RESUMO
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.
Assuntos
Doença/genética , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Animais , Variação Biológica da População/genética , Humanos , Fatores de Iniciação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Estresse Fisiológico/genética , Serina-Treonina Quinases TOR/genéticaRESUMO
Generative artificial intelligence (AI) is a burgeoning field with widespread applications, including in science. Here, we explore two paradigms that provide insight into the capabilities and limitations of Chat Generative Pre-trained Transformer (ChatGPT): its ability to (i) define a core biological concept (the Central Dogma of molecular biology); and (ii) interpret the genetic code.
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Inteligência Artificial , Código Genético , Biologia MolecularRESUMO
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, 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.
Assuntos
Genoma Humano/genética , RNA não Traduzido/classificação , Terminologia como Assunto , Humanos , RNA não Traduzido/genéticaRESUMO
The eukaryotic initiation factor eIF5A is a translation factor that, unusually, has been assigned functions in both initiation and elongation. Additionally, it is implicated in transcription, mRNA turnover and nucleocytoplasmic transport. Two eIF5A isoforms are generated from distinct but related genes. The major isoform, eIF5A1, is considered constitutive, is abundantly expressed in most cells, and is essential for cell proliferation. The second isoform, eIF5A2, is expressed in few normal tissues but is highly expressed in many cancers and has been designated a candidate oncogene. Elevated expression of either isoform carries unfavorable prognostic implications for several cancers, and both have been advanced as cancer biomarkers. The amino acid hypusine, a presumptively unique eIF5A post-translational modification, is required for most known eIF5A functions and it renders eIF5A susceptible to inhibitors of the modification pathway as therapeutic targets. eIF5A has been shown to regulate a number of gene products specifically, termed the eIF5A regulon, and its role in translating proline-rich sequences has recently been identified. A model is advanced that accommodates eIF5A in both the initiation and elongation phases of translation. We review here the biochemical functions of eIF5A, the relationship of its isoforms with human cancer, and evolving clinical applications. This article is part of a Special Issue entitled: Translation and Cancer.
Assuntos
Regulação da Expressão Gênica , Neoplasias/metabolismo , Proteínas Oncogênicas/metabolismo , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos , Lisina/análogos & derivados , Lisina/genética , Lisina/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Oncogênicas/genética , Fatores de Iniciação de Peptídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteínas de Ligação a RNA/genética , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Natural language-based generative artificial intelligence (AI) has become increasingly prevalent in scientific research. Intriguingly, capabilities of generative pre-trained transformer (GPT) language models beyond the scope of natural language tasks have recently been identified. Here we explored how GPT-4 might be able to perform rudimentary structural biology modeling. We prompted GPT-4 to model 3D structures for the 20 standard amino acids and an α-helical polypeptide chain, with the latter incorporating Wolfram mathematical computation. We also used GPT-4 to perform structural interaction analysis between nirmatrelvir and its target, the SARS-CoV-2 main protease. Geometric parameters of the generated structures typically approximated close to experimental references. However, modeling was sporadically error-prone and molecular complexity was not well tolerated. Interaction analysis further revealed the ability of GPT-4 to identify specific amino acid residues involved in ligand binding along with corresponding bond distances. Despite current limitations, we show the capacity of natural language generative AI to perform basic structural biology modeling and interaction analysis with atomic-scale accuracy.
RESUMO
Natural language-based generative artificial intelligence (AI) has become increasingly prevalent in scientific research. Intriguingly, capabilities of generative pre-trained transformer (GPT) language models beyond the scope of natural language tasks have recently been identified. Here we explored how GPT-4 might be able to perform rudimentary structural biology modeling. We prompted GPT-4 to model 3D structures for the 20 standard amino acids and an α-helical polypeptide chain, with the latter incorporating Wolfram mathematical computation. We also used GPT-4 to perform structural interaction analysis between the anti-viral nirmatrelvir and its target, the SARS-CoV-2 main protease. Geometric parameters of the generated structures typically approximated close to experimental references. However, modeling was sporadically error-prone and molecular complexity was not well tolerated. Interaction analysis further revealed the ability of GPT-4 to identify specific amino acid residues involved in ligand binding along with corresponding bond distances. Despite current limitations, we show the current capacity of natural language generative AI to perform basic structural biology modeling and interaction analysis with atomic-scale accuracy.
