Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012; Galactose-1-phosphate uridylyltransferase (GALT) transfers a UMP group from UDP-glucose to Gal1P in the second step of the Leloir pathway for galactose metabolism. Pathogenic mutations in the GALT gene cause deficient or absent activity of the enzyme and result in Classical Galactosemia. Mutational analysis of 27 Portuguese patients confirmed Q188R as the prevalent molecular defect (≈60%), and revealed the intronic variation c.820+13a>g (IVS8+13a>g) as the second most frequent mutation, accounting for 12.5% of the mutant alleles. In silico analysis revealed that the presence of the c.820+13a>g variation activates a cryptic splicing donor site and seems to create a strong ESE motif for the binding of the SRSF1 protein. A minigene-based approach was used to analyze the effects of this presumptive pre-mRNA splicing mutation. The pSPL3 vector containing either the genomic wild-type or mutant fragments was transfected into COS-7 and Hek293 cell lines. 24h after transfection, RNA was purified and amplified by RT-PCR. Direct sequencing analysis of the reaction products clearly showed that c.820+13a>g favors the next GT dinucleotide (c.820+14_15) to be used as a new 5’ splice donor site...
In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood. In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence. Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG. Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing. Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation. This activation is sequence-specific and unidirectional. However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG. Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals. Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC.
cDNA clones coding for human plasma membrane Ca2+ pump isoforms have been isolated from a fetal skeletal muscle cDNA library. Compared with the sequence of a teratoma cDNA-encoded pump these clones specify isoforms that contain either 29- or 38-amino acid insertions within the calmodulin-binding region. Replacement of two basic arginine residues by an aspartic acid and a glutamine residue could influence the binding of calmodulin to these isoforms. RNase mapping shows that RNA species containing the 29-residue-encoding insertion are particularly abundant in skeletal muscles. The sequences coding for the insertions are present on a single 154-base-pair exon, as demonstrated by an analysis of the corresponding genomic region, and they are included in their respective mRNAs by alternative splicing involving the differential usage of two internal "cryptic" donor splice sites in the presence of a nearby canonical one. Inclusion of the complete 154-base-pair exon results in an mRNA coding for a pump protein with a shorter C-terminal amino acid sequence that lacks a consensus site for phosphorylation by the cAMP-dependent kinase. Exclusion, inclusion, or partial inclusion of the same exon can thus lead to the production of four different mRNAs from a single gene. When expressed as protein...
Steingrimsdottir, H; Rowley, G; Dorado, G; Cole, J; Lehmann, A R
Fonte: PubMedPublicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 25/03/1992Português
Relevância na Pesquisa
A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA.
The class I myosin, Myo1b, is a calmodulin- and actin-associated molecular motor widely expressed in mammalian tissues. Analytical ultracentrifugation studies indicate that Myo1b purified from rat liver has a Stokes radius of 6.7 nm and a sedimentation coefficient, s20,w, of 7.0 S with a predicted molar mass of 213 kg/mol. These results indicate that Myo1b is monomeric and consists primarily of a splice variant having five associated calmodulins. Molecular modeling based on the analytical ultracentrifugation studies are supported by electron microscopy studies that depict Myo1b as a single-headed, tadpole-shaped molecule with outer dimensions of 27.9 × 4.0 nm. Above a certain Myo1b/actin ratio, Myo1b bundles actin filaments presumably by virtue of a second actin-binding site. These studies provide new information regarding the oligomeric state and morphology of Myo1b and support a model in which Myo1b cross-links actin through a cryptic actin-binding site.
den Hollander, Anneke I.; Koenekoop, Robert K.; Yzer, Suzanne; Lopez, Irma; Arends, Maarten L.; Voesenek, Krysta E. J.; Zonneveld, Marijke N.; Strom, Tim M.; Meitinger, Thomas; Brunner, Han G.; Hoyng, Carel B.; van den Born, L. Ingeborgh; Rohrschneider
Fonte: The American Society of Human GeneticsPublicador: The American Society of Human Genetics
Leber congenital amaurosis (LCA) is one of the main causes of childhood blindness. To date, mutations in eight genes have been described, which together account for ∼45% of LCA cases. We localized the genetic defect in a consanguineous LCA-affected family from Quebec and identified a splice defect in a gene encoding a centrosomal protein (CEP290). The defect is caused by an intronic mutation (c.2991+1655A→G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 messenger RNA. This mutation was detected in 16 (21%) of 76 unrelated patients with LCA, either homozygously or in combination with a second deleterious mutation on the other allele. CEP290 mutations therefore represent one of the most frequent causes of LCA identified so far.
