RESUMEN

Nicotine, a naturally occurring tobacco alkaloid responsible for tobacco addiction, has long been considered non-carcinogenic. However, emerging evidence suggests that nicotine may possess carcinogenic properties in mice and could be a potential carcinogen in humans. This review aims to summarize the potential molecular mechanisms underlying nicotine-induced carcinogenesis, with a specific focus on epigenetic regulation and the activation of nicotinic acetylcholine receptors (nAChRs) in addition to genotoxicity and excess reactive oxygen species (ROS). Additionally, we explore a novel hypothesis regarding nicotine's carcinogenicity involving the downregulation of stem-loop binding protein (SLBP), a critical regulator of canonical histone mRNA, and the polyadenylation of canonical histone mRNA. By shedding light on these mechanisms, this review underscores the need for further research to elucidate the carcinogenic potential of nicotine and its implications for human health.

Asunto(s)

Nicotina , Receptores Nicotínicos , Humanos , Ratones , Animales , Nicotina/toxicidad , Histonas/metabolismo , Epigénesis Genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Carcinogénesis/inducido químicamente , Transducción de Señal , ARN Mensajero/metabolismo

RESUMEN

Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

Asunto(s)

Leishmania , Proteínas de Unión a Caperuzas de ARN/metabolismo , Histonas/metabolismo , Proteómica , ARN Mensajero/metabolismo , Ciclo Celular , Unión Proteica

RESUMEN

Electronegative clusters (ENCs) made up of acidic residues and/or phosphorylation sites are the most abundant repetitive sequences in RNA-binding proteins. Previous studies have indicated that ENCs inhibit RNA binding for structured RNA-binding domains (RBDs). However, this is not the case for the unstructured RBD in histone pre-mRNA stem-loop binding protein (SLBP). The SLBP RBD contains 70 amino acids and is followed by a phosphorylatable ENC. ENC phosphorylation increases RNA-binding affinity of SLBP to the sub-picomolar range. In this study, we use NMR and molecular dynamics simulations to elucidate the mechanism for this tight binding. Our NMR data demonstrate that the ENC transiently folds apo SLBP into an RNA-bound resembling state. We find that in the RNA-bound state, the phosphorylated ENC interacts with the loop region opposite to the RNA-binding site. This allosteric interaction stabilizes the complex and therefore enhances RNA binding. To evaluate the generality of our findings, we graft an ENC onto endoribonuclease homolog 1's first double-stranded RNA-binding motif (DRBM1), an unstructured RBD that shares no homology with SLBP. We find that the engineered ENC increases the folded species of DRBM1 and inhibits RNA binding. On the contrary, introducing basic residues to DRBM1 makes the domain more unfolded, enhances RNA binding, and mitigates the inhibitory effect of the engineered ENC. In summary, our study suggests that ENCs promote folding of unstructured RNA-binding domains, and their effects on RNA binding depend on the electropositive charges on the RBD surface.

Asunto(s)

Histonas , Proteínas Nucleares , Histonas/metabolismo , Proteínas Nucleares/química , Factores de Escisión y Poliadenilación de ARNm/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Sitios de Unión , Unión Proteica

RESUMEN

The use of electronic-cigarettes (e-cigs) has increased substantially in recent years, particularly among the younger generations. Liquid nicotine is the main component of e-cigs. Previous studies have shown that mice exposed to e-cig aerosols developed lung adenocarcinoma and bladder hyperplasia. These findings implicated a potential role for e-cig aerosols and nicotine in cancer development, although the underlying mechanisms are not fully understood. Here we report that exposure to liquid nicotine or nicotine aerosol generated from e-cig induces downregulation of Stem-loop binding protein (SLBP) and polyadenylation of canonical histone mRNAs in human bronchial epithelial cells and in mice lungs. Canonical histone mRNAs typically do not end in a poly(A) tail and the acquisition of such a tail via depletion of SLBP has been shown to causes chromosome instability. We show that nicotine-induced SLBP depletion is reversed by an inhibitor of α7-nicotinic acetylcholine receptors (α7-nAChR) or siRNA specific for α7-nAChR, indicating a nAChR-dependent reduction of SLBP by nicotine. Moreover, PI3K/AKT pathway is activated by nicotine exposure and CK2 and probably CDK1, 2 kinases well known for their function for SLBP phosphorylation and degradation, are shown to be involved, α7-nAChR-dependently, in nicotine-induced SLBP depletion. Importantly, nicotine-induced anchorage-independent cell growth is attenuated by inhibition of α7-nAChR and is rescued by overexpression of SLBP. We propose that the SLBP depletion and polyadenylation of canonical histone mRNAs via activation of α7-nAChR and a series of downstream signal transduction pathways are critical for nicotine-induced cell transformation and potential carcinogenesis.

