RESUMO
Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or S. cerevisiae model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.
Assuntos
Proteínas de Saccharomyces cerevisiae , Spliceossomos , Humanos , Spliceossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Splicing de RNA , Íntrons , Ribonucleoproteína Nuclear Pequena U1/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.
Assuntos
Proteínas Nucleares/metabolismo , Splicing de RNA , Receptores de LDL/genética , Colesterol/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Fígado/metabolismo , Mutação , Proteínas Nucleares/genética , Receptores de LDL/metabolismo , Spliceossomos/metabolismoRESUMO
Inadequate reference databases in RNA-seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high-quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica, a widely-used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity. This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during low-temperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low-temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to low-temperature stress, suggesting a unique co-regulatory mechanism governing transcription and splicing in the context of low-temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica.
Assuntos
Processamento Alternativo , Temperatura Baixa , Poaceae , Processamento Alternativo/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poaceae/genética , Estresse Fisiológico/genética , Transcriptoma/genéticaRESUMO
Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type-specific alternative splicing patterns.
Assuntos
Splicing de RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Spliceossomos/genética , Adulto , Pareamento de Bases , Sequência de Bases , Fracionamento Celular/métodos , Éxons , Feto , Células HEK293 , Humanos , Íntrons , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Especificidade de Órgãos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Spliceossomos/química , Spliceossomos/metabolismoRESUMO
Variants in genes encoding core components of the spliceosomes are associated with craniofacial syndromes, collectively called craniofacial spliceosomopathies. SNRPE encodes a core component of pre-mRNA processing U-rich small nuclear ribonuclear proteins (UsnRNPs). Heterozygous variants in SNRPE have been reported in six families with isolated hypotrichosis simplex in addition to one case of isolated non syndromic congenital microcephaly. Here, we report a patient with a novel blended phenotype of microcephaly and congenital atrichia with multiple congenital anomalies due to a de novo intronic SNRPE deletion, c.82-28_82-16del, which results in exon skipping. As discussed within, this phenotype, which we propose be named SNRPE-related syndromic microcephaly and hypotrichosis, overlaps other craniofacial splicesosomopathies.
Assuntos
Anormalidades Múltiplas , Hipotricose , Microcefalia , Humanos , Microcefalia/diagnóstico , Microcefalia/genética , Microcefalia/complicações , Fenótipo , Alopecia/complicações , Hipotricose/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Proteínas Centrais de snRNP/genéticaRESUMO
Stentor coeruleus is a ciliate known for its regenerative ability. Recent genome sequencing reveals that its spliceosomal introns are exceptionally small. We wondered whether the multimegadalton spliceosome has any unique characteristics for removal of the tiny introns. First, we analyzed intron features and identified spliceosomal RNA/protein components. We found that all snRNAs are present, whereas many proteins are conserved but slightly reduced in size. Some regulators, such as Serine/Arginine-rich proteins, are noticeably undetected. Interestingly, while most parts of spliceosomal proteins, including Prp8's positively charged catalytic cavity, are conserved, regions of branching factors projecting to the active site are not. We conjecture that steric-clash avoidance between spliceosomal proteins and a sharply looped lariat might occur, and splicing regulation may differ from other species.
Assuntos
Cilióforos , Precursores de RNA , Arginina/metabolismo , Cilióforos/genética , Íntrons/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Serina/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
The spliceosome is a multi-subunit RNA-protein complex involved in the removal of non-coding segments (introns) from between the coding regions (exons) in precursors of messenger RNAs (pre-mRNAs). Intron removal proceeds via two transesterification reactions, occurring between conserved sequences at intron-exon junctions. A tightly regulated, hierarchical assembly with a multitude of structural and compositional rearrangements posed a great challenge for structural studies of the spliceosome. Over the years, X-ray crystallography dominated the field, providing valuable high-resolution structural information that was mostly limited to individual proteins and smaller sub-complexes. Recent developments in the field of cryo-electron microscopy allowed the visualisation of fully assembled yeast and human spliceosomes, providing unprecedented insights into substrate recognition, catalysis, and active site formation. This has advanced our mechanistic understanding of pre-mRNA splicing enormously.
