SEARCH · PubMed Health

Results for “RNA Splicing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,171 records · Page 65Linked to original sources

PubMed2002

A comparative analysis of vertebrate sex determination.

Sex determination in vertebrates is controlled by a variety of mechanisms. We compared the expression of SF1, DAX1, DMRT1, SOX9 and AMH during gonadogenesis in the mouse, chicken and alligator embryo. In contrast to the expression profile of Sf1 in mouse embryos, chicken and alligator embryos show higher levels of Sf1 expression in the developing ovaries compared to testes. This may reflect the higher level of sex hormone synthesis in the ovary compared to the testis in chickens and alligators. The DAX1 gene has a similar expression profile in all three vertebrate species but appears to have different gene structure. As in mouse, DMRT1 was expressed at very high levels in the chicken and alligator male gonad. The male-specific up-regulation of SOX9 expression appears to be a common feature in all three vertebrates. In the chicken and alligator AMH is expressed prior to SOX9, suggesting that in these species SOX9 cannot initiate AMH expression as it does in mammals. SOX9 acts at multiple points in the vertebrate testis pathway but it appears that only some of these functions have been conserved through evolution.

Animals↗

PubMed2002

Concerted regulation of gonad differentiation by transcription factors and growth factors.

It is well known that signals from growth factors regulate gene transcription thus initiating certain steps of cellular and tissue differentiation during development. In gonad differentiation several transcription factors have been identified as the genes underlying human diseases displaying gonadal defects and as the genes necessary for gonad differentiation as demonstrated by gene disruption studies. In addition, one of the growth factors, WNT4, is known to be involved in gonadal differentiation. However, it remains unclear which gene is directly downstream of the WNT4 signal. We have recently demonstrated that Dax1 (NR0B1) gene transcription is significantly up-regulated by the presence of SF1 (NR5A1). Functional analysis showed that DAX1 acts as a repressor against SF1 through direct interaction between the repeated sequences at the N-terminus of DAX1 and a ligand-binding domain of SF1. Considering that the expressions of these factors during gonad differentiation show a sexually dimorphic pattern, it is likely that the Dax1 gene transcription is up-regulated by WNT4 signal and thereafter DAX1 suppresses the genes downstream of SF1 such as Amh and steroidogenic genes in female gonads.

Animals↗

PubMed1993

Direct detection of HIV-1 RNA in epidermal Langerhans cells of HIV-infected patients.

Human Langerhans cells (LC) are bone marrow-derived, HLA-DR+, CD1a+, and CD4+ dendritic antigen-presenting cells found in stratified squamous epithelia. As other members of the dendritic leukocyte family, to which they belong, LC have been reported as targets for HIV-1 infection. The aim of the present study was to investigate whether HIV-1 RNA is expressed in epidermal LC of HIV-1-infected patients. Bulk epidermal cell (EC) suspensions were prepared from skin of nine recently deceased AIDS patients and 11 seronegative controls. Purified LC (94 +/- 4% HLA-DR+ cells with no CD3+ cells, as assessed by flow microfluorimetry analysis) and LC-depleted EC were obtained by immunomagnetic separation using an anti-CD1a monoclonal antibody. Samples were analyzed for the presence of HIV-1 RNA by reverse transcription of a spliced mRNA region of the tat gene, followed by polymerase chain reaction amplification. HIV-1-spliced RNA was detected in LC from 6 of 9 patients examined, whereas LC-depleted EC fractions from the same patients were all negative. The results indicate that epidermal LC from HIV-seropositive patients actively transcribe HIV-1 proviral DNA, further supporting the hypothesis that HIV productively infected LC could serve as a reservoir of the virus in the epidermis and as a source for the infection of T lymphocytes.

Adult↗

PubMed2003

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia.

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (</= 10% of control) platelet content of GPIIb-IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3'-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both.

Alternative Splicing↗

PubMed2005

Regulation of protein activity with small-molecule-controlled inteins.

Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts. Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision. These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins. Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs. This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing. This conditional activation was also found to occur in a dose-dependent manner. Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands. The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.

Allosteric Site↗

PubMed1989

Beta-thalassemia due to two novel nucleotide substitutions in consensus acceptor splice sequences of the beta-globin gene.

We have identified two novel RNA-splicing mutations affecting a critical nucleotide (nt) in the acceptor consensus sequences at both the IVS-1/exon 2 and IVS-2/exon 3 junctions of the human beta-globin gene. Both mutations are single nt substitutions, T to G and C to A, at position -3 adjacent to the invariant AG dinucleotide. For the IVS-2/exon 3 mutation abnormal splicing into the cryptic splice site at IVS-2 nt 579 is documented. Identification of these two mutations provides further support for the importance of the location of specific nucleotides within the consensus sequences in splice site selection and RNA processing.

Base Sequence↗

PubMed1988

Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins A1 and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.

HeLa Cells↗

PubMed2005

Formation of the CRS2-CAF2 group II intron splicing complex is mediated by a 22-amino acid motif in the COOH-terminal region of CAF2.

CRS2-associated factors 1 and 2 (CAF1 and CAF2) are closely related proteins that function in concert with chloroplast RNA splicing 2 (CRS2) to promote the splicing of specific sets of group II introns in maize chloroplasts. The CRS2-CAF complexes bind tightly to their cognate group II introns in vivo, with the CAF subunit determining the intron specificity of the complex. In this work we show that the CRS2-CAF complexes are stable in the absence of their intron targets and that CRS2 binds a 22 amino acid motif in the COOH-terminal region of CAF2 that is conserved in CAF1. Yeast two-hybrid assays and co-fractionation studies using recombinant proteins show that this motif is both necessary and sufficient to bind CRS2. The 22-amino acid motif is predicted to form an amphipathic helix whose hydrophobic surface is conserved between CAF1 and CAF2. We propose that this surface binds the hydrophobic patch on the surface of CRS2 previously shown to be necessary for the interaction between CRS2 and CAF2.

Amino Acid Motifs↗

PubMed2025

A six-repeat PPR protein WPR directly binds target RNAs and coordinates chloroplast RNA processing via dual recruitment of MORF1, MORF8b, and CAF2 proteins in rice.

Pentatricopeptide repeat (PPR) proteins are key regulators of organelle RNA metabolism in plants, yet their precise mechanisms in chloroplast RNA processing remain unclear. Here, we identify WPR, a unique P-type PPR protein in rice (Oryza sativa L.), as a critical factor in chloroplast RNA splicing and editing. A ~112-kb chromosomal inversion upstream of WPR causes an albino panicle rachis phenotype (wpr mutant), while complete loss of WPR function leads to seedling lethality. WPR deficiency disrupts the splicing of multiple group II introns (atpF, ndhA, ndhB, petB, rpl2, and rps12) and impairs RNA editing in transcripts such as ndhA, ndhB, ndhG, rps14, and ycf3. Electrophoretic mobility shift assay (EMSA) data confirm that WPR directly binds to precursor mRNAs of atpF, ndhA, petB, rpl2, and rps12. Strikingly, WPR interacts with both RNA editing factors (MORF1, MORF8b) and the splicing factor CAF2, but not with other PPR proteins targeting the same transcripts. Unlike most PPR proteins, WPR contains only six PPR repeats, which is the fewest among all functionally characterized rice PPR proteins. With few informative repeats, WPR likely possesses a broad, low-specificity RNA-binding activity. Moreover, WPR may act on chloroplast RNA maturation by recruiting MORFs and CAF2 rather than other PPR proteins, highlighting a novel regulatory mode in which P-type PPR protein may act as an RNA-binding scaffold to integrate diverse RNA-processing machineries. This study advances the understanding of PPR protein diversity and provides new insights into the molecular mechanisms of chloroplast RNA processing in rice.

Oryza↗

PubMed1997

Functional and sequence analysis of splicing defective nrdB mutants of bacteriophage T4 reveal new bases and a new sub-domain required for group I intron self-splicing.

