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PubMed2006

Nuclear hypertrophy reflects increased biosynthetic activities in myocytes of human hypertrophic hearts.

BACKGROUND: The nucleus of the myocytes in human hypertrophic hearts is characterized by its bizarre shape and widespread clumping of chromatin. The functional significance has not been determined. METHODS AND RESULTS: Left ventricular (LV) endomyocardial biopsies obtained from patients with dilated cardiomyopathy (DCM, n=23), postmyocarditis (n=13), hypertrophic cardiomyopathy (HCM, n=21), apical hypertrophic cardiomyopathy (APH, n=11) and hypertensive heart disease (HHD, n=11), and from nonhypertrophic hearts (controls, n=14) were examined. Myocyte size and LV mass index were similar among the hypertrophic hearts, but the nuclear hypertrophy score (grade 0-3) was highest in hearts with systolic failure (DCM and postmyocarditis) and higher in those without it (HCM, APH, and HHD), compared with controls. So were biosynthetic activities such as DNA repair/synthesis, immunohistochemically assessed by proliferating cell nuclear antigen, transcription activity by spliceosome component of 35 kDa, and translation efficiency by 70 kDa S6 protein kinase. There were significant correlations between nuclear hypertrophy and each biosynthetic activity. Additionally, most of the proliferating cell nuclear antigen-positive nuclei co-expressed oxidative DNA damage markers. CONCLUSION: A link is suggested between structural alteration and molecular biological events in the nuclei of myocytes from human hypertrophic hearts; the nuclear hypertrophy reflects increased biosynthetic activities of DNA repair/synthesis, transcription, and translation efficiency.

Aged↗

PubMed2005

The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding.

Prp24 is an essential yeast U6 snRNP protein with four RNA recognition motifs (RRMs) that facilitates the association of U4 and U6 snRNPs during spliceosome assembly. Genetic interactions led to the proposal that RRMs 2 and 3 of Prp24 bind U6 RNA, while RRMs 1 and 4 bind U4 RNA. However, the function of each RRM has yet to be established through biochemical means. We compared the binding of recombinant full-length Prp24 and truncated forms lacking RRM 1 or RRM 4 with U6 RNA. Contrary to expectations, we found that the N-terminal segment containing RRM 1 is important for high-affinity binding to U6 RNA and for discrimination between wild-type U6 RNA and U6 with point mutations in the 3' intramolecular stem-loop. In contrast, deletion of RRM 4 and the C terminus did not significantly alter the affinity for U6 RNA, but resulted in the formation of higher order Prp24.U6 complexes. Truncation and internal deletion of U6 RNA mapped three Prp24-binding sites, with the central site providing most of the affinity for Prp24. A newly identified temperature-sensitive lethal point mutation in RRM 1 is exacerbated by mutations in the U6 RNA telestem, as is a mutation in RRM 2, but not one in RRM 3. We propose that RRMs 1 and 2 of yeast Prp24 bind the same central site in U6 RNA that is bound by the two RRMs of human Prp24, and that RRMs 3 and 4 bind lower affinity flanking sites, thereby restricting the stoichiometry of Prp24 binding.

Amino Acid Sequence↗

PubMed2005

Identification and analysis of U5 snRNA variants in Drosophila.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5' segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3' hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5' half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

Amino Acid Sequence↗

PubMed2006

Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment.

The deposition of proteins onto newly spliced mRNAs has far reaching consequences for their subsequent metabolism. We affinity-purified spliced human mRNPs under physiological conditions from HeLa nuclear extract and present the first comprehensive inventory of their protein composition as determined by mass spectrometry. Several proteins previously not known to be mRNP-associated were detected, including the DEAD-box helicases DDX3, DDX5, and DDX9, and the ELG, hNHN1, BCLAF1, and TRAP150 proteins. The association of some of the newly identified mRNP proteins was shown to be splicing-dependent, but not to require EJC formation. Initial recruitment of EJC proteins to the spliceosome did not require an EJC binding platform at the -20/24 region of the 5' exon. Finally, while recruitment of EJC proteins and stable EJC formation were not dependent on the cap binding complex, several of the newly identified mRNP proteins required the latter for their association with mRNPs. These results provide novel insights into the composition of spliced mRNPs and the requirements for the association of mRNP proteins with the newly spliced mRNA.

Base Sequence↗

PubMed2004

Pseudouridines in and near the branch site recognition region of U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing in Xenopus oocytes.

