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 775 records · Page 43Linked to original sources

PubMed2026

CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and zygotic genome activation during goat embryogenesis.

Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.

Animals↗

PubMed1984

Lariat RNA's as intermediates and products in the splicing of messenger RNA precursors.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

Adenoviruses, Human↗

PubMed1981

A possible regulatory mechanism in RNA processing and its implication for posttranscriptional sequence control during differentiation of cell function.

1. This paper is concerned with the possible molecular mechanism for RNA processing including posttranscriptional sequence control underlying the differentiation of cell functions. 2. It was previously postulated that intramolecular double-stranded hairpin structures present at 5'- and 3'-terminal regions of a 'pre-mRNA' are key elements for RNA splicing [24]. 3. In this paper the possibility is considered that the splicing of 'pre-mRNA' can be regulated in such a way that the formation of the proper double-stranded hairpin structures is prevented by the binding of low-molecular-weight nuclear RNA (LnRNA) to the terminal regions and/or to the nucleotide sequences around the exon-intron and intron-exon joint sites of the 'pre-mRNA' molecules. 4. Complementarity assessment of nucleotide sequences of rat preproinsulin 'pre-mRNA' and rat LnRNA, i.e. Ul, showed that Ul RNA is capable of forming stable double-standard intermolecular structures around the joint sites of preproinsulin 'pre-mRNA' and may prevent the formation of intramolecular double-stranded structures required for RNA splicing. This may imply a regulatory (inhibitory) role for Ul RNA in the processing of 'pre-mRNA'. 5. A possible regulatory role of LnRNA in RNA splicing is discussed in relation to the determination of the mRNA population to be translated in the cytoplasm during differentiation of cell functions.

Animals↗

PubMed1998

A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron.

In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.

Alleles↗

PubMed1991

Nickel mutagenesis: alteration of the MuSVts110 thermosensitive splicing phenotype by a nickel-induced duplication of the 3' splice site.

We investigated DNA damage caused by carcinogenic metals in a murine sarcoma virus (MuSV)-based mutagenicity assay in which mutations targeted to v-mos expression can be selected. Nickel chloride treatment of NRK cells (termed 6m2 cells) infected with MuSVts110, a retrovirus conditionally defective in viral RNA splicing and cell transformation, caused the outgrowth of transformed "revertants" with changes in the MuSVts110 RNA splicing phenotype. Cadmium and chromium treatment of 6m2 cells resulted in the selection of a second class of revertants with what appeared to be frameshift mutations allowing the translation of a readthrough gag-mos protein. In both classes of metal-induced revertants, viral gene expression was distinct from that observed in revertants arising in untreated 6m2 cultures, arguing that metal treatment did not simply enhance the rate of spontaneous reversion. In one representative nickel revertant line the operative nickel-induced mutation affecting MuSVts110 RNA splicing was a duplication of 70 bases surrounding the 3' splice site. The effect of this mutation was to direct splicing to the most downstream of the duplicated 3' sites and concomitantly relax its characteristic thermosensitivity. These data establish the mutagenic potential of nickel and provide the first example of a defined nickel-induced mutation in a mammalian gene.

Animals↗

PubMed1996

SR protein splicing factors interact with the Rous sarcoma virus negative regulator of splicing element.