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron. In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analyzed for both sequence and functional changes. These mutants cluster towards the ends in regions of conserved structural elements of the intron. These 23 mutants have single base changes at 14 different sites. Interestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron. A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron structure. The mutation sites in pairing regions P3, P7, P8, P9 and between P6[3'] and P7[5'] are identical to changes found in the well studied td (encoding dTMP synthase) intron. However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrdB intron and disrupt self-splicing. A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in this pairing, thus defining a new sub-domain essential for RNA splicing. Like the td intron, the mutation site in P9 of the nrdB intron is a hot spot for mutations, but unlike td, the nrdB intron does not show a mutational hot spot in the P6[5'] region. Our molecular dissection of the nrdB intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3' splice site selection, cleavage and exon ligation.

Bacteriophage T4↗

PubMed1996

Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency.

The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences. By using chemical and enzymatic methods to probe the RNA secondary structure and site-directed mutagenesis, we established the complete secondary structure of the U3A snoRNA precursor. This is the first determination of the complete secondary structure of an RNA spliced in a spliceosome. The peculiar cruciform structure of the U3A snoRNA 3'-terminal region is formed in the precursor RNA and the conserved Boxes B and C are accessible for binding the U3 snoRNP proteins. The intron forms a highly folded structure with a long central stem-loop structure that brings the 5' box and the branch site together. This is in agreement with the idea that secondary structure interactions are necessary for efficient splicing of long introns in yeast. The 3' splice site is in a bulged loop and the branch site sequence is single-stranded. Surprisingly, the 5' splice site is involved in a 6-base pair interaction. We used in vitro splicing experiments to show that, despite a noncanonical branch site sequence and a base paired 5' splice site, transcripts that mimic the authentic pre-U3A snoRNA are spliced very efficiently in vitro. Sequestering the 5' splice site in a more stable structure had a negative effect on splicing, which was partially compensated by converting the branch site sequence into a canonical sequence. Analysis of spliceosomal complex formation revealed a cumulative negative effect of a base pair interaction at the 5' splice site and of a deviation to the consensus sequence at the branch site on the efficiency of spliceosome formation in vitro.

Base Composition↗

PubMed2004

Crystal structure of a group I intron splicing intermediate.

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.

Azoarcus↗

PubMed1994

Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect.

While the majority of metazoan genes and those of the DNA viruses which infect them contain introns which require RNA splicing, herpes simplex virus type 1 (HSV-1) encodes relatively few spliced products. We previously showed that the HSV-1 immediate-early protein ICP27 decreased the levels of spliced target mRNAs in transfections and spliced cellular mRNAs during infection, suggesting that ICP27 may function in impairing host cell splicing. Here, we show that during infections with the wild type, but not in infections with an ICP27 viral mutant termed 27-LacZ, precursor RNA accumulated for a virus transcript which contained introns. Pre-mRNA accumulation in the nucleus was greater than that in the cytoplasm, indicating that splicing rather than transport was affected. Furthermore, splicing of a beta-globin pre-mRNA substrate was inhibited in nuclear extracts from wild-type-infected cells but not in extracts from cells infected with 27-LacZ. The inhibitory activity in extracts from wild-type-infected cells was able to reduce the splicing efficiency of competent extracts in biochemical complementation assays. ICP27 appeared to be responsible for this decrease, because the splicing activity of an extract from cells infected with an ICP27 ts mutant was significantly reduced after incubation of the extract at the permissive temperature to allow renaturation of the conformationally defective ICP27 protein. These results strongly suggest that HSV-1 infection inhibits host cell splicing through the action of ICP27.

Cells, Cultured↗

PubMed1993

The conserved pre-mRNA splicing factor U2AF from Drosophila: requirement for viability.

