cis-acting sequences involved in exon selection in the chicken beta-tropomyosin gene.

The chicken beta-tropomyosin gene contains an internal pair of mutually exclusive exons (6A and 6B) that are selected in a tissue-specific manner. Exon 6A is incorporated in fibroblasts and smooth muscle cells, whereas exon 6B is skeletal muscle specific. In this study we show that two different regions in the intron between the two mutually exclusive exons are important for this specific selection in nonmuscle cells. Sequences in the 3' end of the intron have a negative effect in the recognition of the 3' splice site, while sequences in the 5' end of the intron have a positive effect in the recognition of the 5' splice site. First, sequences in exon 6B as well as in the intron upstream of exon 6B are both able to inhibit splicing when placed in a heterologous gene. The sequences in the polypyrimidine stretch region contribute to splicing inhibition of exons 5 or 6A to 6B through a mechanism independent of their implication in the previously described secondary structure around exon 6B. Second, we have identified a sequence of 30 nucleotides in the intron just downstream of exon 6A that is essential for the recognition of the 5' splice site of exon 6A. This is so even after introduction of a consensus sequence into the 5' splice site of this exon. Deletion of this sequence blocks splicing of exon 6A to 6B after formation of the presplicing complex. Taken together, these results suggest that both the mutually exclusive behavior and the choice between exons 6A and 6B of the chicken beta-tropomyosin gene are trans regulated.     

Molecular evolution of type VI intermediate filament proteins

Background  

Tanabin, transitin and nestin are type VI intermediate filament (IF) proteins that are developmentally regulated in frogs, birds and mammals, respectively. Tanabin is expressed in the growth cones of embryonic vertebrate neurons, whereas transitin and nestin are found in myogenic and neurogenic cells. Another type VI IF protein, synemin, is expressed in undifferentiated and mature muscle cells of birds and mammals. In addition to an IF-typical α-helical core domain, type VI IF proteins are characterized by a long C-terminal tail often containing distinct repeated motifs. The molecular evolution of type VI IF proteins remains poorly studied.     

Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts

Human immunodeficiency virus type 1 (HIV-1) exonic splicing silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of splicing by ESSV. Possible splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.     

An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p

Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA. By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core. Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch. In vitro, the switch requires both Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing.     

New insight into the Nox4 subcellular localization in HEK293 cells: first monoclonal antibodies against Nox4

Nox4, a member of Nox family of NADPH oxidase expressed in nonphagocytic cells, is a major source of reactive oxygen species in many cell types. But understanding of the role of Nox4 in the production of ROS and of regulation mechanism of oxidase activity is largely unknown. This study reports for the first time the generation and characterization of 5 mAbs against a recombinant Nox4 protein (AA: 206-578). Among 5 novel mAbs, 3 mAbs (8E9, 5F9, 6B11) specifically recognized Nox4 protein in HEK293 transfected cells or human kidney cortex by western blot analysis; mAb 8E9 reacted with intact tet-induced T-REx™ Nox4 cells in FACS studies. The other 2 mAbs 10B4 and 7C9 were shown to have a very weak reactivity after purification. Immunofluorescence confocal microscopy showed that Nox4 localized not only in the perinuclear and endoplasmic reticulum regions but also at the plasma membrane of the cells which was further confirmed by TIRF-microscopy. Epitope determination showed that mAb 8E9 recognizes a region on the last extracellular loop of Nox4, while mAbs 6B11 and 5F9 are directed to its cytosolic tail. Contrary to mAb 6B11, mAb 5F9 failed to detect Nox4 at the plasma membrane. Cell-free oxidase assays demonstrated a moderate but significant inhibition of constitutive Nox4 activity by mAbs 5F9 and 6B11. In conclusion, 5 mAbs raised against Nox4 were generated for the first time. 3 of them will provide powerful tools for a structure/function relationship of Nox4 and for physiopathological investigations in humans.     

RS domain-splicing signal interactions in splicing of U12-type and U2-type introns

Serine-arginine (SR) proteins are general metazoan splicing factors that contain an essential arginine/serine-rich (RS) domain. On typical U2-type introns, RS domains contact the branchpoint and 5' splice site to promote base-pairing with U small nuclear RNAs (snRNAs). Here we analyze the role of SR proteins in splicing of U12-type introns and in the second step of U2-type intron splicing. We show that RS domains contact the branchpoint and 5' splice site of a U12-type intron. On a U2-type intron, we find that the RS domain contacts the site of the U6 snRNA-5' splice site interaction during the first step of splicing and shifts to contact the site of the U5 snRNA-exon 1 interaction during the second step. Our results reveal alternative interactions between the RS domain and 5' splice site region that coincide with remodeling of the spliceosome between the two catalytic steps.     

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.     