Assuntos
Inteligência Artificial , Modelos Moleculares , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Conformação Proteica , COVID-19/virologia , Aminoácidos/químicaRESUMO
We recently identified the snaR family of small non-coding RNAs that associate in vivo with the nuclear factor 90 (NF90/ILF3) protein. The major human species, snaR-A, is an RNA polymerase III transcript with restricted tissue distribution and orthologs in chimpanzee but not rhesus macaque or mouse. We report their expression in human tissues and their evolution in primates. snaR genes are exclusively in African Great Apes and some are unique to humans. Two novel families of snaR-related genetic elements were found in primates: CAS (catarrhine ancestor of snaR), limited to Old World Monkeys and apes; and ASR (Alu/snaR-related), present in all monkeys and apes. ASR and CAS appear to have spread by retrotransposition, whereas most snaR genes have spread by segmental duplication. snaR-A and snaR-G2 are differentially expressed in discrete regions of the human brain and other tissues, notably including testis. snaR-A is up-regulated in transformed and immortalized human cells, and is stably bound to ribosomes in HeLa cells. We infer that snaR evolved from the left monomer of the primate-specific Alu SINE family via ASR and CAS in conjunction with major primate speciation events, and suggest that snaRs participate in tissue- and species-specific regulation of cell growth and translation.
Assuntos
Evolução Molecular , Pequeno RNA não Traduzido/genética , Elementos Alu , Animais , Sequência de Bases , Linhagem Celular , Linhagem Celular Transformada , Cercopithecidae/genética , Citoplasma/química , Duplicação Gênica , Genômica , Células HeLa , Hominidae/genética , Humanos , Dados de Sequência Molecular , Família Multigênica , Pequeno RNA não Traduzido/análise , Pequeno RNA não Traduzido/metabolismo , Ribossomos/química , Distribuição TecidualRESUMO
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 â RNA â 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 Genomics > Ribonomics RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Translation > Mechanisms.
Assuntos
Edição de Genes , RNA , RNA/genéticaRESUMO
Progranulin (also known as granulin/epithelin precursor, GEP) is composed of seven granulin/epithelin repeats (granulins) and functions both as a full-length protein and as individual granulins. It is a secretory protein but a substantial amount of GEP is found inside cells, some in complexes with positive transcription elongation factor b (P-TEFb). GEP and certain granulins interact with the cyclin T1 subunit of P-TEFb, and with its HIV-1 Tat co-factor, leading to repression of transcription from the HIV promoter. We show that GEP lacking the signal peptide (GEPspm) remains inside cells and, like wild-type GEP, interacts with cyclin T1 and Tat. GEPspm represses transcription from the HIV-1 promoter at the RNA level. Granulins that bind cyclin T1 are phosphorylated by P-TEFb in vivo and in vitro on serine residues. GEPspm and those granulins that interact with cyclin T1 also inhibit transcription from cellular cad and c-myc promoters, which are highly dependent on P-TEFb, but not from the PCNA promoter. In addition, GEPspm and granulins repress transcriptional activation by VP16 or c-Myc, proteins that bind and recruit P-TEFb to responsive promoters. These data suggest that intracellular GEP is a promoter-specific transcriptional repressor that modulates the function of cellular and viral transcription factors.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Aspartato Carbamoiltransferase/genética , Sítios de Ligação , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Ciclina T/genética , Di-Hidro-Orotase/genética , Regulação para Baixo , Genes myc , Granulinas , HIV-1/genética , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Complexos Multiproteicos , Células NIH 3T3 , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Progranulinas , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Transativadores/genética , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
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.
Assuntos
Antígenos de Neoplasias/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Catálise , Ensaio de Desvio de Mobilidade Eletroforética , RNA Helicases/metabolismoRESUMO
BACKGROUND: Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture. RESULTS: Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression. CONCLUSION: At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Piridonas/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Ciclopirox , Deferiprona , Humanos , Leucócitos Mononucleares/virologia , Lisina/metabolismo , Oxigenases de Função Mista/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
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-gamma 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.
