Biomedical subjects

Xiang-Dong Fu

Publications and source records attributed to Xiang-Dong Fu.

8 recordsLinked to original sources

PubMed2005

ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle.

The transition from juvenile to adult life is accompanied by programmed remodeling in many tissues and organs, which is key for organisms to adapt to the demand of the environment. Here we report a novel regulated alternative splicing program that is crucial for postnatnal heart remodeling in the mouse. We identify the essential splicing factor ASF/SF2 as a key component of the program, regulating a restricted set of tissue-specific alternative splicing events during heart remodeling. Cardiomyocytes deficient in ASF/SF2 display an unexpected hypercontraction phenotype due to a defect in postnatal splicing switch of the Ca(2+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) transcript. This failure results in mistargeting of the kinase to sarcolemmal membranes, causing severe excitation-contraction coupling defects. Our results validate ASF/SF2 as a fundamental splicing regulator in the reprogramming pathway and reveal the central contribution of ASF/SF2-regulated CaMKIIdelta alternative splicing to functional remodeling in developing heart.

Alternative Splicing↗

PubMed2004

Towards a splicing code.

A combination of experimental and bioinformatics approaches leads Burge and colleagues (Wang et al., 2004 [this issue of Cell]) to a global view of how an RNA segment may be selected or avoided in mature mRNAs due to biased distributions of exonic enhancers and silencers, a process vital for genome evolution, developmental control, and disease onset.

Algorithms↗

PubMed2004

Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart.

Many genetic diseases are caused by mutations in cis-acting splicing signals, but few are triggered by defective trans-acting splicing factors. Here we report that tissue-specific ablation of the splicing factor SC35 in the heart causes dilated cardiomyopathy (DCM). Although SC35 was deleted early in cardiogenesis by using the MLC-2v-Cre transgenic mouse, heart development appeared largely unaffected, with the DCM phenotype developing 3-5 weeks after birth and the mutant animals having a normal life span. This nonlethal phenotype allowed the identification of downregulated genes by microarray, one of which was the cardiac-specific ryanodine receptor 2. We showed that downregulation of this critical Ca2+ release channel preceded disease symptoms and that the mutant cardiomyocytes exhibited frequency-dependent excitation-contraction coupling defects. The implication of SC35 in heart disease agrees with a recently documented link of SC35 expression to heart failure and interference of splicing regulation during infection by myocarditis-causing viruses. These studies raise a new paradigm for the etiology of certain human heart diseases of genetic or environmental origin that may be triggered by dysfunction in RNA processing.

Animals↗

PubMed2004

A versatile assay for high-throughput gene expression profiling on universal array matrices.

We report a flexible, sensitive, and quantitative gene-expression profiling system for assaying more than 400 genes, with three probes per gene, for 96 samples in parallel. The cDNA-mediated annealing, selection, extension and ligation (DASL) assay targets specific transcripts, using oligonucleotides containing unique address sequences that can hybridize to universal arrays. Cell-specific gene expression profiles were obtained using this assay for hormone-treated cell lines and laser-capture microdissected cancer tissues. Gene expression profiles derived from this assay were consistent with those determined by qRT-PCR. The DASL assay has been automated for use with a bead-based 96-array matrix system. The combined high-throughput assay and readout system is accurate and efficient, and can cost-effectively profile the expression of hundreds of genes in thousands of samples.

Androgens↗

PubMed2003

Processive phosphorylation of alternative splicing factor/splicing factor 2.

SR proteins, named for their multiple arginine/serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF/SF2. By using the start-trap strategy, we monitored the progress curve for ASF/SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF/SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme-substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF/SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains "locked" onto SRPK1 during RS domain modification.

Adenosine Triphosphate↗

PubMed2003

Phosphorylation-dependent and -independent nuclear import of RS domain-containing splicing factors and regulators.

SR proteins and related RS domain-containing polypeptides are an important class of splicing regulators in higher eukaryotic cells. The RS domain facilitates nuclear import of SR proteins and mediates protein-protein interactions during spliceosome assembly; both functions appear to subject to regulation by phosphorylation. Previous studies have identified two nuclear import receptors for SR proteins, transportin-SR1 and transportin-SR2. Here we show that transportin-SR1 and transportin-SR2 are the alternatively spliced products of the same gene and that transportin-SR2 is the predominant transcript in most cells and tissues examined. While both receptors import typical SR proteins in a phosphorylation-dependent manner, they differentially import the RS domain-containing splicing regulators hTra2alpha and hTra2beta in different phosphorylation states. We suggest that differential regulation of nuclear import may serve as a mechanism for homeostasis of RS domain-containing splicing factors and regulators in the nucleus and for selective cellular responses to signaling.

Alternative Splicing↗

PubMed2002

Profiling alternative splicing on fiber-optic arrays.

The human transcriptome is marked by extensive alternative mRNA splicing and the expression of many closely related genes, which may be difficult to distinguish using standard microarray techniques. Here we describe a sensitive and specific assay for parallel analysis of mRNA isoforms on a fiber-optic microarray platform. The method permits analysis of mRNA transcripts without prior RNA purification or cDNA synthesis. Using an endogenously expressed viral transcript as a model, we demonstrated that the assay readily detects mRNA isoforms from as little as 10-100 pg of total cellular RNA or directly from a few cells. Multiplexed analysis of human cancer cell lines revealed differences in mRNA splicing and suggested a potential autocrine mechanism in the development of choriocarcinomas. Our approach may be useful in the large-scale analysis of the role of alternative splicing in development and disease.

Alternative Splicing↗

PubMed2002

Rev inhibition strongly affects intracellular distribution of human immunodeficiency virus type 1 RNAs.

To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.

Active Transport, Cell Nucleus↗