light, and analyzed by SDS-polyacrylamide gel electrophoresis. A prominent M(r) 35,000 radiolabeled protein(s) with the following characteristics was identified: (i) binds selectively to beta 1- and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind to either alpha 1b-adrenergic receptor mRNA, which does not undergo agonist induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and (iv) displays binding to receptor mRNA that can be competed by RNA harboring destabilizer sequences that are AU-rich and AUUUA pentamer-rich. The abundance of the M(r) 35,000 RNA-binding protein selective for beta-adrenergic receptor message, a factor we term beta ARB protein, varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA.