pre-mRNA Splicing Factors Research Articles

Structural and functional analysis of RNA and TAP binding to SF2/ASF

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded beta-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix alpha1, together with amino acids from strand beta2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.

Crystal structure of the C-terminal domain of splicing factor Prp8 carrying retinitis pigmentosa mutants.

Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an alpha/beta structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn(2+) coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn(2+)-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Identification of photoreceptor genes affected by PRPF31 mutations associated with autosomal dominant retinitis pigmentosa.

Several ubiquitously expressed genes encoding pre-mRNA splicing factors have been associated with autosomal dominant retinitis pigmentosa (adRP), including PRPF31, PRPF3 and PRPF8. Molecular mechanisms by which defects in pre-mRNA splicing factors cause photoreceptor degeneration are not clear. To investigate the role of pre-mRNA splicing in photoreceptor gene expression and function, we have begun to search for photoreceptor genes whose pre-mRNA splicing is affected by mutations in PRPF31. Using an immunoprecipitation-coupled-microarray method, we identified a number of transcripts associated with PRPF31-containing complexes, including peripherin/RDS, FSCN2 and other photoreceptor-expressed genes. We constructed minigenes to study the effects of PRPF31 mutations on the pre-mRNA splicing of these photoreceptor specific genes. Our experiments demonstrated that mutant PRPF31 significantly inhibited pre-mRNA splicing of RDS and FSCN2. These observations suggest a functional link between ubiquitously expressed and retina-specifically expressed adRP genes. Our results indicate that PRPF31 mutations lead to defective pre-mRNA splicing of photoreceptor-specific genes and that the ubiquitously expressed adRP gene, PRPF31, is critical for pre-mRNA splicing of a subset of photoreceptor genes. Our results provide an explanation for the photoreceptor-specific phenotype of PRPF31 mutations.

Mutations in the Gene Coding for the Pre-mRNA Splicing Factor,PRPF31, in Patients with Autosomal Dominant Retinitis Pigmentosa

Retinitis pigmentosa is a clinically and genetically heterogeneous disorder. It is characterized by progressive degeneration of the peripheral retina, leading to night blindness and loss of the peripheral visual field. PRPF31 is one of four pre-mRNA splicing factors identified as causing autosomal dominant retinitis pigmentosa, with incomplete penetrance being the unique feature associated with mutations in this gene. The purpose of this study was to identify PRPF31 mutations in a cohort of 118 cases of autosomal dominant retinitis pigmentosa and determine the genotype-phenotype correlation emerging from the spectrum of mutations in this gene. Probands with autosomal dominant retinitis pigmentosa underwent ophthalmic evaluation. Blood samples were obtained, genomic DNA was isolated, and PRPF31 exons along with adjacent splice junctions were amplified by PCR and screened by direct sequencing. In the 118 individuals with autosomal dominant retinitis pigmentosa, six mutations were identified, of which four were novel. One previously known splice site mutation was identified in two other apparently unrelated families. Mutations in PRPF31 causing adRP were present in nearly 5% of a mixed U.K. population. The age of onset and the severity of the disease varied with different mutations. In addition, individuals carrying the same mutation showed a range of phenotypic variation, suggesting the involvement of other modifying genes.

Structural characterization of the fission yeast U5.U2/U6 spliceosome complex

The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA. The megadalton-sized spliceosome is composed of four small nuclear RNPs and additional pre-mRNA splicing factors. The formation of an active spliceosome involves a series of regulated steps that requires the assembly and disassembly of large multiprotein/RNA complexes. The dynamic nature of the pre-mRNA splicing reaction has hampered progress in analyzing the structure of spliceosomal complexes. We have used cryo-electron microscopy to produce a 29-A density map of a stable 37S spliceosomal complex from the genetically tractable fission yeast, Schizosaccharomyces pombe. Containing the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors, this complex closely resembles in vitro purified mammalian C complex. The density map reveals an asymmetric particle, approximately 30 x 20 x 18 nm in size, which is composed of distinct domains that contact each other at the center of the complex.