The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. The dU2AF50 protein is closely related to its mammalian counterpart and contains three carboxyl-terminal ribonucleoprotein consensus sequence RNA binding domains and an amino-terminal arginine- and serine-rich (R/S) domain. Recombinant dU2AF50 protein complements mammalian splicing extracts depleted of U2AF activity. Germline transformation of Drosophila with the dU2AF50 complementary DNA rescues a lethal mutation, establishing that the dU2AF50 gene is essential for viability. R/S domains have been found in numerous metazoan splicing factors, but their function is unknown. The mutation in Drosophila U2AF will allow in vivo analysis of a conserved R/S domain-containing general splicing factor.

Amino Acid Sequence↗

PubMed1990

A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1.

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

AMP Deaminase↗

PubMed1992

Calcium pump isoforms: diversity, selectivity and plasticity. Review article.

Ca2+ pumps are essential for removing cytosolic Ca2+ either across the plasma membrane (PM) or into internal organelles such as the sarcoplasmic reticulum (SR). Four genes (PMCA1, PMCA2, PMCA3 and PMCA4) have been reported to encode the PM Ca2+ pumps and three (SERCA1, SERCA2 and SERCA3) to encode the SR Ca2+ pumps. The PM Ca2+ pumps are stimulated by calmodulin, the SR Ca2+ pumps encoded by SERCA1 and SERCA2 are stimulated by phospholamban while the product of SERCA3 may be regulated directly by cAMP-dependent protein kinase. Alternative splicing of the primary transcripts of several of these genes has been reported to occur in a tissue selective manner and for others to alter during ontogeny. For the PM Ca2+ pump, alternative RNA splicing may result in isoforms with altered cyclic nucleotide dependent protein kinase sensitivity. The diversity in distribution of Ca2+ pump isoforms and their regulatory factors when coupled with different Ca2+ entry mechanisms allows for tissue selectivity and plasticity in stimulus-response coupling. The roles of various Ca2+ pump isoforms, the rationale behind their tissue selective expression and the plasticity in this expression are among the new challenges to researchers in this field.

Animals↗

PubMed1990

Distribution and consensus of branch point signals in eukaryotic genes: a computerized statistical analysis.

An intermediate stage in the process of eukaryotic RNA splicing is the formation of a lariat structure. It is anchored at an adenosine residue in intron between 10 and 50 nucleotides upstream of the 3' splice site. A short conserved sequence (the branch point sequence) functions as the recognition signal for the site of lariat formation. It has been generally assumed that the branch point is recognized mainly by the presence of its unique sequence where the lariat is formed. However, the known branch point consensus sequence is found to be distributed nearly randomly throughout the gene sequence with only a slightly higher frequency in the expected lariat region. Further, the known consensus sequence is found to be clearly inadequate to specify branch points. These observations have implications for understanding the mechanism of branch point recognition in the process of splicing, and the possible evolution of the branch point signal.

Animals↗

PubMed1994

Aberrant splicing of the COL4A5 gene in patients with Alport syndrome.

A variety of mutations have been identified in the X-linked type IV collagen alpha 5 chain (COL4A5) gene in patients with Alport syndrome. A substantial number of these mutations were predicted to have an effect on RNA splicing. For 4 such mutations in our group of patients the effect of the DNA mutation on the COL4A5 mRNA structure and stability was analysed. An alteration of the invariant splice acceptor site of intron 41 resulted in a shift of the actual splicing to either a cryptic splice site within exon 42 or the normal splice site in the next intron. A single base substitution of the final nucleotide of exon 48 resulted in the removal of the entire exon. Two frameshift mutations, a 10 basepair duplication in exon 49 and a single base deletion in exon 41, were incorporated in the mRNA as such and resulted in a stretch of missense codons terminated by a premature stop codon. Exon skipping was occasionally observed in these samples, but not reproducibly in every experiment. In healthy controls exon skipping was never detected. Analysis of female carriers revealed that in only one case was the stability of the mutated mRNA reduced in comparison with the normal transcript. The extent to which the non-collagenous domain was predicted to be deleted correlated with the severeness of the disease.

Base Sequence↗