Virtually all uridines in the branch site recognition region (BSRR) of vertebrate U2 are converted into pseudouridines after initial transcription. Here, we report a functional analysis of these modified nucleotides using the Xenopus oocyte reconstitution system. Using site-specific (32)P-labeling and TLC, we show that U2 pseudouridylation occurs much faster in the BSRR than in the 5'-terminal region. To functionally dissect the pseudouridines in the BSRR, we replaced each uridine with 5-fluorouridine (unmodifiable nucleotide) using site-specific RNase H cleavage directed by 2'-O-methyl-RNA-DNA chimeras followed by three-piece ligation. Whereas in vitro transcribed U2 containing no 5-fluorouridines rescued splicing in U2-depleted oocytes, no rescue was observed with U2 RNA containing 5-fluorouridines introduced into the BSRR. Additionally, U2 RNA containing 5-fluorouridines in the BSRR specifically inhibited pseudouridylation in the BSRR of in vitro transcribed U2 injected at a later time, although pseudouridylation in the 5'-end region was not affected. Our reconstitution results indicated that prior injection into U2-depleted oocytes with U2 RNA containing 5-fluorouridines in the BSRR almost completely abrogated the ability of in vitro transcribed U2 to rescue splicing, whereas full rescue was obtained with either cellular U2 or U2 containing pseudouridines in the BSRR. Further analyses using glycerol-gradient and native gel electrophoresis indicated that U2 RNAs lacking the BSRR pseudouridines do not participate in the assembly of the functionally active 17S U2 snRNP and the spliceosome. We conclude that the BSRR pseudouridines of vertebrate U2 are required for complete snRNP assembly and pre-mRNA splicing in Xenopus oocytes.

Animals↗

PubMed2004

In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei.

Studies over the past years indicate that there is extensive coupling between nuclear export of mRNA and pre-mRNA processing. Here, we visualized the distribution of exon junction complex (EJC) proteins and RNA export factors relative to sites of abundant pre-mRNA synthesis in the nucleus. We analyzed both HeLa cells infected with adenovirus and murine erythroleukemia (MEL) cells stably transfected with the human beta-globin gene. Using in situ hybridization and confocal microscopy, we observe accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1, and Magoh) and core spliceosome components (U snRNPs) at sites of transcription. This suggests that EJC proteins bind stably to pre-mRNA cotranscriptionally. No concentration of the export factors NXF1/TAP, p15, and Dbp5 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. These results also suggest that binding of EJC proteins to the mRNA is not sufficient to recruit TAP-p15, consistent with recent findings showing that the EJC does not play a crucial role in mRNA export. Contrasting to the results obtained in MEL cells expressing normal human beta-globin transcripts, mutant pre-mRNAs defective in splicing and 3'end processing do not colocalize with SRm160, REF, UAP56, or Sm proteins. This shows that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNAs, arguing that transcription per se is not sufficient for the stable assembly of the EJC.

Adenoviridae↗

PubMed2004

Multiple links between transcription and splicing.

Transcription and pre-mRNA splicing are extremely complex multimolecular processes that involve protein-DNA, protein-RNA, and protein-protein interactions. Splicing occurs in the close vicinity of genes and is frequently cotranscriptional. This is consistent with evidence that both processes are coordinated and, in some cases, functionally coupled. This review focuses on the roles of cis- and trans-acting factors that regulate transcription, on constitutive and alternative splicing. We also discuss possible functions in splicing of the C-terminal domain (CTD) of the RNA polymerase II (pol II) largest subunit, whose participation in other key pre-mRNA processing reactions (capping and cleavage/polyadenylation) is well documented. Recent evidence indicates that transcriptional elongation and splicing can be influenced reciprocally: Elongation rates control alternative splicing and splicing factors can, in turn, modulate pol II elongation. The presence of transcription factors in the spliceosome and the existence of proteins, such as the coactivator PGC-1, with dual activities in splicing and transcription can explain the links between both processes and add a new level of complexity to the regulation of gene expression in eukaryotes.

Alternative Splicing↗

PubMed2005

A mutational analysis of U12-dependent splice site dinucleotides.

Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5' A residue can splice to any 3' residue, although C is preferred. A 5' G residue can splice to 3' G or U residues with a preference for G. Little or no splicing was observed to 3' A or C residues. A 5' U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5' U to 3' U produced detectable spliced products. The dependence of 3' splice site activity on the identity of the 5' residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5' splice site and the next to last position of the 3' splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3' splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3' splice site distance of 11-12 nucleotides appears to be the same for both classes.

Animals↗

PubMed2002

An ATP-dependent step is required for the translocation of microinjected precursor mRNA into nuclear speckles.