Retroviral replication requires that a portion of the primary transcripts generated from proviral DNA be spliced to serve as mRNA for the envelope protein and in Rous sarcoma virus as src mRNA. However, a substantial amount of full-length RNA must be maintained in an unspliced form, as the unspliced RNA serves both as mRNA for structural proteins and virion-associated enzymatic proteins and as genomic RNA for progeny virions. The extent of viral RNA splicing must be finely controlled, since only a narrow range in the ratio of unspliced RNA to spliced RNA is tolerated for optimal replication. A number of cis-acting sequences within the RNA of Rous sarcoma virus play a role in preserving a large pool of unspliced RNA. One such sequence, the negative regulator of splicing (NRS), is of interest because it blocks splicing but is not located near any of the splice junctions. To better understand how this novel element blocks splicing at a distance, we set out to identify host cell factors that interact specifically with this inhibitory sequence. In this study, proteins from nuclear extracts with molecular masses of 26, 36, 44, and 55 kDa were shown by UV cross-linking assays to bind the NRS preferentially. One of them, p55, was also detected in a specific complex with SR protein electrophoretic mobility shift assay. All but p55 have biochemical properties consistent with SR protein splicing factors, and some, but not all, of the total SR proteins purified from HeLa cells cross-link specifically to the NRS. The strongest cross-linking SR protein is SRp30a/b, which is composed of the splicing factors SF2/ASF and SC35. The NRS specifically binds bacterially expressed SF2/ASF, whereas nonfunctional mutants do not. Data indicating that the 36-kDa protein which cross-links in nuclear extracts is SF2/ASF are presented. The data indicate that factors normally required for RNA splicing may be exploited by retroviruses to block splicing.

Avian Sarcoma Viruses↗

PubMed2001

Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing.

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3' splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem-loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem-loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3' A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

3' Untranslated Regions↗

PubMed2003

In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.

Aspergillus nidulans↗

PubMed1999

A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y heterozygote.

BACKGROUND & AIMS: Most patients with genetic hemochromatosis are homozygous for a single mutation of the HFE gene (C282Y). There is a second mutation, H63D, but its role in iron overload is less conclusive. The aim of this study was to investigate the basis of iron overload in a patient with classical hemochromatosis who was only heterozygous for C282Y and negative for H63D. METHODS: Genotype for the C282Y, H63D, and S65C mutations of HFE was determined in patient RFH, his family members, and 365 controls. The HFE gene was sequenced in patient RFH. Allele-specific reverse-transcription polymerase chain reaction was performed to investigate RNA splicing. Allele frequency was determined by allele-specific oligonucleotide hybridization. RESULTS: The patient is compound heterozygous for C282Y and a novel splice site mutation (IVS3 + 1G --> T). His sister has an identical genotype and elevated serum ferritin and transferrin saturation. The novel mutation functionally alters messenger RNA splicing, causing obligate skipping of exon 3. However, the IVS3 + 1G --> T mutation was found to be rare and was not detected in 630 control European chromosomes. CONCLUSIONS: IVS3 + 1G --> T in the compound heterozygous state with C282Y results in iron overload that can progress to a severe phenotype of classical hemochromatosis. The demonstration of IVS3 +1G --> T highlights the possibility of other rare HFE mutations, particularly in C282Y heterozygotes with iron overload.

Adult↗

PubMed2005

Dopamine neurons express multiple isoforms of the nuclear receptor nurr1 with diminished transcriptional activity.

Nurr1 (NR4A2) is an orphan nuclear receptor required for the development and maintenance of the dopaminergic phenotype in neurons of the ventral midbrain. This study demonstrates that multiple splice variants of nurr1 are produced in rat and human dopamine neurons. Formed by alternative RNA splicing in exon 7, nurr1a has a truncated carboxy-terminus, nurr1b has an internal deletion in the ligand-binding domain and nurr1c, newly identified in this study, has a novel carboxy-terminus produced by a frame shift downstream of the splice junction. Alternative RNA splicing in exon 3 produces the isoform known as the transcriptionally-inducible nuclear receptor (TINUR), lacking the amino-terminus. Nurr2 and the newly identified nurr2c are produced by utilization of both exon 3 and exon 7 alternative splice sites. In rat midbrain, variants other than full-length nurr1 constitute 20-35% of NR4A2 transcripts. Transfection studies in dopaminergic SK-N-AS cells demonstrate that nurr1a, nurr1b, nurr1c and TINUR have significantly reduced transcriptional activities compared with full-length nurr1, while nurr2 and nurr2c are inactive. Furthermore, in these experiments, nurr2 and nurr2c both act as dominant negatives. Production of these nurr1 variants in vivo as demonstrated here could represent a novel regulatory mechanism of nurr1 transcriptional activity and therefore, dopaminergic phenotype.