Leber congenital amaurosis (LCA) is a severe hereditary retinal dystrophy responsible for congenital or early-onset blindness. The most common disease-causing mutation (>10%) is located deep in intron 26 of the CEP290 gene (c.2991+1655A>G). It creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. In the present study, we show that the use of antisense oligonucleotides (AONs) allow an efficient skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients. These data support the feasibility of an AON-mediated exon skipping strategy to correct the aberrant splicing.
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease that is frequently caused by a de novo point mutation at position 1824 in LMNA. This mutation activates a cryptic splice donor site in exon 11, and leads to an in-frame deletion within the prelamin A mRNA and the production of a dominant-negative lamin A protein, known as progerin. Here we show that primary HGPS skin fibroblasts experience genome-wide correlated alterations in patterns of H3K27me3 deposition, DNA-lamin A/C associations, and, at late passages, genome-wide loss of spatial compartmentalization of active and inactive chromatin domains. We further demonstrate that the H3K27me3 changes associate with gene expression alterations in HGPS cells. Our results support a model that the accumulation of progerin in the nuclear lamina leads to altered H3K27me3 marks in heterochromatin, possibly through the down-regulation of EZH2, and disrupts heterochromatin–lamina interactions. These changes may result in transcriptional misregulation and eventually trigger the global loss of spatial chromatin compartmentalization in late passage HGPS fibroblasts.
The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern...
Insertional mutagenesis screens have provided thousands of mutant alleles for analysing genes of varied functions in Drosophila melanogaster. We here document mechanisms of insertional mutagenesis by a LINE element, the I factor, by determining the molecular structure of RNAs produced from two alleles of the white gene of D.melanogaster, wIR1 and wIR6. These alleles result from insertion of the I factor into introns of the gene. We show that sequences present within the element direct aberrant splicing and termination events. When the I factor is inserted within the white first intron it may lead to the use of a cryptic 3' splice site which does not contain the dinucleotide AG. This splicing gives rise to a chimeric messenger RNA whose synthesis is controlled differently in tissues where the mutated gene is expressed. When the I factor is inserted within the white last intron it induces synthesis of truncated mRNAs. These results provide, for the first time, mechanisms for I factor insertional mutagenesis. They are discussed in the more general context of RNA processing in Drosophila and the evolution of eukaryotic gene introns.
It has recently been argued that pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe may be more similar to splicing in metazoan species than in the budding yeast Saccharomyces cerevisiae. In this report we show that, contrary to this assumption, the conserved sequence element 5'-CTPu APy-3' found in all S. pombe introns 6-18 nucleotides upstream of the 3' splice site is, like the TACTAAC box in S. cerevisiae, indispensable for efficient splicing. The conserved adenine residue of this sequence is used for branch formation and point mutations introduced into the CTPuAPy sequence abolish splicing and seem not to result in the recruitment of cryptic branch sites. We also show that an S. cerevisiae intron is correctly excised in S. pombe whereby the TACTAAC box is used in branch formation.
Most eukaryotic messenger RNAs are capped, spliced, and polyadenylated via
co-transcriptional processes that are coupled to each other and to the
transcription machinery. Coordination of these processes ensures correct RNA
maturation and provides for the diversity of the transcribed isoforms. Thus,
RNA processing is a chain of events in which the completion of one event is
coupled to the initiation of the next one. In this context, the relationship
between splicing and polyadenylation is an important aspect of gene regulation.
We have found that cryptic polyadenylation signals are widely distributed over
the intron sequences of Drosophila melanogaster. As shown by
analyzing the distribution of genes arranged in a nested pattern, where one
gene is fully located within an intron of another gene, overlapping of putative
polyadenylation signals is a fairly common event affecting about 17% of all
genes. Here we show that polyadenylation signals are silenced within introns:
the poly(A) signal is utilized in the exonic but not in the intronic regions of
the transcript. The transcription does not end within the introns, either in a
transient reporter system or in the genomic context, while deletion of the
5'-splice site restores their functionality. According to a full
Drosophila transcriptome analysis...