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. In a previous study (Melcák et al., 2001), it has been shown that the pre-spliceosomal assembly on microinjected splicing-competent precursor mRNA takes place in the speckles, and it has been suggested that the targeting of RNA into speckes consists of two interdependent steps, namely the diffusion process, followed by the energy-dependent translocation of RNA into the speckles. In the present study, we confirm the existence of these two steps and show that this latter translocation is ATP dependent.

Adenosine Triphosphate↗

PubMed2025

Global Changes in Gene Expression and Splicing in Alcoholic Liver Disease.

Alcohol use disorder is a widespread illness commonly leading to alcoholic liver disease (ALD) and cirrhosis with an increased incidence of hepatocellular carcinoma (HCC), but the mechanisms of alcohol-related oncogenesis in the liver are incompletely understood. We tested the hypothesis that ALD predisposes to HCC via dysregulation of splicing. RNA sequencing was performed on liver biopsies from patients with different stages of ALD: early alcoholic steatohepatitis (eASH), non-severe alcoholic hepatitis (nsAH), and severe alcoholic hepatitis (sAH); furthermore, explants were collected from patients who underwent liver transplantation due to sAH (exAH). We found that alcohol caused widespread changes in transcriptome in all stages of ALD: among ~ 58,000 analyzed genomic features, ~ 4,900 were altered in eASH, ~ 9,100 - in nsAH, 14,100 - in sAH, and ~ 14,300 - in exAH. We observed thousands of missplicing events in all hepatic conditions, with mutually exclusive exons (MEE) being the most common event and exon skipping (ES) - second most common event. Analysis of ~ 600,000 exons revealed that ALD is associated with a genome-wide effect on exon expression, with ~ 50,000 exons being differentially expressed in eASH, ~ 130,000 - in nsAH, ~ 150,000 - in sAH, and ~ 120,000 - in exAH. To determine whether alcohol directly perturbs splicing, we subjected rats to alcohol vapor for 7 weeks and found that the expression of multiple snRNAs was drastically decreased, while expression of splicing factors was not affected. Screening of oncogenes and tumor suppressors, commonly involved in HCC pathogenesis, revealed that ALD affected the hepatic expression and/or splicing of most of these cancer-related genes. In summary, it appears that alcohol causes profound genome-wide changes in gene expression and splicing in the liver, likely via affecting the spliceosome. This results in altered expression and missplicing of key oncogenes and tumor suppressors involved in HCC, suggesting a novel mechanism of oncogenesis in the liver of patients with ALD.

alcohol use disorder↗

PubMed1999

Dynamic redistribution of nuclear matrix proteins by adenovirus infection.

We analyzed the redistribution of nuclear proteins, PML protein, Ku70/Ku80, a putative spliceosome associated protein, pNMM102 and a nucleolar proliferating antigen, p120 after adenovirus 5 (Ad5) infection using immunofluorescent staining. These proteins remained after in situ fractionation. PML was located at irregular bars 6 h after infection from fine dots of uninfected cells. Distribution pattern of PML was not changed throughout the course of infection. Internal nuclear matrix network composed of Ku protein became much coarser in interphase cells at 12 h after infection. Ku protein was clumped at 18 h after infection, where EIA protein was colocalized. Speckles and interconnecting fibrils recognized by monoclonal antibody NMM102 disappeared early after infection, and reappeared at 18 h after infection in various patterns. The number and staining intensity of p120 containing domains increased markedly in early replication phase, and their shape became irregular with a few fine dots. A few Ad5 infected cells revealed diffuse nucleoplasmic as well as nucleolar p120 in late replication phase. Redistribution of four different nuclear matrix proteins by Ad5 infection indicates that the nuclear matrix is dynamically involved in gene expression.

Adenoviridae Infections↗

PubMed2006

Models of systemic lupus erythematosus: development of autoimmunity following peptide immunizations of noninbred pedigreed rabbits.

Reported in this study are the initial results from studies to develop rabbit models of systemic lupus erythematosus (SLE) by immunizations using two distinct peptides on branched polylysine backbones (multiple Ag peptide)-peptides. Eleven rabbits received a peptide from the Sm B/B' spliceosomal complex previously shown to be immunogenic in rabbits, and 13 rabbits received a peptide from the rabbit N-methyl-d-aspartate receptor NR2b. All 24 animals in different generations of pedigreed, noninbred rabbits produced peptide-specific responses. Anti-nuclear autoantibody responses, including anti-dsDNA, were seen in 17 of 24 rabbits. To date, two rabbits have been observed to have seizure-like events and a third nystagmus. A model for eliciting development of SLE in genetically related yet heterogeneous rabbits may more closely resemble development of human SLE than do some models in inbred mice. Through selective breeding, it may also ultimately provide additional information about the genetics and etiology of SLE and serve as a model for assessing new treatment options.

