Abstract
The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often ...restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid–binding proteins YB-1 and human Tra2-β1. Here, we show that hTra2-β1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-β1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-β1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis. (Cancer Res 2006; 66(9): 4774-80)
Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein ...phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.
Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in tauopathies, which include Alzheimer's disease (AD). Tau is a ...microtubule‐associated protein whose transcript undergoes alternative splicing in the brain. Exon 10 encodes one of four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau protein isoforms containing four microtubule‐binding repeats (4R) whereas exclusion leads to isoforms containing only three repeats (3R). The ratio between 3R and 4R isoforms is tightly controlled via alternative splicing in the human adult nervous system and distortion of this balance results in neurodegeneration. Previous studies showed that several splicing regulators, among them hTRA2‐beta1 and CLK2, regulate exon 10 alternative splicing. Like most splicing factors, htra2‐beta and clk2 pre‐mRNAs are regulated by alternative splicing. Here, we investigated whether human postmortem brain tissue of AD patients reveal differences in alternative splicing patterns of the tau, htra2‐beta, presenilin 2 and clk2 genes when compared with age‐matched controls. We found that the splicing patterns of all four genes are altered in affected brain areas of sporadic AD patients. In these affected areas, the amount of mRNAs of tau isoforms including exon 10, the htra2‐beta1 isoform and an inactive form of clk2 are significantly increased. These findings suggest that a misregulation of alternative splicing seems to contribute to sporadic AD.
Alternative splicing is one of the most important mechanisms to generate a large number of mRNA and protein isoforms from the surprisingly low number of human genes. Unlike promoter activity, which ...primarily regulates the amount of transcripts, alternative splicing changes the structure of transcripts and their encoded proteins. Together with nonsense-mediated decay (NMD), at least 25% of all alternative exons are predicted to regulate transcript abundance. Molecular analyses during the last decade demonstrate that alternative splicing determines the binding properties, intracellular localization, enzymatic activity, protein stability and posttranslational modifications of a large number of proteins. The magnitude of the effects range from a complete loss of function or acquisition of a new function to very subtle modulations, which are observed in the majority of cases reported. Alternative splicing factors regulate multiple pre-mRNAs and recent identification of physiological targets shows that a specific splicing factor regulates pre-mRNAs with coherent biological functions. Therefore, evidence is now accumulating that alternative splicing coordinates physiologically meaningful changes in protein isoform expression and is a key mechanism to generate the complex proteome of multicellular organisms.
The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to ...certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2- beta 1. Here, we show that hTra2- beta 1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2- beta 1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2- beta 1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis. (Cancer Res 2006; 66(9): 4774-80)
We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are ...two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300
bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.
Alternative splicing is an important regulatory mechanism of mammalian gene expression. The alternative splicing database (ASD) consortium is systematically collecting and annotating data on ...alternative splicing. We present the continuation and upgrade of the ASD T. A. Thanaraj, S. Stamm, F. Clark, J. J. Riethoven, V. Le Texier, J. Muilu (2004) Nucleic Acids Res. 32, D64-D69 that consists of computationally and manually generated data. Its largest parts are AltSplice, a value-added database of computationally delineated alternative splicing events. Its data include alternatively spliced introns/exons, events, isoform splicing patterns and isoform peptide sequences. AltSplice data are generated by examining gene-transcript alignments. The data are annotated for various biological features including splicing signals, expression states, (SNP)-mediated splicing and cross-species conservation. AEdb forms the manually curated component of ASD. It is a literature-based data set containing sequence and properties of alternatively spliced exons, functional enumeration of observed splicing events, characterization of observed splicing regulatory elements, and a collection of experimentally clarified minigene constructs. ASD includes a workbench, which is an analysis tool that enables users to carry out splicing related analysis such as characterization of introns for various splicing signals, identification of splicing regulatory elements on a given RNA sequence, prediction of putative exons and prediction of putative translation start codons. The different ASD modules are integrated and can be accessed through user-friendly interfaces and visualization tools. ASD data has been integrated with Ensembl genome annotation project as a Distributed Annotation System (DAS) resource and can be viewed on Ensembl genome browser. The ASD resource is presented at (http://www.ebi.ac.uk/asd).
Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome ...research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to ...certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.
In this work, I performed a genome wide analysis and systematical collection of alternative exons. It resulted in the improvement of a database of alternatively spliced exons collected from ...literature (AEdb) for ASD (Alternative Splicing Database). Based on this data, a custom splice array covering around 300 splicing factors was constructed. This created a platform for further research on alternative splicing and splicing factors.An increasing number of diseases are associated with alternative splicing. These diseases can be caused by mutation in regulatory sequences of the pre-mRNA or by changes in the concentration of trans-acting factors. Among them, we concentrated on the Alzheimer’s Disease (AD) and breast cancer. We found that the regulation of the CD44 gene by tra2-beta1 was associated with tumor progression and metastasis in breast cancer.In sporadic AD patients, the amount of mRNAs of tau isoforms including exon 10, the htra2-beta1 isoform and an inactive form of clk2 are significantly increased. This suggests that a mis-regulation of alternative splicing contributes to sporadic AD.Previous research shows that TRA2-BETA1, an important SR-like splicing factor, accumulates in the cytosol under cellular stress conditions. Yeast two hybrid studies showed that TRA2-BETA1 directly binds to RPL3, a protein of the large ribosomal subunit that plays a role in peptidyltransferase center formation. To identify the regulation of translation by tra2-beta1, we confirmed the interaction between TRA2-BETA1 and RPL3 using in vitro pull down assays with recombinant proteins. Using sucrose gradient fractionation, TRA2-BETA1 was found to co-sediments with ribosomes and polysome fractions. Furthermore, CLIP (RNA Cross-Linking and Immuno: Precipitation) of TRA2-BETA1 shows that most of the tra2-beta1 targets from cytoslic RNA is ribosomal RNA. The CLIP targets were localized mainly on the large subunit of the ribosome, near the RPL3 binding sites in the 28S rRNA that is closed to RPL3 binding site. In functional assay, TRA2-BETA1 with an inserted nuclear export signal strongly activates luciferase reporter constructs that contain a TRA2-BETA1 binding motif. This stimulation is regulated by the dephosphorylation in its PP1 (Protein Phosphatase 1) binding site which is localized between RRM (RNA Recognition Motif) and the second RS domain. In summary, we demonstrated that tra2-beta1 has a novel role in translation regulation in addition to its general function in splicing.
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