Asunto(s)

Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa 7 , Animales , Transformación Celular Neoplásica , Regulación hacia Abajo , Histonas/metabolismo , Humanos , Ratones , Nicotina/toxicidad , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo

RESUMEN

Pre-mRNA processing of the replication-dependent canonical histone mRNAs requires an endonucleolytic cleavage immediately after a conserved stem loop structure which occurs before RNA Pol II encounters any poly(A) signal. Thus, in contrast to all other eukaryotic mRNAs, the canonical histone mRNAs are not polyadenylated in their 3' ends. The binding of stem-loop binding protein (SLBP) to the stem loop structure of the histone mRNAs is required for this process. SLBP is also involved in regulation of histone mRNA nuclear export, degradation, and translation. Depletion of SLBP has been shown to induce polyadenylation of histone mRNAs and alteration of histone protein levels, which are considered to contribute to the observed aberrant cell cycle progress and genomic instability resulting from the loss of SLBP function. Recent studies have demonstrated that some heavy metal carcinogens, including arsenic and nickel, can induce the loss of SLBP and the gain of polyadenylation of canonical histone mRNAs. Polyadenylated canonical histone H3 can result in abnormal transcription, cell cycle arrest, genomic instability, and cell transformation, which links SLBP depletion and subsequent histone mRNA misprocessing to cancer. This review seeks to briefly summarize what is known about regulation of SLBP expression, consequences of SLBP depletion, its roles in cancer-related end points, with particular focus on metal-induced SLBP depletion and the potential of SLBP depletion as a new mechanism for metal-induced carcinogenesis.

Asunto(s)

Carcinogénesis/inducido químicamente , Metales Pesados/efectos adversos , Proteínas Nucleares/efectos de los fármacos , Factores de Escisión y Poliadenilación de ARNm/efectos de los fármacos , Animales , Humanos

RESUMEN

Arsenic pollution impacts health of millions of people in the world. Inorganic arsenic is a carcinogenic agent in skin and lung cancers. The stem-loop binding protein (SLBP) binds to the stem-loop of the canonical histone mRNA and regulates its metabolism during cell cycle. Our previous work has shown arsenic induces ubiquitin-proteasome dependent degradation of SLBP and contributes to lung cancer. In this study, we established the first comprehensive SLBP interaction network by affinity purification-mass spectrometry (AP-MS) analysis, and further demonstrated arsenic enhanced the association between SLBP and a crucial chaperone complex containing heat shock proteins (HSPs) and ERp44. Strikingly, knockdown of these proteins markedly rescued the protein level of SLBP under arsenic exposure conditions, and abolished the increasing migration capacity of BEAS-2B cells induced by arsenic. Taken together, our study provides a potential new mechanism that a chaperone complex containing HSPs and ERp44 attenuates the stability of SLBP under both normal and arsenic exposure conditions, which could be essential for arsenic-induced high cell migration.

Asunto(s)

Arsénico , Arsénico/toxicidad , Proteínas de Choque Térmico , Humanos , Proteínas de la Membrana , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Unión Proteica , Estabilidad Proteica , Proteómica , Factores de Escisión y Poliadenilación de ARNm

RESUMEN

Stem-loop binding protein (SLBP) binds a stem-loop structure of the mRNA, which is important for the stability of histone mRNAs and translation process. In the present study, two slbp cDNAs (Ecslbp1 and Ecslbp2) were cloned from a protogynous hermaphroditic orange-spotted grouper, Epinephelus coioides. Ecslbp1 cDNA contained a 678 base pair (bp) open reading frame (ORF), encoding a predicted polypeptide of 225 amino acids. Ecslbp2 cDNA contained a 1041 bp, encoding a predicted protein of 346 amino acids. The result of real-time PCR revealed that Ecslbp2 mRNA was exclusively detected in the ovary. Moreover, it was found to be restricted to oocytes according to in situ hybridization (ISH) analysis. Ecslbp2 was found to be hardly detected in gonia and significantly increase in the cytoplasm of primary-growth stage oocytes, but decreased during the process of vitellogenesis. Interestingly, Ecslbp2 expression centralized as a perinuclear speckle in early-primary-growth stage oocytes, which appeared to form into the Balbiani body (Bb) in late-primary-growth stage oocytes. These data indicated that Ecslbp2 might play an important role in the process of oocyte development, and could serve as an oocyte-specific molecular marker for the study of ovary development and sex reversal in groupers.

