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PDF - 109K, Fig. S5. EF-P signals in target protein sequences.

PDF - 468K, Fig. S1. Inhibition of DOHH by ciclopirox and deferiprone. Fig. S2. Cell cycle analysis. Fig. S3. Effects of CPX and DEF on cell size and morphology. Fig. S4. Effect of CPX and DEF on HeLa cell proteins analyzed by 2D gel electrophoresis.

PDF - 74K, Table S1. Identification of drug-dependent spots.

Francis Crick advanced two distinct but interrelated fundamental principles of molecular biology: (1) the Sequence Hypothesis and (2) the Central Dogma. The Sequence Hypothesis defines biological information transfer as the residue-by-residue transfer of sequence information between nucleic acids and to proteins. This is commonly summarized as DNA ➔ RNA ➔ protein and is colloquially referred to as the Central Dogma. More specifically, however, the Central Dogma expounded by Crick included a critical restriction, stipulating that "once sequential information has passed into protein it cannot get out again." Under this definition, the Central Dogma has stood the test of time despite challenges. In principle, a violation of the Central Dogma could transpire through synthetic biology or by natural occurrence. To address these possibilities, we draw insights from existing modes of information transfer in protein synthesis and from synthetic Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) gene-editing. We introduce a three-part evaluation scheme, which we apply to the CRISPR/Cas9 system and the more recent CRISPR prime editing system. Potential mechanisms by which engineered sequence editing systems might violate the Central Dogma are considered. We conclude that although information transfer in protein synthesis and CRISPR gene-editing remain within the bounds of the Central Dogma, the underlying mechanisms point toward an avenue of synthetic biology that could directly violate the Central Dogma. Finally, we speculate on some of the theoretical and practical implications of a protein-derived information transfer system. This article is categorized under: RNA Evolution and GenomicsRibonomics RNA Interactions with Proteins and Other MoleculesProtein-RNA Interactions: Functional Implications TranslationMechanisms.

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Research on non‐coding RNA (ncRNA) is a rapidly expanding field. Providing an official gene symbol and name to ncRNA genes brings order to otherwise potential chaos as it allows unambiguous communication about each gene. The HUGO Gene Nomenclature Committee (HGNC, http://www.genenames.org) is the only group with the authority to approve symbols for human genes. The HGNC works with specialist advisors for different classes of ncRNA to ensure that ncRNA nomenclature is accurate and informative, where possible. Here, we review each major class of ncRNA that is currently annotated in the human genome and describe how each class is assigned a standardised nomenclature., Working with experts in the field, the HUGO Gene Nomenclature Committee describes the standardized nomenclature assigned to all major classes of ncRNA currently annotated in the human genome.

Protein synthesis involves a complex machinery comprising numerous proteins and RNAs joined by noncovalent interactions. Its function is to link long chains of amino acids into proteins with precise sequences as encoded by the genome. Regulation of protein synthesis, called translational control, occurs both at a global level and at specific messenger RNAs (mRNAs). To understand how translation is regulated, knowledge of the molecular structures and kinetic interactions of its components is needed. This review focuses on the targets of translational control and the mechanisms employed.

Advances in sequencing and high-throughput techniques have provided an unprecedented opportunity to interrogate human diseases on a genome-wide scale. The list of disease-causing mutations is expanding rapidly, and mutations affecting mRNA translation are no exception. Translation (protein synthesis) is one of the most complex processes in the cell. The orchestrated action of ribosomes, tRNAs and numerous translation factors decodes the information contained in mRNA into a polypeptide chain. The intricate nature of this process renders it susceptible to deregulation at multiple levels. In this Review, we summarize current evidence of translation deregulation in human diseases other than cancer. We discuss translation-related diseases on the basis of the molecular aberration that underpins their pathogenesis (including tRNA dysfunction, ribosomopathies, deregulation of the integrated stress response and deregulation of the mTOR pathway) and describe how deregulation of translation generates the phenotypic variability observed in these disorders.

As obligate intracellular parasites, virus reproduction requires host cell functions. Despite variations in genome size and configuration, nucleic acid composition, and their repertoire of encoded functions, all viruses remain unconditionally dependent on the protein synthesis machinery resident within their cellular hosts to translate viral messenger RNAs (mRNAs). A complex signaling network responsive to physiological stress, including infection, regulates host translation factors and ribosome availability. Furthermore, access to the translation apparatus is patrolled by powerful host immune defenses programmed to restrict viral invaders. Here, we review the tactics and mechanisms used by viruses to appropriate control over host ribosomes, subvert host defenses, and dominate the infected cell translational landscape. These not only define aspects of infection biology paramount for virus reproduction, but continue to drive fundamental discoveries into how cellular protein synthesis is controlled in health and disease.

Cancer etiology is influenced by alterations in protein synthesis that are not fully understood. In this study, we took a novel approach to investigate the role of the eukaryotic translation initiation factor eIF5A in human cervical cancers, where it is widely overexpressed. eIF5A contains the distinctive amino acid hypusine, which is formed by a posttranslational modification event requiring deoxyhypusine hydroxylase (DOHH), an enzyme that can be inhibited by the drugs ciclopirox and deferiprone. We found that proliferation of cervical cancer cells can be blocked by DOHH inhibition with either of these pharmacologic agents, as well as by RNA interference–mediated silencing of eIF5A, DOHH, or another enzyme in the hypusine pathway. Proteomic and RNA analyses in HeLa cervical cancer cells identified two groups of proteins in addition to eIF5A that were coordinately affected by ciclopirox and deferiprone. Group 1 proteins (Hsp27, NM23, and DJ-1) were downregulated at the translational level, whereas group 2 proteins (TrpRS and PRDX2) were upregulated at the mRNA level. Further investigations confirmed that eIF5A and DOHH are required for Hsp27 expression in cervical cancer cells and for regulation of its key target IκB and hence NF-κB. Our results argue that mature eIF5A controls a translational network of cancer-driving genes, termed the eIF5A regulon, at the levels of mRNA abundance and translation. In coordinating cell proliferation, the eIF5A regulon can be modulated by drugs such as ciclopirox or deferiprone, which might be repositioned to control cancer cell growth. Cancer Res; 74(2); 552–62. ©2013 AACR.

Electrophilic trisubstituted ethylenes, ring-substituted ethyl 2-cyano-3-phenyl-2-propenoates, RPhCH˭C(CN)CO2C2H5 (where R is 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2-F, 3-F, 4-F, 2-Cl-4-F, 2-Cl-6-F, 3-Cl-4-F) were prepared and copolymerized with styrene. The monomers were synthesized by the piperidine catalyzed Knoevenagel condensation of ring-substituted benzaldehydes and ethyl cyanoacetate, and characterized by CHN analysis, IR, 1H and 13C-NMR. All the ethylenes were copolymerized with styrene (M1) in solution with radical initiation (ABCN) at 70°C. The composition of the copolymers was calculated from nitrogen analysis, and the structures were analyzed by IR, 1H and 13C-NMR. The order of relative reactivity (1/r 1) for the monomers is 4-Br (7.74) > 2-Br (1.62) > 3-Cl (1.56) > 3-Br (1.39) > 4-F (0.97) > 3-F (0.86) = 4-Cl (0.86) > 2-Cl-6-F (0.57) > 3-Cl-4-F (0.51) > 2-Cl (0.49) > 2-F (0.42) > 2-Cl-4-F (0.37). Relatively high Tg of the copolymers in comparison with that of polystyrene indicates a decrease i...

The heterodimeric nuclear factor (NF) 90/NF45 complex (NF90/NF45) binds nucleic acids and is a multifunctional regulator of gene expression. Here we report that depletion of NF90/NF45 restores the expression of the p53 and p21 proteins in cervical carcinoma cells infected with high-risk human papillomaviruses (HPVs). Knockdown of either NF90 or NF45 by RNA interference led to greatly elevated levels of p53 and p21 proteins in HPV-derived HeLa and SiHa cells but not in other cancerous or normal cell lines. In HeLa cells, p21 messenger-RNA (mRNA) increased concomitantly but the level of p53 mRNA was unaffected. RNA interference directed against p53 prevented the induction of both proteins. These results indicated that the upregulation of p21 is due to p53-dependent transcription, whereas p53 is regulated post-transcriptionally. Proteasome-mediated turnover of p53 is accelerated by the HPV E6 and cellular E6AP proteins. We therefore examined the hypothesis that this pathway is regulated by NF90/NF45. Indeed, depletion of NF90 attenuated the expression of E6 RNA and inhibited transcription from the HPV early promoter, revealing a new role for NF90/NF45 in HPV gene expression. The transcription inhibition was largely independent of the reduction of P-TEFb (positive transcription elongation factor b) levels caused by NF90 depletion. Consistent with p53 derepression, NF90/NF45-depleted HeLa cells displayed elevated poly ADP-ribose polymerase (PARP) cleavage and susceptibility to camptothecin-induced apoptosis. We conclude that high-risk strains of HPV utilize the cellular NF90/NF45 complex for viral E6 expression in infected cervical carcinoma cell lines. Interference with NF90/NF45 function could assist in controlling cervical carcinoma.

Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased γ-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process.

The small NF90 associated RNA (snaR) family of small noncoding RNAs (ncRNA) appears to have evolved from retrotransposon ancestors at or soon after pivotal stages in primate evolution. snaRs are thought to be derived from a FLAM C-like (free left Alu monomer) element through multiple short insertion/deletion (indel) and nucleotide (nt) substitution events. Tracing snaR’s complex evolutionary history through primate genomes led to the recent discovery of two novel retrotransposons: the Alu/snaR related (ASR) and catarrhine ancestor of snaR (CAS) elements. ASR elements are present in the genomes of Simiiformes, CAS elements are present in Old World Monkeys and apes, and snaRs are restricted to the African Great Apes (Homininae, including human, gorilla, chimpanzee and bonobo). Unlike their ancestors, snaRs have disseminated by multiple rounds of segmental duplication of a larger encompassing element. This process has produced large tandem gene arrays in humans and possibly precipitated the accelerated evolution of snaR. Furthermore, snaR segmental duplication created a new form of chorionic gonadotropin β subunit (CGβ) gene, recently classified as Type II CGβ, which has altered mRNA tissue expression and can generate a novel short peptide.

Spectral Compression Positioning™ (SCP) is an innovative method for deriving positioning, navigation, and timing (PNT) information from Signals of Opportunity (SoOps). The method enables precise PNT in GNSS obstructed environments providing expanded coverage, reliability, and accuracy. This paper introduces the core principles of SCP-based PNT and explores both autonomous and network-based implementation architectures. Providing precise, cost-effective, and autonomous positioning information for navigating indoors or in GPS obstructed environments is a technical challenge that remains largely unsolved. Advances in hybrid Wi-Fi/A-GPS positioning and inertial technologies have yet to deliver meter-level accuracy without the need for custom infrastructure or extensive RF fingerprinting. SCP technology offers a unique alternative approach to current RF-based positioning techniques that readily exploits terrestrial and orbital Signals of Opportunity without the requirement for purpose built infrastructure or a priori knowledge of the signal source location and clock states.

Protein synthesis and its regulation are central to all known forms of life and impinge on biological arenas as varied as agriculture, biotechnology, and medicine. Otherwise known as translation and translational control, these processes have been investigated with increasing intensity since the middle of the 20th century, and in increasing depth with advances in molecular and cell biology. We review the origins of the field, focusing on the underlying concepts and early studies of the cellular machinery and mechanisms involved. We highlight key discoveries and events on a timeline, consider areas where current research has engendered new ideas, and conclude with some speculation on future directions for the field.

Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70–80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3′ untranslated region (3′UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3′UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3′UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-γ in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3′UTR in CHO cells.

We identified a novel family of human noncoding RNAs by in vivo cross-linking to the nuclear factor 90 (NF90) protein. These small NF90-associated RNAs (snaRs) are transcribed by RNA polymerase III and display restricted tissue distribution, with high expression in testis and discrete areas of the brain. The most abundant human transcript, snaR-A, interacts with the cell's transcription and translation systems. snaR genes have evolved in African Great Apes (human, chimpanzee, and gorilla) and some are unique to humans. We traced their ancestry to the Alu SINE (short interspersed nucleotide element) family, via two hitherto unreported sets of short genetic elements termed ASR (Alu/snaR-related) and CAS (Catarrhine ancestor of snaR). This derivation entails a series of internal deletions followed by expansions. The evolution of these genes coincides with major primate speciation events: ASR elements are found in all monkeys and apes, whereas CAS elements are limited to Old World monkeys and apes. In contrast to ASR and CAS elements, which are retrotransposons, human snaR genes are predominantly located in three clusters on chromosome 19 and have been duplicated as part of a larger genetic element. Insertion of the element containing snaR-G into a gene encoding a chorionic gonadotropin beta subunit generated new hormone genes in African Great Apes.