Assuntos
Precursores de RNA/metabolismo , Spliceossomos/metabolismo , Catálise , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Íntrons/genética , Íntrons/fisiologia , Precursores de RNA/genética , Spliceossomos/genética , Spliceossomos/ultraestruturaRESUMO
Pre-mRNA splicing is a dynamic, multistep process that is catalyzed by the RNA (ribonucleic acid)-protein complex called the spliceosome. The spliceosome contains a core set of RNAs and proteins that are conserved in all organisms that perform splicing. In higher organisms, peptidyl-prolyl isomerase H (PPIH) directly interacts with the core protein pre-mRNA processing factor 4 (PRPF4) and both integrate into the pre-catalytic spliceosome as part of the tri-snRNP (small nuclear RNA-protein complex) subcomplex. As a first step to understand the protein interactions that dictate PPIH and PRPF4 function, we expressed and purified soluble forms of each protein and formed a complex between them. We found two sites of interaction between PPIH and the N-terminus of PRPF4, an unexpected result. The N-terminus of PRPF4 is an intrinsically disordered region and does not adopt secondary structure in the presence of PPIH. In the absence of an atomic resolution structure, we used mutational analysis to identify point mutations that uncouple these two binding sites and find that mutations in both sites are necessary to break up the complex. A discussion of how this bipartite interaction between PPIH and PRPF4 may modulate spliceosomal function is included.
Assuntos
Peptidilprolil Isomerase/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Spliceossomos/metabolismo , Calorimetria , Dicroísmo Circular , Clonagem Molecular , Proteínas Intrinsicamente Desordenadas/metabolismo , Peptidilprolil Isomerase/genética , Ligação Proteica , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ressonância de Plasmônio de Superfície , UltracentrifugaçãoRESUMO
To identify potential therapeutic targets for lung cancer, we performed semi-genome-wide shRNA screening combined with the utilization of genome-wide expression and copy number data. shRNA screening targeting 5043 genes in NCI-H460 identified 51 genes as candidates. Pathway analysis revealed that the 51 genes were enriched for the five pathways, including ribosome, proteasome, RNA polymerase, pyrimidine metabolism and spliceosome pathways. We focused on the proteasome pathway that involved six candidate genes because its activation has been demonstrated in diverse human malignancies, including lung cancer. Microarray expression and array CGH data showed that PSMA6, a proteasomal subunit of a 20S catalytic core complex, was highly expressed in lung cancer cell lines, with recurrent gene amplifications in some cases. Therefore, we further examined the roles of PSMA6 in lung cancer. Silencing of PSMA6 induced apoptosis or G2/M cell cycle arrest in cancer cell lines but not in an immortalized normal lung cell line. These results suggested that PSMA6 serves as an attractive target with a high therapeutic index for lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Domínio Catalítico/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Complexo de Endopeptidases do Proteassoma/genética , Células A549 , Idoso , Apoptose/genética , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Amplificação de Genes , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Terapia de Alvo Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR-DNA binding protein 43 kDa (TDP-43) must be elucidated. TDP-43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP-43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylate-rich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre-mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre-mRNA splicing. Although the major class of spliceosomes regulates most pre-mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre-mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases.
Assuntos
Esclerose Lateral Amiotrófica/etiologia , Proteínas de Ligação a DNA/metabolismo , Gêmeos de Corpos Enovelados/metabolismo , Doença dos Neurônios Motores/etiologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Gêmeos de Corpos Enovelados/ultraestrutura , Humanos , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , RNA/metabolismo , Splicing de RNARESUMO
Animal density-dependent experiences have profound effects on reproductive strategies with marked fecundity differences. Migratory locust adopts distinct population density-dependent reproductive strategies to cope with their respective life cycles, but the mechanisms remain poorly understood. Here, we report that Piwi-interacting RNAs (piRNAs) in the locust germline play key roles in this process. We find that the locust Piwi protein Liwi1 and piRNAs are highly expressed in early developing egg chambers in solitarious locusts, which have higher fecundity than gregarious locusts. Approximately 40% of solitarious locust-associated piRNAs map to protein-coding genes. We find that Liwi1/piRNAs facilitate pre-mRNA splicing of oocyte development-related genes, such as oo18 RNA-binding protein (Orb), in the germline by recruiting the splicing factor U2AF35 to piRNA-targeted introns, thereby increasing fecundity. Such piRNA-guided pre-mRNA splicing is also functional in Drosophila and mouse germ cells. We uncover a piRNA-guided splicing mechanism for processing reproduction-related mRNAs and determining animal reproductive strategies.