Asunto(s)

Lubina/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Procesos de Determinación del Sexo , Animales , Lubina/genética , Lubina/metabolismo , Clonación Molecular , Femenino , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sistemas de Lectura Abierta , Especificidad de Órganos , Ovario/crecimiento & desarrollo , Ovario/metabolismo

RESUMEN

Transcription elongation rate influences cotranscriptional pre-mRNA maturation, but how such kinetic coupling works is poorly understood. The formation of nonadenylated histone mRNA 3' ends requires recognition of an RNA structure by stem-loop-binding protein (SLBP). We report that slow transcription by mutant RNA polymerase II (Pol II) caused accumulation of polyadenylated histone mRNAs that extend past the stem-loop processing site. UV irradiation, which decelerates Pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3' processing by slow Pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem-loop fails to fold in transcripts made by slow Pol II, thereby explaining the absence of SLBP and failure to process 3' ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure and suggest a mechanism by which alternative processing could be controlled.

Asunto(s)

Histonas/genética , Procesamiento de Término de ARN 3' , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Elongación de la Transcripción Genética , Células HEK293 , Histonas/metabolismo , Humanos , Cinética , Proteínas Nucleares/metabolismo , Pliegue del ARN , Precursores del ARN/química , ARN Mensajero/química , Transcripción Genética/efectos de la radiación , Rayos Ultravioleta , Factores de Escisión y Poliadenilación de ARNm/metabolismo

RESUMEN

Histones are evolutionarily conserved proteins that together with DNA constitute eukaryotic chromatin in a defined stoichiometry. Core histones are dynamic scaffolding proteins that undergo a myriad of post-translational modifications, which selectively engage chromosome condensation, replication, transcription and DNA damage repair. Cullin4-RING ubiquitin E3 ligases are known to hold pivotal roles in a wide spectrum of chromatin biology ranging from chromatin remodeling and transcriptional repression, to sensing of cytotoxic DNA lesions. Our recent work uncovers an unexpected function of a CRL4 ligase upstream of these processes in promoting histone biogenesis. The CRL4WDR23 ligase directly controls the activity of the stem-loop binding protein (SLBP), which orchestrates elemental steps of canonical histone transcript metabolism. We demonstrate that non-proteolytic ubiquitination of SLBP ensures sufficient histone reservoirs during DNA replication and is vital for genome integrity and cellular fitness.

Asunto(s)

Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Humanos , Secuencias Invertidas Repetidas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitinación

RESUMEN

In prokaryotic cells, genomic DNA forms an aggregated structure with various nucleoid-associated proteins (NAPs). The functions of genomic DNA are cooperatively modulated by NAPs, of which HU is considered to be one of the most important. HU binds double-stranded DNA (dsDNA) and serves as a structural modulator in the genome architecture. It plays important roles in diverse DNA functions, including replication, segregation, transcription and repair. Interestingly, it has been reported that HU also binds single-stranded DNA (ssDNA) regardless of sequence. However, structural analysis of HU with ssDNA has been lacking, and the functional relevance of this binding remains elusive. In this study, we found that ssDNA induced a significant change in the secondary structure of Thermus thermophilus HU (TtHU), as observed by analysis of circular dichroism spectra. Notably, this change in secondary structure was sequence specific, because the complementary ssDNA or dsDNA did not induce the change. Structural analysis using nuclear magnetic resonance confirmed that TtHU and this ssDNA formed a unique structure, which was different from the previously reported structure of HU in complex with dsDNA. Our data suggest that TtHU undergoes a distinct structural change when it associates with ssDNA of a specific sequence and subsequently exerts a yet-to-be-defined function.

RESUMEN

Stem-loop-binding proteins (SLBPs) have been revealed to interact with stem-loop of histones, and oocyte-specific and oocyte-preferential SLBP2 have been identified in vertebrates including Xenopus (S. tropicalis) and B. taurus to play a key role in histone translation regulation, but no oocyte-specific SLBPs have been characterized in fish. Here, we have identified and characterized the first fish oocyte-specific SLBP2 in Carassius gibelio. Its full-length cDNA contains 975 bp ORF encoding 324 amino acids. Firstly, the polyclonal antibody specific to C. gibelio SLBP2 was prepared. Then, RT-PCR analysis and Western blot detection revealed its oocyte-specific and dynamic expression pattern during oogenesis and embryogenesis of C. gibelio. Moreover, in situ hybridization and immunofluorescence localization observed its abundant expression in cortical alveolar stage oocytes and dynamic distribution in different stage oocytes. Altogether, our current data suggest that C. gibelio SLBP2 might play significant role in the early oogenesis and oocyte growth.