Biochemistry and molecular biology occupy a unique place in the medical school curriculum. They are frequently studied prior to medical school and are fundamental to the teaching of biomedical sciences in undergraduate medical education. These two circumstances, and the trend toward increased integration among the disciplines, have led to reconsideration of biochemistry instruction in many medical schools. We conducted a survey to explore the evolving trends in biochemistry education. A broad diversity was evident in parameters including course content, faculty, governance, prerequisites, and teaching methods. Notably, sharp differences were apparent between freestanding biochemistry courses and those in which biochemistry is integrated with other subjects. Furthermore, the data imply a likely trend toward increased integration of biochemistry with other disciplines in the medical school curriculum.

Topoisomerase IIalpha interacts with numerous nuclear factors, through which it is engaged in diverse nuclear events such as DNA replication, transcription and the formation or maintenance of heterochromatin. We previously reported that topoisomerase IIalpha interacts with RNA helicase A (RHA), consistent with a recent view that topoisomerases and helicases function together. Intrigued by our observation that the RHA-topoisomerase IIalpha interaction is sensitive to ribonuclease A, we explored whether the RHA-topoisomerase IIalpha interaction can be recapitulated in vitro using purified proteins and a synthetic RNA. This work led us to an unexpected finding that an RNA-binding activity is intrinsically associated with topoisomerase IIalpha. Topoisomerase IIalpha stably interacted with RNA harboring a 3'-hydroxyl group but not with RNA possessing a 3'-phosphate group. When measured in decatenation and relaxation assays, RNA binding influenced the catalytic function of topoisomerase IIalpha to regulate DNA topology. We discuss a possible interaction of topoisomerase IIalpha with the poly(A) tail and G/U-rich 3'-untranslated region (3'-UTR) of mRNA as a key step in transcription termination.

Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA- and RNA-binding proteins together with the less-studied protein NF45. These complexes have been implicated in gene regulation, but little is known about their cellular roles and whether they are redundant or functionally distinct. We show that heterodimeric core complexes, NF90-NF45 and NF110-NF45, exist within larger complexes that are more labile and contain multiple NF90/110 isoforms and additional proteins. Depletion of the NF45 subunit by RNA interference is accompanied by a dramatic decrease in the levels of NF90 and NF110. Reciprocally, depletion of NF90 but not of NF110 greatly reduces the level of NF45. Coregulation of NF90 and NF45 is a posttranscriptional phenomenon, resulting from protein destabilization in the absence of partners. Depletion of NF90-NF45 complexes retards cell growth by inhibition of DNA synthesis. Giant multinucleated cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is depleted. This study identified NF45 as an unstable regulatory subunit of NF90-NF45 complexes and uncovered their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct from NF110 and is more important for cell growth.

Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein implicated in multiple cellular functions, but with few identified RNA partners. Using in vivo cross-linking followed by immunoprecipitation, we discovered a family of small NF90-associated RNAs (snaR). These highly structured non-coding RNAs of approximately 117 nucleotides are expressed in immortalized human cell lines of diverse lineages. In human tissues, they are abundant in testis, with minor distribution in brain, placenta and some other organs. Two snaR subsets were isolated from human 293 cells, and additional species were found by bioinformatic analysis. Their genes often occur in multiple copies arranged in two inverted regions of tandem repeats on chromosome 19. snaR-A is transcribed by RNA polymerase III from an intragenic promoter, turns over rapidly, and shares sequence identity with Alu RNA and two potential piRNAs. It interacts with NF90's double-stranded RNA-binding motifs. snaR orthologs are present in chimpanzee but not other mammals, and include genes located in the promoter of two chorionic gonadotropin hormone genes. snaRs appear to have undergone accelerated evolution and differential expansion in the great apes.