Animals↗

PubMed2006

The splicing factor PSF is part of a large complex that assembles in the absence of pre-mRNA and contains all five snRNPs.

PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4 degrees C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.

Alternative Splicing↗

PubMed1999

Genomic organization of a voltage-gated Na+ channel in a hydrozoan jellyfish: insights into the evolution of voltage-gated Na+ channel genes.

Voltage-gated Na+ channels are responsible for fast propagating action potentials. The structurally simplest animals known to contain rapid, transient, voltage-gated currents carried exclusively by Na+ ions are the Cnidaria. The Cnidaria are thought to be close to the origin of the metazoan radiation and thus are pivotal organisms for studying the evolution of the Na+ channel gene. Here we describe the genomic organization of the Na+ channel alpha subunit, PpSCN1, from the hydrozoan jellyfish, Polyorchis penicillatus. We show that most of the 20 intron sites in this diploblast are conserved in mammalian Na+ channel genes, with some even shared by Ca2+ channels. One of these conserved introns is spliced by a rare U 12-type spliceosome. Such conservation places the origin of the primary exon arrangement of Na+ channels and different intron splicing mechanisms to at least the common ancestors of diploblasts and triploblasts, approximately 600 million-1 billion years ago.

Alternative Splicing↗

PubMed2000

ATP can be dispensable for prespliceosome formation in yeast.

The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure.

Adenosine Triphosphate↗

PubMed2000

Chromosome activities in cleaving mouse embryos to construct prenucleolar bodies orienting an intranuclear chromosome-chromosome configuration.

Most chromosomes of cleaving mouse embryos formed spherical structures called as prenucleolar bodies. Embryos stained with fluorescing dyes selective for AT-rich regions of DNA indicated that chromosome centromeres were organized by prenucleolar bodies and that chromosomes at pre-prophase of the cleavage division started to condense with their centromere regions aligned towards the peripheries of spheres. Embryo chromosomes as well as prenucleolar bodies were shown to bind a monoclonal antibody to spliceosomal snRNAs, which are contrasted to the nucleoli or chromosomes of somatic cells. More than 10 prenucleolar bodies were found in the pronuclei at the earliest stage after fertilization and 40 of them at most, diploid number of chromosomes of the mouse, in the nuclei of the 2-cell embryos. The number was decreased later in the cell cycles and at the 4-cell stage. Hence, every chromosome of cleaving mouse embryos is highly active in constructing prenucleolar bodies and behaves during the interphase until mitosis as a unit of the body; either as a single unit or as a member of the units fused. In other words, the prenucleolar bodies function as centers orienting chromosome-chromosome configuration in the fertilized eggs.

Adenine↗

PubMedDate not supplied

Higher processing rates of Alu-containing sequences in kidney tumors and cell lines with overexpressed Alu-mRNAs.

Tumor cell growth and differentiation involve several molecular mechanisms that control gene expression and define specific genomic molecular profiles in cancer cells. Among these mechanisms, it has been shown that Alu-repetitive sequences are capable of regulating gene expression at transcriptional and posttranscriptional levels, and also of modulating cellular growth, differentiation and tumor suppression. Furthermore, repetitive sequences have also been implicated in alternative RNA splicing, although the specific mechanisms involved remain unknown. Nonetheless, exactly what the involvement of Alu-containing sequences in tumor cell growth and differentiation is or to what extent they might be related to tumorigenesis or to alternative splicing is not yet clear. In order to address some of these issues, we analyzed the level of expression of Alu-containing sequences in renal tumors and cell lines and their association with immunoprecipitated ribonucleoprotein splicing complexes in nuclear RNA fractions. Over-expression of Alu-containing sequences was detected in the poly(A)-RNA fractions of all analyzed tumors and cell lines. Furthermore, Alu-sequences were associated with tumor cell growth and differentiation and found overexpressed in purified small nuclear ribonucleoprotein fractions. Overall, our results suggest the involvement of Alu-sequences in the overexpression of Alu-containing-mRNAs in human tumors, and also higher processing rates of Alu-containing sequences at the spliceosome associated with tumor cell growth and differentiation.

Adenocarcinoma, Clear Cell↗

PubMed1995

U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone.

U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.

Animals↗