Effect of bleomycin and cisplatin on the expression profile ofSRA1, a novel member of pre-mRNA splicing factors, in HL-60 human promyelocytic leukemia cells

Recently, a new member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors, SRA1 (SR-A1), has been discovered and cloned by members of our group, the gene for which was found to be overexpessed in a series of human tumors. In the present study, we investigated the significance of alterations at the mRNA expression levels of the SRA1 gene after treatment of HL-60 human promyelocytic leukemia cells with the anticancer drugs cisplatin and bleomycin. The kinetics of apoptosis and cell toxicity were investigated by DNA laddering and the MTT and trypan blue assays, respectively. Total RNA was extracted and cDNA was prepared by reverse transcription. The splicing-related genes SRA1 and SC35, as well as the apoptosis-related gene BCL2 (Bcl-2), were amplified by PCR using gene-specific primers. The results showed that mRNA levels of SRA1 were up-regulated upon treatment with the antibiotic bleomycin, whereas they were down-regulated by treatment of HL-60 human promyelocytic leukemia cells with cisplatin. Our results support the hypothesis that mRNA expression analysis of SRA1 may serve as a new prospective molecular marker, playing an important role in chemotherapy outcome in human leukemia.

Alternative Conformations at the RNA-binding Surface of the N-terminal U2AF65 RNA Recognition Motif

The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF65) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF65 must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF65 variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF65. Here, the X-ray structure of the N-terminal RRM domain of U2AF65 (RRM1) is described at 1.47 Å resolution in the absence of RNA. Notably, RNA-binding by U2AF65 selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the β2/β3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF65 to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.

Nuclear structure in early mouse embryos: A comparative ultrastructural and immunocytochemical study with special emphasis on the “2-cell block in vitro”

The embryos from many outbred and inbred strains of mice are arrested at the late 2-cell stage when cultured in vitro in simple culture media. This phenomenon is referred to as the “2-cell block in vitro”. The ultrastructural morphology of the nuclei of the blocked embryos is not yet well described. In the present paper we documented the results of a comparative study on the nuclei of mouse embryos, both normally developing and arrested at the 2-cell stage. The blocked embryos maintain the morphological integrity of their nuclei. Main nuclear domains (nucleolus precursor bodies, interchromatin granule clusters, perichromatin granules, and perichromatin fibrils), typical for the control embryos, are observed in the blocked ones. A number and morphological characteristics of these nuclear substructures are not changed significantly in the blocked embryos. At the same time, RNA polymerase II and pre-mRNA splicing factors are redistributed in the nucleus of the blocked embryos. Although something goes to show that nuclear organization of the blocked embryos differ from that of the control, we could not reveal in the blocked embryos distinct signs of degeneration which might characterize aged or dying cells. Our data confirm a peculiar functional state of the 2-cell blocked embryos.

Splicing Regulation in Neurologic Disease

The importance of alternative splicing in the regulation of diverse biological processes is reflected in the growing list of human diseases associated with known or suspected splicing defects. It is becoming evident that alternative splicing plays a particularly important role in neurologic disease, which is perhaps not surprising given the important role splicing plays in generating complexity and function in the brain. This review considers the evidence that defects in regulation of splicing may underlie many types of human neurologic diseases.

Systematic identification of factors involved in post-transcriptional processes in wheat grain

Post-transcriptional processing of primary transcripts can significantly affect both the quantity and the structure of mature mRNAs and the corresponding protein products. It is an important mechanism of gene regulation in animals, yeast and plants. Here we have investigated the interactive networks of pre-mRNA processing factors in the developing grain of wheat (Triticum aestivum), one of the world's major food staples. As a first step we isolated a homologue of the plant specific AtRSZ33 splicing factor, which has been shown to be involved in the early stages of embryo development in Arabidopsis. Real-time PCR showed that the wheat gene, designated TaRSZ38, is expressed mainly in young, developing organs (flowers, root, stem), and expression peaks in immature grain. In situ hybridization and immunodetection revealed preferential abundance of TaRSZ38 in mitotically active tissues of the major storage organ of the grain, the endosperm. The protein encoded by TaRSZ38 was subsequently used as a starting bait in a two-hybrid screen to identify additional factors in grain that are involved in pre-mRNA processing. Most of the identified proteins showed high homology to known splicing factors and splicing related proteins, supporting a role for TaRSZ38 in spliceosome formation and 5' site selection. Several clones were selected as baits in further yeast two-hybrid screens. In total, cDNAs for 16 proteins were isolated. Among these proteins, TaRSZ22, TaSRp30, TaU1-70K, and the large and small subunits of TaU2AF, are wheat homologues of known plant splicing factors. Several, additional proteins are novel for plants and show homology to known pre-mRNA splicing, splicing related and mRNA export factors from yeast and mammals.

Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells

Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

The Relationship between Intranuclear Mobility of the NF-κB Subunit p65 and Its DNA Binding Affinity

It has been hypothesized that the main determinant of the intranuclear mobility of transcription factors is their ability to bind DNA. In the present study, we have extensively tested the relationship between the intranuclear mobility of the NF-kappaB subunit p65 and binding to its consensus target sequence. The affinity of p65 for this binding site is altered by mutation of specific acetylation sites, so these mutants provide a model system to study the relationship between specific DNA binding affinity and intranuclear mobility. DNA binding affinity was measured in vitro using an enzyme-linked immunosorbent assay-based method, and intranuclear mobility was measured using the fluorescence recovery after photobleaching technique on yellow fluorescent protein-tagged p65 constructs. A negative correlation was observed between DNA binding affinity and intranuclear mobility of p65 acetylation site mutants. However, moving the yellow fluorescent protein tag from the C terminus of p65 to the N terminus resulted in an increased mobility but did not significantly affect DNA binding affinity. Thus, all changes in DNA binding affinity produce alterations in mobility, but not vice versa. Finally, a positive correlation was observed between mobility and the randomness of the intranuclear distribution of p65. Our data are in line with a model in which the intranuclear mobility and distribution of a transcription factor are determined by its affinity for specific DNA sequences, which may be altered by protein-protein interactions.

Epitope Tagging of Proteins at the Native Chromosomal Loci of Genes in Mice and in Cultured Vertebrate Cells

Adding epitope tags to proteins is an important method for biochemical analyses and is generally accomplished in metazoan cells using ectopically expressed, tagged trans-genes. In Saccharomyces cerevisiae, the addition of epitope tags to proteins is easily achieved at the genomic locus of a gene of interest due to the high efficiency of homologous recombination in that organism. Most metazoan cells do not exhibit this high homologous recombination efficiency, and therefore trans-genes with in-frame epitope tags are used. Although epitope tagged trans-genes have proven useful, replacing the native promoter with a heterologous promoter introduces numerous artifactual possibilities. These include overexpression, which can lead to promiscuous interactions, and the loss of native transcriptional control, which in live animals often leads to developmental defects and embryonic lethality. We describe an efficient method that overcomes the problems encountered using epitope tagged trans-genes by introducing the epitope tag into the native chromosomal gene locus in vertebrate cells, embryonic stem cells and live mice. These tagged proteins are physically associated with the expected relevant particles, and highly sensitive as shown by co-purification of homologues of the yeast pre-mRNA splicing factors Prp38p and Prp39p, not previously shown to be associated with metazoan snRNPs. These techniques will enhance the validity of conclusions made regarding epitope-tagged proteins and improve our understanding of proteomic dynamics in cultured vertebrate cells and live animals.

Structural Basis for Polypyrimidine Tract Recognition by the Essential Pre-mRNA Splicing Factor U2AF65

The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.