Animals↗

PubMed1990

Structure and chromosomal location of the human gene encoding cartilage matrix protein.

Cartilage matrix protein (CMP) is a major component of the extracellular matrix of nonarticular cartilage. The structure and chromosomal location of the human gene encoding CMP was determined by molecular cloning analysis. We used a partial chicken CMP cDNA probe to isolate three overlapping human genomic clones. From one of these clones, a probe containing 2 human CMP exons was isolated and used to map the gene to chromosome 1p35 and to screen a human retina cDNA library. Two overlapping cDNA clones were isolated. The predicted protein sequence of 496 amino acids includes a 22-residue signal peptide and a 474-residue mature protein of Mr 51,344. The human CMP gene and polypeptide are strikingly similar to the chicken CMP gene and polypeptide. Human CMP is 79% identical to chicken CMP and contains two homologous domains separated by an epidermal growth factor-like domain. One potential N-glycosylation site is conserved between the two species. The human CMP gene spans 12 kilobase pairs with 8 exons and 7 introns which are similar in size to those of the chicken CMP gene. Both RNA splice junctions of intron G in the human and chicken CMP genes are nonconforming to the consensus splice sequences. This suggests that the CMP gene utilizes a new RNA splicing mechanism.

Amino Acid Sequence↗

PubMed2006

Generation of interleukin-17 receptor-like protein (IL-17RL) in prostate by alternative splicing of RNA.

BACKGROUND: Interleukin-17 receptor-like protein (IL-17RL) expressed in prostate tissues changes with advanced cancers due to extensive alternative splicing, which affects the final protein. Predominant IL-17RL splice isoform variants have not been identified, hindering functional studies. METHODS: A cDNA library of IL-17RL transcripts was arrayed onto nylon membranes. Individual transcript exon structures were determined by successively probing membranes with exon-specific oligonucleotides. The most common variants were transiently over-expressed in 293T cells. RESULTS: We detected >90 different IL-17RL isoforms. Three most abundant isoforms account for approximately half the total transcripts; the full-length variant just over 11%. Surprisingly, most alternative splicing does not alter the reading frame of the full-length molecule; therefore, resulting proteins vary mostly in N-terminal domains. CONCLUSIONS: IL-17RL exists as multiple isoforms due to extensive alternative splicing. We identified the most abundant splices in prostate tissue and established a technique to investigate changes in RNA IL-17RL splicing that occur in advanced cancers.

Alternative Splicing↗

PubMed1999

Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase activity by the intron RNA.

Group II introns encode reverse transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3' exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62.

Amino Acid Sequence↗

PubMed1997

Trans-activation of group II intron splicing by nuclear U5 snRNA.

Similarities between RNA splicing during autocatalytic excision of group II introns and pre-mRNA processing led to the hypothesis that group II introns might be the evolutionary predecessors of spliceosomal small nuclear RNAs. The ID3 subdomain stem-loop structure of group II introns, the proposed analogue of the spliceosomal U5 snRNA, is thought to be essential for 5' splice site recognition and anchoring of the free 5' exon. Using the group II intron bI1 we have analysed the role of ID3 in splicing. In the absence of ID3 the 5' splice site was recognized accurately and efficiently, but exon anchoring was greatly reduced. This step was restored in the presence of RNA fragments consisting of either the terminal stem-loop structure of ID3 or spliceosomal U5 snRNA. This suggests that the predominant role of both RNAs is to anchor the 5' exon during exon ligation. Furthermore, as U5 complements for the loss of ID3, a similar network of structural RNAs may form the catalytic core of both group II introns and spliceosomes.

Binding Sites↗

PubMed2006

Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles.

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.

Alternative Splicing↗

PubMed1997

Single-channel properties of recombinant AMPA receptors depend on RNA editing, splice variation, and subunit composition.