The cellular positive transcription elongation factor b (P-TEFb), containing cyclin T1 and cyclin-dependent kinase 9 (CDK9), interacts with the human immunodeficiency virus, type 1 (HIV-1) regulatory protein Tat to enable viral transcription and replication. Cyclin T1 is an unusually long cyclin and is engaged by cellular regulatory proteins. Previous studies showed that the granulin/epithelin precursor (GEP) binds the histidine-rich region of cyclin T1 and inhibits P-TEFb function. GEP is composed of repeats that vary in sequence and properties. GEP also binds directly to Tat. Here we show that GEP and some of its constituent granulin repeats can inhibit HIV-1 transcription via Tat without directly binding to cyclin T1. The interactions of granulins with Tat and cyclin T1 differ with respect to their binding sites and divalent cation requirements, and we identified granulin repeats that bind differentially to Tat and cyclin T1. Granulins DE and E bind Tat but do not interact directly with cyclin T1. These granulins are present in complexes with Tat and P-TEFb in which Tat forms a bridge between the cellular proteins. Granulins DE and E repress transcription from the HIV-1 LTR and gene expression from the viral genome, raising the possibility of developing granulin-based inhibitors of viral infection.

RNA duplexes have been catapulted into the spotlight by the discovery of RNA interference and related phenomena. But double-stranded and highly structured RNAs have long been recognized as key players in cell processes ranging from RNA maturation and localization to the antiviral response in higher organisms. Penetrating insights into the metabolism and functions of such RNAs have come from the identification and study of proteins that contain the double-stranded-RNA-binding motif.

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.

Like other HIV-1 (human immunodeficiency virus type 1) proteins, Tat undergoes rapid mutation and occurs in numerous sequence variants in nature. Virus isolated from patients often has defects in Tat that lower its activity. The levels of P-TEFb, an essential cellular cofactor for Tat, are elevated by T-cell activation. To test the hypothesis that stimulation of P-TEFb levels might compensate for attenuation of Tat activity, we generated Tat constructs with a range of transactivation function. Transactivation by the Tat mutants correlated with their ability to bind to P-TEFb in vitro. Treatment of U937 cells with the phorbol ester PMA (phorbol myristate acetate) induced P-TEFb and stimulated Tat transactivation for alleles with basal transcription activity above a threshold (>5% compared to wild-type). Highly active alleles (>66% of wild-type) were stimulated to a lesser extent than those with activity in the intermediate range. Thus, attenuation of Tat function, in concert with low levels of P-TEFb activity, could serve to keep the virus in a latent state in quiescent cells yet permit viral replication after cell activation.

The interferon-inducible, double-stranded RNA (dsRNA)-activated protein kinase, PKR, plays key roles in regulation of cell growth and differentiation, and has been postulated as a tumor suppressor. Downstream effectors of PKR include the translation initiation factor, eIF2alpha, and the transcription factor, NF-kappaB. We found elevated levels of PKR protein, dsRNA-dependent PKR autophosphorylation activity, and phosphorylated eIF2alpha in melanoma cells compared to nontransformed melanocytes in culture. Treatment with interferon-alpha2b further induced PKR expression and activity. Immunohistochemical analysis of primary melanomas demonstrated minimal PKR immunoreactivity, but melanoma lymph node metastases expressed a high level of PKR protein. Furthermore, analysis of colon cancer specimens revealed that transformation from normal mucosa to adenomas and carcinomas was coincident with an increase in PKR expression. These data do not support the concept of PKR as a classic tumor suppressor but instead suggest that PKR upregulation occurs at defined steps in cancer progression, probably as a cellular response to neoplasia.

Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

Nonsense-mediated mRNA decay (NMD) couples protein synthesis to mRNA turnover. It eliminates defective transcripts and controls the abundance of certain normal mRNAs. Our study establishes a connection between NMD and the translation factor eIF5A (eukaryotic initiation factor 5A) in human cells. eIF5A modulates the synthesis of groups of proteins (the eIF5A regulon), and undergoes a distinctive two-step post-translational modification (hypusination) catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. We show that expression of NMD-susceptible constructs was increased by depletion of the major eIF5A isoform, eIF5A1. NMD was also attenuated when hypusination was inhibited by RNA interference with either of the two eIF5A modifying enzymes, or by treatment with the drugs ciclopirox or deferiprone which inhibit deoxyhypusine hydroxylase. Transcriptome analysis by RNA-Seq identified human genes whose expression is coordinately regulated by eIF5A1, its modifying enzymes, and the pivotal NMD factor, Upf1. Transcripts encoding components of the translation system were highly represented, including some encoding ribosomal proteins controlled by alternative splicing coupled to NMD (AS-NMD). Our findings extend and strengthen the association of eIF5A with NMD, previously inferred in yeast, and show that hypusination is important for this function of human eIF5A. In addition, they advance drug-mediated NMD suppression as a therapeutic opportunity for nonsense-associated diseases. We propose that regulation of mRNA stability contributes to eIF5A's role in selective gene expression.

Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.

CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.

Highly structured and double-stranded (ds) RNAs are adaptable and potent biochemical entities. They interact with dsRNA-binding proteins (RBPs), the great majority of which contain a sequence called the dsRNA-binding motif (dsRBM). This approximately 70-amino-acid sequence motif forms a tertiary structure that interacts with dsRNA, with partially duplexed RNA and, in some cases, with RNA-DNA hybrids, generally without obvious RNA sequence specificity. At least nine families of functionally diverse proteins contain one or more dsRBMs. The motif also participates in complex formation through protein-protein interactions.

Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.

The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat, the transactivator of transcription, dramatically increases the production of viral RNA (for a review, see reference 26). This effect is dependent on the transactivation response element (TAR) located downstream of the promoter in the HIV long terminal repeat (LTR). TAR is an RNA element, and TAR RNA binds directly to Tat (17, 72). Although Tat may also exert a stimulatory effect on transcriptional initiation, its predominant effect in vivo appears to be at the level of transcriptional elongation (42, 46, 47). In one model, Tat serves to promote the formation of highly processive RNA polymerase complexes at the HIV LTR. In the absence of Tat, HIV-directed transcripts tend to terminate prematurely at apparently random sites (45). This model is supported by experiments conducted with cell-free transcription systems (28, 44, 50, 51). A unique feature of the stimulation of HIV-directed transcription in vitro is the observation that it is preferentially inhibited by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), an adenosine analogue that targets RNA polymerase II (pol II)-mediated elongation in vitro and in vivo (5, 51). Efforts to uncover the mechanism of Tat action have included extensive searches for cellular components that interact with Tat in vivo and in vitro. This approach disclosed structural and functional interactions with several general transcription factors as well as other proteins with as-yet-unknown roles in transcription (8, 23, 38, 41, 43). Outstanding among these Tat-interacting proteins is a novel Tat-associated protein kinase, TAK, found by Herrmann and Rice (35, 36). This kinase is able to phosphorylate the carboxy-terminal repeat domain (CTD) of pol II and is highly sensitive to DRB. The mammalian CTD consists of a series of 52 heptad repeats, YSPTSPS, located on the large subunit of pol II. It participates in gene expression at several levels and appears to coordinate the machinery of transcription and posttranscriptional RNA processing (37, 55, 57). The CTD can be extensively phosphorylated at multiple sites, predominantly on serine but also on threonine and tyrosine residues (11). Its phosphorylation plays a dominant role in transcription regulation: the hyperphosphorylated form of the large subunit (IIo) is associated with transcription elongation complexes, while only the hypomodified form (IIa) can assemble into the preinitiation complex (60). During the transition from initiation to elongation, or soon after, IIa is converted to IIo (10). The role of the CTD in transcription is not entirely understood, however, and it is possible that more than one round of phosphorylation is required, perhaps with intervening dephosphorylation. The CTD is essential for cell viability in yeast and for transcription from TATA-less promoters in vitro but is nonessential for TATA-containing promoters in vitro (1, 48, 59, 70). It is required for Tat transactivation (7, 61, 75). At least 10 kinases which can phosphorylate the CTD in vitro are known, and 3 of them have an established connection with the transcription machinery. These are CDK7/cyclin H, subunits of the CDK-activating kinase (CAK) complex, which is a subassembly of transcription factor TFIIH; CDK8/cyclin C, a component of the pol II holoenzyme; and CDK9/cyclin T, now identified as components of the positive transcription elongation factor P-TEFb. P-TEFb was first recognized in Drosophila Kc cell extract as an activity needed to overcome abortive elongation and allow formation of long transcripts (53). P-TEFb is distinguished from other transcription factors by its sensitivity to very low doses of DRB (52). It differs from TFIIH, which exhibits less sensitivity to DRB, in that TFIIH functions in initiation and promoter clearance while P-TEFb has the attributes of an elongation factor (18, 53, 54, 62). The possibility that TAK corresponds to human P-TEFb was suggested by the functional similarity between the stimulation of elongation by Tat and the shift to productive elongation by P-TEFb. Circumstantial support for this inference was provided by the observations that both P-TEFb and TAK are CTD kinases and that low concentrations of DRB inhibit P-TEFb, TAK, and the Tat effect (35, 51–53). Conclusive evidence came when Zhu et al. (80) cloned the catalytic subunit of Drosophila P-TEFb and showed that its human homologue (PITALRE) is identical to the kinase subunit of TAK. PITALRE, now called CDK9 (67, 73), was first cloned by Grana et al. (29) as a CDC2-related kinase of unknown function. Most of the kinases in this family have cyclin-regulating subunits, and the cyclin partners of human and Drosophila CDK9 were recently cloned and studied (66). The human cyclins T1, T2a, and T2b were all identified as functional partners of CDK9, and almost all of the CDK9 in HeLa nuclear extracts is associated with either cyclin T1 or T2 (67). Cyclin T1 can also bind directly to the activation domain of Tat (73). Several additional proteins are also associated with CDK9 in immunocomplexes (24, 78, 80), suggesting that human P-TEFb is a multiprotein complex or that there are other CDK9-containing complexes in addition to P-TEFb. It is also likely that Tat binds other factors in addition to CDK9/cyclin T (79). The gene for human cyclin T1 maps to chromosome 12, and this chromosome is necessary for a strong Tat effect on transcription from the HIV LTR in mouse and Chinese hamster ovary (CHO) cells (73). Transfection of CHO and mouse cells with the cyclin T1 gene overcomes the poor Tat response that was previously noticed in these cells (73). The species specificity of the Tat response is also partially complemented in mouse cells harboring human chromosome 6, but the factor(s) responsible is unknown. Our study addresses the interaction of CDK9/cyclin T1 with Tat and its CTD substrate. We demonstrate that human Tat-associated P-TEFb contains cyclin T1 and that rodent P-TEFb is deficient in binding to Tat. Human cyclin T1 as part of the P-TEFb complex in CHO cells complements this deficiency and thus contributes to HIV-1 species specificity. We also show that human TAK is part of a large complex that phosphorylates serine 5 in the CTD heptad and is itself phosphorylated on serine and threonine residues. Our data suggest that TAK and CAK differentially phosphorylate the CTD substrate.