Asunto(s)

Carpas/fisiología , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Oocistos/metabolismo , Oogénesis , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Animales , Carpas/embriología , Secuencia Conservada , Desarrollo Embrionario , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Masculino , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oocistos/citología , Especificidad de Órganos , Filogenia , Transporte de Proteínas , ARN Mensajero/metabolismo , Alineación de Secuencia , Especificidad de la Especie , Testículo/metabolismo , Factores de Escisión y Poliadenilación de ARNm/química , Factores de Escisión y Poliadenilación de ARNm/genética

RESUMEN

The high-mobility group (HMG) domain containing proteins regulate transcription, DNA replication and recombination. They adopt L-shaped folds and are structure-specific DNA binding motifs. Here, I define the L-motif super-family that consists of DNA-binding HMG-box proteins and the L-motif of the histone mRNA binding domain of stem-loop binding protein (SLBP). The SLBP L-motif and HMG-box domains adopt similar L-shaped folds with three α-helices and two or three small hydrophobic cores that stabilize the overall fold, but have very different and distinct modes of nucleic acid recognition. A comparison of the structure, dynamics, protein-protein and nucleic acid interactions, and regulation by PTMs of the SLBP and the HMG-box L-motifs reveals the versatile and diverse modes by which L-motifs utilize their surfaces for structure-specific recognition of nucleic acids to regulate gene expression.

Asunto(s)

Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Dominios HMG-Box/genética , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Secuencias de Aminoácidos/genética , Proteínas de Unión al ADN/química , Humanos , Secuencias Invertidas Repetidas/genética , Proteínas Nucleares/química , Conformación de Ácido Nucleico , Fosforilación , Conformación Proteica , Pliegue de Proteína , Proteínas de Unión al ARN/química , Factores de Escisión y Poliadenilación de ARNm/química

RESUMEN

The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.

Asunto(s)

Proteínas de Drosophila/biosíntesis , Desarrollo Embrionario , Histonas/genética , Poliadenilación/genética , Factores de Escisión y Poliadenilación de ARNm/biosíntesis , Animales , Citoplasma/genética , Drosophila , Proteínas de Drosophila/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Mutación , Nucleoplasminas/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Fase S/genética , Factores de Escisión y Poliadenilación de ARNm/genética

RESUMEN

Phosphorus-31 ((31)P) NMR can be used to characterize the structure and dynamics of phosphorylated proteins. Here, I use (31)P NMR to report on the chemical nature of a phosphothreonine that lies in the RNA binding domain of SLBP (stem-loop binding protein). SLBP is an intrinsically disordered protein and phosphorylation at this threonine promotes the assembly of the SLBP-RNA complex. The data show that the (31)P chemical shift can be a good spectroscopic probe for phosphate-coupled folding and binding processes in intrinsically disordered proteins, particularly where the phosphate exhibits torsional strain and is involved in a network of hydrogen-bonding interactions.

RESUMEN

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3'-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.

Asunto(s)

Proteínas de Unión al ARN/biosíntesis , Receptores Notch/metabolismo , Retina/embriología , Pez Cebra/embriología , Animales , Diferenciación Celular , Proliferación Celular , Quimiocina CXCL12/biosíntesis , Proteínas Fluorescentes Verdes , Histonas/genética , Mutación , Neurogénesis , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Retina/citología , Transducción de Señal/genética , Células Madre , Ubiquitina-Proteína Ligasas/genética , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética

RESUMEN

The wide array of vital functions that RNA performs is dependent on its ability to dynamically fold into different structures in response to intracellular and extracellular changes. RNA-binding proteins regulate much of this activity by targeting specific RNA structures or motifs. One of these structures, the 3-way RNA junction, is characteristically found in ribosomal RNA and results from the RNA folding in cis, to produce three separate helices that meet around a central unpaired region. Here we demonstrate that 3-way junctions can also form in trans as a result of the binding of microRNAs in an unconventional manner with mRNA by splinting two non-contiguous regions together. This may be used to reinforce the base of a stem-loop motif being targeted by an RNA-binding protein. Trans interactions between non-coding RNA and mRNA may be used to control the post-transcriptional regulatory code and suggests a possible role for some of the recently described transcripts of unknown function expressed from the human genome.