STABILIZED1, a Stress-Upregulated Nuclear Protein, Is Required for Pre-mRNA Splicing, mRNA Turnover, and Stress Tolerance in Arabidopsis

In plants, many gene transcripts are very unstable, which is important for the tight control of their temporal and spatial expression patterns. To identify cellular factors controlling the stability of unstable mRNAs in plants, we used luciferase imaging in Arabidopsis thaliana to isolate a recessive mutant, stabilized1-1 (sta1-1), with enhanced stability of the normally unstable luciferase transcript. The sta1-1 mutation also causes the stabilization of some endogenous gene transcripts and has a range of developmental and stress response phenotypes. STA1 encodes a nuclear protein similar to the human U5 small ribonucleoprotein-associated 102-kD protein and to the yeast pre-mRNA splicing factors Prp1p and Prp6p. STA1 expression is upregulated by cold stress, and the sta1-1 mutant is defective in the splicing of the cold-induced COR15A gene. Our results show that STA1 is a pre-mRNA splicing factor required not only for splicing but also for the turnover of unstable transcripts and that it has an important role in plant responses to abiotic stresses.

Specific Role of the SR Protein Splicing Factor B52 in Cell Cycle Control in Drosophila

E2F and retinoblastoma tumor suppressor protein pRB are important regulators of cell proliferation; however, the regulation of these proteins in vivo is not well understood. In Drosophila there are two E2F genes, an activator, de2f1, and a repressor, de2f2. The loss of de2f1 gives rise to the G(1)/S block accompanied by the repression of E2F-dependent transcription. These defects can be suppressed by mutation of de2f2. In this work, we show that the de2f1 mutant phenotype is rescued by the loss of the pre-mRNA splicing factor SR protein B52. Mutations in B52 restore S phase in clones of de2f1 mutant cells and phenocopy the loss of the de2f2 function. B52 acts upstream of de2f2 and plays a specific role in regulation of de2f2 pre-mRNA splicing. In B52-deficient cells, the level of dE2F2 protein is severely reduced and the expression of dE2F2-dependent genes is deregulated. Reexpression of the intronless copy of dE2F2 in B52-deficient cells restores the dE2F2-mediated repression. These results uncover a previously unrecognized role of the splicing factor in maintaining the G(1)/S block in vivo by specific regulation of the dE2F2 repressor function.

Crystallization and preliminary X-ray analysis of a U2AF65variant in complex with a polypyrimidine-tract analogue by use of protein engineering

The large subunit of the essential pre-mRNA splicing factor U2 auxiliary factor (U2AF65) binds the polypyrimidine tract near the 3' splice site of pre-mRNA introns and directs the association of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) of the spliceosome with the pre-mRNA. Protein engineering, in which the flexible linker region connecting tandem RNA-recognition motifs (RRMs) within the U2AF65 RNA-binding domain was partially deleted, allowed successful crystallization of the protein-nucleic acid complex. Cocrystals of a U2AF65 variant with a deoxyuridine dodecamer diffract X-rays to 2.9 angstroms resolution and contain one complex per asymmetric unit.

The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that snRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that snRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in snRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new snRNPs--and perhaps regenerated snRNPs after splicing--is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.

The roles of cyclin‐dependent kinase 11 (CDK11) during mammalian development

CDK11 gene encodes two proteins, p110 and p58. CDK11p110 proteins are part of large-molecular-weight complexes containing RNA polymerase II, transcriptional elongation and general pre-mRNA splicing factors, suggesting they couple transcription and pre-mRNA splicing. CDK11p58 is expressed only during G2/M from an internal ribosome entry site (IRES) present in the mRNA encoding CDK11p110 and appears to regulate mitosis. To examine the in vivo role of CDK11p110/p58 kinases, we generated CDK11p110/p58-null mice and found CDK11p110/p58deficiency results in early embryonic lethality and apoptosis between E3.5 and E4.0. Cells within CDK11p110/p58−/− embryos exhibit both proliferative defects and a mitotic arrest. These results indicated that the CDK11p110/p58kinases are essential for cellular viability and normal early embryonic development. CDK11p110/p58 T cell-specific knockout mice generated by using Cre/loxP system show defects in T cell development. Deletion of CDK11p110/p58 in thymocytes results in a block at the double negative (DN) stage due to the defective pre-TCR signaling, impaired proliferation and apoptosis. CDK11p110/p58 deficiency in thymocytes also affects alternative splicing of CD45. Taken together, these results indicate that CDK11 protein kinases play important roles in transcription, splicing and mitotic regulation. (Supported by NIH grants GM44088, CA0211765, and ALSAC)