The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.

RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G+C-rich sequence context, but a run of five T residues in a G+C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAI promoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNA I and VA RNA II , that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNA I in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNA II is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNA II in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNA II , and with an unidentified protein, p97, which did bind VA RNA II . Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNA II > VA RNA I > single-stranded RNA. Furthermore, VA RNA II is more effective than VA RNA I as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNA II .

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

The adenovirus E1A 243R oncoprotein is capable of transactivating the expression of the human proliferating cell nuclear antigen (PCNA) promoter. Mutational analysis of the E1A 243R protein suggested that both its p300/CBP- and p107-binding regions are required for optimal induction of the PCNA promoter (C. Kannabiran, G. F. Morris, C. Labrie, and M. B. Mathews, J. Virol. 67:425–437, 1993). We show that overexpression of p107 antagonizes the induction of PCNA by E1A 243R in transient expression assays. This inhibition is largely independent of p107’s ability to interact with E1A 243R, because p107 mutants unable to bind to E1A 243R retain the ability to repress the E1A-activated PCNA promoter. Electrophoretic mobility shift assays with the PCNA promoter detected the presence of p107 in one of the major DNA-protein complexes, EH1, formed with HeLa cell nuclear extracts. Promoter mutations that disrupt the formation of complex EH1 abrogated p107’s ability to reverse E1A 243R-induced PCNA expression. The same mutations characterize a sequence important for the binding of transcription factor RFX1 (C. Labrie, G. F. Morris, and M. B. Mathews, Nucleic Acids Res. 23:3732–3741, 1995), implying that p107 antagonizes E1A 243R-induced PCNA expression through this RFX1-binding site. Our data are suggestive of a novel cooperative mechanism for transactivation of PCNA expression, in which E1A 243R relieves transcriptional repression exerted by p107 on the promoter.

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.