Enhanced expression of the cKi-ras proto-oncogene in a bone marrow-derived mouse cell line, 416B, has been shown to be associated with the integration of Friend viral DNA into the cellular gene. Here we report the results of experiments designed to clarify the molecular mechanism responsible for the cKi-ras overexpression. Based on primer extension analyses and DNA sequencing of cKi-ras cDNA clones, we have obtained evidence that the 416B cells contain viral-host chimaeric transcripts that initiate within the 3' long terminal repeat (LTR) of the integrated provirus. Processing of the transcripts from the rearranged cKi-ras gene includes an unexpected splicing event associated with the fortuitous creation of a cryptic donor splice site at the junction between the proviral and cellular DNA sequences. These data demonstrate that enhanced cKi-ras expression in the 416B cells results from a retroviral promoter insertion mechanism of transcriptional activation.
Interferons (IFNs) are key regulators for both innate and adaptive immune responses. By screening ENU-mutagenized mice, we identified a pedigree- P117 which displayed impaired response to type I, but not type II, IFNs. Through inheritance test, genetic mapping and sequencing, we found a T to A point mutation in the 5' splice site of STAT2 intron 4–5, leading to cryptic splicing and frame shifting. As a result, the expression of STAT2 protein was greatly diminished in the mutant mice. Nonetheless, a trace amount of functional STAT2 protein was still detectable and was capable of inducing, though to a lesser extent, IFNα-downstream gene expressions, suggesting that P117 is a STAT2 hypomorphic mutant. The restoration of mouse or human STAT2 gene in P117 MEFs rescued the response to IFNα, suggesting that the mutation in STAT2 is most likely the cause of the phenotypes seen in the pedigree. Development of different subsets of lymphocytes appeared to be normal in the mutant mice except that the percentage and basal expression of CD86 in splenic pDC and cDC were reduced. In addition, in vitro Flt3L-dependent DC development and TLR ligand-mediated DC differentiation were also defective in mutant cells. These results suggest that STAT2 positively regulates DC development and differentiation. Interestingly...
S1 nuclease mapping is commonly used to analyze transcription and processing of unlabelled RNAs. However, the S1 protocol that appears best suited to demonstrate splicing of a particular RNA (using an intronless probe that is 5' end-labelled in the downstream exon) is not diagnostic as expected. Rather, both intron-containing RNA and intronless RNA confer protection of probe across the splice juncture. To unambiguously demonstrate correctly spliced RNAs that begin at a specific initiation site, we present a procedure in which unspliced RNA molecules are first cleaved by RNase H following annealing to an intronic DNA fragment and the remaining RNA is then subjected to S1 analysis using an intronless probe present in vast excess. Only spliced, correctly initiated transcripts can protect the probe across the splice junction and up to residue +1. This RNase H/S1 method provides a broadly applicable technique with which to demonstrate splicing and initiation of a variety of transcripts, especially ones from transfected genes that can arise both from the normal and from activated cryptic initiation sites.
We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.
Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5′ donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth.
Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by dramatic premature aging. Classic HGPS is caused by a de novo point mutation in exon 11 (residue 1824, C → T) of the LMNA gene, activating a cryptic splice donor and resulting in a mutant lamin A (LA) protein termed “progerin/LAΔ50” that lacks the normal cleavage site to remove a C-terminal farnesyl group. During interphase, irreversibly farnesylated progerin/LAΔ50 anchors to the nuclear membrane and causes characteristic nuclear blebbing. Progerin/LAΔ50's localization and behavior during mitosis, however, are completely unknown. Here, we report that progerin/LAΔ50 mislocalizes into insoluble cytoplasmic aggregates and membranes during mitosis and causes abnormal chromosome segregation and binucleation. These phenotypes are largely rescued with either farnesyltransferase inhibitors or a farnesylation-incompetent mutant progerin/LAΔ50. Furthermore, we demonstrate that small amounts of progerin/LAΔ50 exist in normal fibroblasts, and a significant percentage of these progerin/LAΔ50-expressing normal cells are binucleated, implicating progerin/LAΔ50 as causing similar mitotic defects in the normal aging process. Our findings present evidence of mitotic abnormality in HGPS and may shed light on the general phenomenon of aging.