Assuntos
Gafanhotos , Precursores de RNA , Animais , Drosophila , Íntrons/genética , Camundongos , Precursores de RNA/genética , Splicing de RNA , RNA Interferente Pequeno , Reprodução/genéticaRESUMO
In mammals, the testis-specific serine/threonine kinase (TSSK) is essential for spermatogenesis and male fertility. TSSK4 belongs to the family of the testis-specific serine/threonine-protein kinase (TSSK), with a crucial role in spermatogenesis. This study aimed to analyze the variable spliceosome of the TSSK4 gene in the yak for understanding the regulatory function of the TSSK4 spliceosome in yak testis development using PCR amplification and cloning techniques. The GST pull-down was used for pulling down the protein interacting with TSSK4, and then the protein interacting with TSSK4 was identified using LC-MS/MS. The results of the PCR amplification demonstrated multiple bands of the TSSK4 gene in the yak. The cloning and sequencing yielded a total of six alternative spliceosomes, which included only two alternative spliceosomes before sexual maturity and four alternative spliceosomes after sexual maturity. The sub-cells of the alternative spliceosomes were found to localize in the nucleus before sexual maturity and in the cytoplasm after sexual maturity. The LC-MS/MS analysis of the alternative spliceosome with the highest expression after sexual maturity yielded a total of 223 interacting proteins. The enrichment analysis of the 223 interacting proteins revealed these proteins participate in biological processes, cell composition, and molecular functions. The KEGG analysis indicated that the TSSK4-interacting protein participates in the estrogen signaling pathways, tight junctions, endoplasmic reticulum protein processing, and other signaling pathways. This study cloned the six alternative spliceosomes of the TSSK4 gene laying the foundation for studying the function of each spliceosome in the future.
RESUMO
In this review I focus on the role of splicing in long non-coding RNA (lncRNA) life. First, I summarize differences between the splicing efficiency of protein-coding genes and lncRNAs and discuss why non-coding RNAs are spliced less efficiently. In the second half of the review, I speculate why splice sites are the most conserved sequences in lncRNAs and what additional roles could splicing play in lncRNA metabolism. I discuss the hypothesis that the splicing machinery can, besides its dominant role in intron removal and exon joining, protect cells from undesired transcripts.
Assuntos
RNA Longo não Codificante , Splicing de RNA , RNA Longo não Codificante/genéticaRESUMO
SF3B1 is the largest subunit of the Spliceosome Factor 3b (SF3B) complex and part of the U2 small nuclear ribosomal protein. It functions as an important part of spliceosomal assembly, converting precursor messenger RNA (mRNA) to mRNA ready for ribosomal translation. Mutations of SF3B1 are commonly seen in myelodysplastic syndromes with ring sideroblasts (MDS-RS)and MDS/myeloproliferative neoplasm (MPN-RS-T). These mutations are typically heterozygous missense substitutions, of which, 55% involve K700E. MDS-RS and MDS/MPN-RS-T usually carry a more favourable prognosis than other subtypes of MDS. SF3B1 itself does not influence survival in these conditions, but does correlate with increased thrombotic risk. Mutated SF3B1 is present in 9%-15% of chronic lymphocytic leukaemia cases and on its own correlates with improved responsiveness to ibrutinib, but is associated with additional adverse genetic abnormalities including TP53 and ATM mutations, which traditionally confer adverse outcomes.
Assuntos
Biomarcadores Tumorais/genética , Eritroblastos/patologia , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Predisposição Genética para Doença , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/terapia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/terapia , Fenótipo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Prognóstico , Conformação Proteica , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
The peptidyl prolyl isomerases (PPI) of the cyclophilin type are distributed throughout human cells, including eight found solely in the nucleus. Nuclear cyclophilins are involved in complexes that regulate chromatin modification, transcription, and pre-mRNA splicing. This review collects what is known about the eight human nuclear cyclophilins: peptidyl prolyl isomerase H (PPIH), peptidyl prolyl isomerase E (PPIE), peptidyl prolyl isomerase-like 1 (PPIL1), peptidyl prolyl isomerase-like 2 (PPIL2), peptidyl prolyl isomerase-like 3 (PPIL3), peptidyl prolyl isomerase G (PPIG), spliceosome-associated protein CWC27 homolog (CWC27), and peptidyl prolyl isomerase domain and WD repeat-containing protein 1 (PPWD1). Each "spliceophilin" is evaluated in relation to the spliceosomal complex in which it has been studied, and current work studying the biological roles of these cyclophilins in the nucleus are discussed. The eight human splicing complexes available in the Protein Data Bank (PDB) are analyzed from the viewpoint of the human spliceophilins. Future directions in structural and cellular biology, and the importance of developing spliceophilin-specific inhibitors, are considered.