Assuntos
Regiões 3' não Traduzidas , Interferon gama/biossíntese , Fatores de Regulação Miogênica/genética , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Cricetinae , Cricetulus , Eritropoetina/genética , Eritropoetina/metabolismo , Expressão Gênica , Humanos , Interferon gama/genética , Modelos Biológicos , Fatores de Regulação Miogênica/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , TransgenesRESUMO
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.
Assuntos
Evolução Molecular , Proteínas do Fator Nuclear 90/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Humanos , Dados de Sequência Molecular , Pan troglodytes/genética , RNA Polimerase III/metabolismo , RNA não Traduzido/química , Alinhamento de Sequência , Distribuição TecidualRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Genoma , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Códon , Citoplasma/metabolismo , Cinética , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
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.
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Biologia Celular/história , Regulação da Expressão Gênica , Biologia Molecular/história , Biossíntese de Proteínas , Animais , História do Século XX , História do Século XXI , Humanos , Oócitos/fisiologia , Reticulócitos/fisiologia , Ouriços-do-Mar/fisiologiaRESUMO
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.
Assuntos
Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Biossíntese de Proteínas , Animais , Humanos , Vírus de Plantas/metabolismo , Processamento Pós-Transcricional do RNA , Ribossomos/metabolismo , Estresse Fisiológico , Proteínas Virais/biossíntese , Viroses/metabolismo , Replicação ViralRESUMO
Positive transcription elongation factor b (P-TEFb) complexes, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1 or T2, are engaged by many cellular transcription regulators that activate or inhibit transcription from specific promoters. The related I-mfa (inhibitor of MyoD family a) and HIC (human I-mfa-domain-containing) proteins function in myogenic differentiation and embryonic development by participating in the Wnt signaling pathway. We report that I-mfa is a novel regulator of P-TEFb. Both HIC and I-mfa interact through their homologous I-mfa domains with cyclin T1 and T2 at two binding sites. One site is the regulatory histidine-rich domain that interacts with CDK9 substrates including RNA polymerase II. The second site contains a lysine and arginine-rich motif that is highly conserved between the two T cyclins. This site overlaps and includes the previously identified Tat/TAR recognition motif of cyclin T1 required for activation of human immunodeficiency virus type 1 (HIV-1) transcription. HIC and I-mfa can serve as substrates for P-TEFb. Their I-mfa domains also bind the activation domain of HIV-1 Tat and inhibit Tat- and P-TEFb-dependent transcription from the HIV-1 promoter. This transcriptional repression is cell-type specific and can operate via Tat and cyclin T1. Genomic and sequence comparisons indicate that the I-mf and HIC genes, as well as flanking genes, diverged from a duplicated chromosomal region. Our findings link I-mfa and HIC to viral replication, and suggest that P-TEFb is modulated in the Wnt signaling pathway.
Assuntos
Ciclinas/química , Ciclinas/metabolismo , Produtos do Gene tat/química , Produtos do Gene tat/metabolismo , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Ciclina T , Quinase 9 Dependente de Ciclina/metabolismo , Ciclinas/genética , Primers do DNA/genética , Evolução Molecular , Duplicação Gênica , Produtos do Gene tat/genética , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Complexos Multiproteicos , Fatores de Regulação Miogênica/genética , Fosforilação , Fator B de Elongação Transcricional Positiva/química , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
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.
RESUMO
Ribonucleoprotein complexes (RNPs) perform a multitude of functions in the cell. Elucidating the composition of such complexes and unraveling their many interactions are current challenges in molecular biology. To stabilize complexes formed in cells and to preclude reassortment of their components during isolation, we employ chemical crosslinking of the RNA and protein moieties. Here we describe the identification of cellular RNAs bound to nuclear factor 90 (NF90), the founder member of a family of ubiquitous double-stranded RNA-binding proteins. Crosslinked RNA-NF90 complexes were immunoprecipitated from stable cell lines containing epitope-tagged NF90 protein isoforms. The bound RNA was released and identified through RNase H digestion and by various gene amplification techniques. We appraise the methods used by altering crosslinking conditions, and the binding profiles of different NF90 protein isoforms in synchronized and asynchronous cells are compared. This study discovers two novel RNA species and establishes NF90 as a multiclass RNA-binding protein, capable of binding representatives of all three classes of RNA.