Non-NMDA glutamate receptor subunits of the AMPA-preferring subfamily combine to form ion channels with heterogeneous functional properties. We have investigated the effects of RNA editing at the Q/R site, splice variation of the "flip/flop" cassette, and multimeric subunit assembly on the single-channel conductance and kinetic properties of the recombinant AMPA receptors formed from GluR2 and GluR4 expressed in HEK 293 cells. We found that AMPA receptor single-channel conductance was dependent on the Q/R site editing state of the subunits comprising the channel. Calcium-permeable (unedited) channels had resolvable single-channel events with main conductance states of 7-8 pS, whereas fully edited GluR2 channels had very low conductances of approximately 300 fS (estimated from noise analysis). Additionally, the flip splice variant of GluR4 conferred agonist-dependent conductance properties reminiscent of those found for a subset of AMPA receptors in cultured cerebellar granule cells. These results provide a description of the single-channel properties of certain recombinant AMPA receptors and suggest that the single-channel conductance may be determined by the expression of edited GluR2 subunits in neurons.

Cell Line↗

PubMed2001

Heterologous, splicing-dependent RNA editing in chloroplasts: allotetraploidy provides trans-factors.

RNA editing is unique among post-transcriptional processes in plastids, as it exhibits extraordinary phylogenetic dynamics leading to species-specific editing site patterns. The evolutionary loss of a site is considered to entail the loss of the corresponding nuclear-encoded site-specific factor, which prevents the editing of foreign, i.e. heterologous, sites. We investigated the editing of short 'spliced' and 'unspliced' ndhA gene fragments from spinach in Nicotiana tabacum (tobacco) in vivo using biolistic transformation. Surprisingly, it turned out that the spinach site is edited in the heterologous nuclear background. Furthermore, only exon-exon fusions were edited, whereas intron-containing messages remained unprocessed. A homologue of the spinach site was found to be present and edited in Nicotiana tomentosiformis, representing the paternal parent, but absent from Nicotiana sylvestris, representing the maternal parent of tobacco. Our data show that: (i) the cis-determinants for ndhA editing are split by an intron; (ii) the editing capacity cannot be deduced from editing sites; and (iii) allopolyploidization can increase the editing capacity, which implies that it can influence speciation processes in evolution.

Base Sequence↗

PubMed2005

The proto-oncoprotein SYT interacts with SYT-interacting protein/co-activator activator (SIP/CoAA), a human nuclear receptor co-activator with similarity to EWS and TLS/FUS family of proteins.

The proto-oncoprotein SYT is involved in the unique translocation t(X;18) found in synovial sarcoma SYT-SSX fusions. SYT has a conserved N-terminal domain (SNH domain) that interacts with the human paralog of Drosophila Brahma (hBRM) and Brahma-related gene 1 (BRG1) chromatin remodeling proteins and a C-terminal transactivating sequence rich in glutamine, proline, glycine, and tyrosine (QPGY domain). Here we reported the isolation of the ribonucleoprotein SYT-interacting protein/co-activator activator (SIP/CoAA), which specifically binds the QPGY domain of SYT and also the SYT-SSX2 translocation fusion. SIP/CoAA is a general nuclear co-activator and an RNA splicing modulator that contains two RNA recognition motifs and multiple hexapeptide repeats. We showed that the region consisting of the hexapeptide motif (YQ domain) is similar to the hexapeptide repeat domain found in EWS and in TLS/FUS family proteins. The YQ domain also resembles the QPGY region of SYT itself and like all these other domains acts as a transcriptional activator in reporter assays. Most interestingly, the last 84 amino acids adjacent to YQ down-modulate by 25-fold the YQ transactivation of the reporter gene, and both domains are important for SIP/CoAA binding to SYT. In addition, SYT acts together with SIP/CoAA in stimulating estrogen and glucocorticoid receptor-dependent transcriptional activation. Activation is hormone-dependent and requires functional hBRM and/or BRG1. The stimulation is strongly reduced if the N-terminal region of hBRM/BRG1 (amino acids 1-211) is deleted. This region encompasses the SNF11 binding domain (amino acids 156-211), which interacts specifically with SYT in vivo and in vitro.

Amino Acid Motifs↗