Assuntos
Núcleo Celular/química , Ciclofilinas/química , Spliceossomos/química , Relação Estrutura-Atividade , Sequência de Aminoácidos , Ciclofilinas/classificação , Ciclofilinas/genética , Ciclofilinas/metabolismo , Humanos , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Conformação Proteica , Splicing de RNA/genética , Spliceossomos/genéticaRESUMO
Single-particle cryo-electron microscopy (EM) is currently gaining attention for the ability to calculate structures that reach sub-5 Å resolutions; however, the technique is more than just an alternative approach to X-ray crystallography. Molecular machines work via dynamic conformational changes, making structural flexibility the hallmark of function. While the dynamic regions in molecules are essential, they are also the most challenging to structurally characterize. Single-particle EM has the distinct advantage of being able to directly visualize purified molecules without the formation of ordered arrays of molecules locked into identical conformations. Additionally, structures determined using single-particle EM can span resolution ranges from very low- to atomic-levels (>30-1.8 Å), sometimes even in the same structure. The ability to accommodate various resolutions gives single-particle EM the unique capacity to structurally characterize dynamic regions of biological molecules, thereby contributing essential structural information needed for the development of molecular models that explain function. Further, many important molecular machines are intrinsically dynamic and compositionally heterogeneous. Structures of these complexes may never reach sub-5 Å resolutions due to this flexibility required for function. Thus, the biochemical quality of the sample, as well as, the calculation and interpretation of low- to mid-resolution cryo-EM structures (30-8 Å) remains critical for generating insights into the architecture of many challenging biological samples that cannot be visualized using alternative techniques.
Assuntos
Microscopia Crioeletrônica/métodos , Modelos Moleculares , Cristalografia por Raios X/métodosRESUMO
INTRODUCTION: Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.
Assuntos
Giardia lamblia/genética , Giardia lamblia/metabolismo , Íntrons/genética , Splicing de RNA/fisiologia , RNA Mensageiro/agonistas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Spliceossomos/genética , Spliceossomos/metabolismo , Biologia Computacional , Giardia lamblia/química , Humanos , Splicing de RNA/genética , RNA Mensageiro/genética , Especificidade da Espécie , Spliceossomos/químicaRESUMO
BACKGROUND: The cyclin E oncogene activates CDK2 to drive cells from G1 to S phase of the cell cycle to commence DNA replication. It coordinates essential cellular functions with the cell cycle including histone biogenesis, splicing, centrosome duplication and origin firing for DNA replication. The two E-cyclins, E1 and E2, are assumed to act interchangeably in these functions. However recent reports have identified unique functions for cyclins E1 and E2 in different tissues, and particularly in breast cancer. FINDINGS: Cyclins E1 and E2 localise to distinct foci in breast cancer cells as well as co-localising within the cell. Both E-cyclins are found in complex with CDK2, at centrosomes and with the splicing machinery in nuclear speckles. However cyclin E2 uniquely co-localises with NPAT, the main activator of cell-cycle regulated histone transcription. Increased cyclin E2, but not cyclin E1, expression is associated with high expression of replication-dependent histones in breast cancers. CONCLUSIONS: The preferential localisation of cyclin E1 or cyclin E2 to distinct foci indicates that each E-cyclin has unique roles. Cyclin E2 uniquely interacts with NPAT in breast cancer cells, and is associated with higher levels of histones in breast cancer. This could explain the unique correlations of high cyclin E2 expression with poor outcome and genomic instability in breast cancer.
RESUMO
PDIP38 (polymerase delta interacting protein 38) was originally discovered as a protein that interacts with DNA polymerase δ and PCNA. PDIP38 is present in multiple intracellular locations and is a multifunctional protein that has been implicated in several diverse cellular functions. We investigated the nuclear localization of PDIP38 in order to gain insights to its response to UV damage. PDIP38 was found to form distinct nuclear foci in response to UV irradiation in several cell lines, including HeLa S3 and A549 cells. However, these foci were not those associated with UV repair foci. Using various markers for different nuclear subcompartments, the UV-induced PDIP38 foci were identified as spliceosomes/nuclear speckles, the storage and assembly sites for mRNA splicing factors. To assess the role of PDIP38 in the regulation of splicing events, the effects of PDIP38 depletion on the UV-induced alternate splicing of MDM2 transcripts were examined by nested RT-PCR. Alternatively spliced MDM2 products were induced by UV treatment but were greatly reduced in cells expressing shRNA targeting PDIP38. These findings indicate that upon UV-induced DNA damage, PDIP38 is translocated to spliceosomes and contributes to the UV-induced alternative splicing of MDM2 transcripts. Similar results were obtained when cells were subjected to transcriptional stresses with actinomycin D or α-amanitin. Taken together, these studies show that PDIP38 is a protein regulated in a dynamic manner in response to genotoxic stress, as evidenced by its translocation to the spliceosomes. Moreover, PDIP38 is required for the induction of the alternative splicing of MDM2 in response to UV irradiation.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA/efeitos da radiação , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Spliceossomos/metabolismo , Raios Ultravioleta , Processamento Alternativo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dactinomicina/toxicidade , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho. Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito. Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados. Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos. Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito.
Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.