A mutational analysis of U12-dependent splice site dinucleotides

Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5' A residue can splice to any 3' residue, although C is preferred. A 5' G residue can splice to 3' G or U residues with a preference for G. Little or no splicing was observed to 3' A or C residues. A 5' U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5' U to 3' U produced detectable spliced products. The dependence of 3' splice site activity on the identity of the 5' residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5' splice site and the next to last position of the 3' splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3' splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3' splice site distance of 11-12 nucleotides appears to be the same for both classes.     

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.     

Hendecad repeat in segment 2A and linker L2 of intermediate filament chains implies the possibility of a right-handed coiled-coil structure

The conformation adopted by intermediate filament chains (IF) has been described in terms of a central rod domain with four, alpha-helical, left-handed coiled-coil segments (1A, 1B, 2A, and 2B) joined by linkers (L1, L12, and L2, respectively). The rod domain is terminated at its N- and C-terminal ends by "globular" head and tail domains, respectively. This analysis, initially undertaken about 20-25 years ago, was based on the recognition of an underlying heptad substructure in the sequence of the rod domain, the presence of which can be directly associated with an alpha-helical coiled-coil structure. In this work, a hendecad sequence motif that is closely related to the heptad repeat but which is nonetheless significantly different from it has been recognized in the primary structure of segments 2A and linker L2. This motif, which is 11 residues long and structurally equivalent to a true heptad plus another heptad with an inclusive stutter, is consistent with the chains adopting a continuous right-handed coiled-coil structure with a long-period pitch length. It is therefore predicted that segment 2 as a whole may have a coiled-coil conformation with both right-handed (2A+L2) and left-handed (2B) regions. The changeover in handedness would be expected to occur at the C-terminal end of linker L2 and N-terminal end of segment 2B.     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection.

The neural cell adhesion molecule (NCAM) gene contains an 801 nt exon that is included preferentially in neuronal cells. We have set up an in vitro splicing system that mimics the neuro-specific alternative splicing profile of NCAM exon 18. Splicing regulation is observed using model pre-mRNAs that contain competing 5' or 3' splice sites, suggesting that distinct pathways regulate NCAM 5' and 3' splice site selection. While inclusion of exon 18 is the predom-inant choice in neuronal cells, an element in the 5' common exon 17 improves exon 17/exon 19 splicing in a neuronal cell line. A similar behavior is observed in vitro as the element can stimulate the 5' splice site of exon 17 or a heterologous 5' splice site. The minimal 32 nt sequence of the exon 17 enhancer consists of purine stretches and A/C motifs. Mutations in the purine stretches compromise the binding of SR proteins and decreases splicing stimulation in vitro. Mutations in the A/C motifs do not affect SR protein binding but reduce enhancing activity. Our results suggest that the assembly of an enhancer complex containing SR proteins in a 5' common exon ensures that NCAM mRNAs lacking exon 18 are made in neuronal cells.     

Alternative splicing of Xenopus alphafast-tropomyosin pre-mRNA during development: identification of determining sequences

The Xenopus alphafast-tropomyosin gene contains in its central part a set of mutually exclusive exons, designated 6A and 6B, which are incorporated into mRNA encoding, respectively, nonmuscle and muscle tropomyosins. In this study, we show that usage of both exons is strictly regulated during development, exon 6A being used in the oocyte and nonmuscle tissues of the embryo, while exon 6B is used in muscle tissues. An approach of transient embryo transgenesis was developed to study the mechanisms involved in the splice site choice during development. We demonstrate that a-tropomyosin minigenes driven by tissue-specific promoters that target gene expression in nonmuscle and muscle tissues recapitulate the splicing pattern of the endogenous gene. A mutational analysis showed that regulation occurred at both exons 6A and 6B in muscle and nonmuscle tissues. In this context, we have identified an element located in the intron downstream of 6A that participates in the recognition of the weak 5' splice site of exon 6A and the repression of exon 6B in nonmuscle cells.     

Identification of a human protein that recognizes the 3'' splice site during the second step of pre-mRNA splicing.

Accurate splicing of precursor mRNAs (pre-mRNAs) requires recognition of the 5' and 3' splice sites at the intron boundaries. Interactions between several splicing factors and the 5' splice site, which occur prior to the first step of splicing, have been well described. In contrast, recognition of the 3' splice site, which is cleaved during the second catalytic step, is poorly understood, particularly in higher eukaryotes. Here, using site-specific photo-crosslinking, we find that the conserved AG dinucleotide at the 3' splice site is contacted specifically by a 70 kDa polypeptide (p70). The p70-3' splice site crosslink has kinetics and biochemical requirements similar to those of splicing, was detected only in the mature spliceosome and occurs subsequent to the first step. Thus, p70 has all the properties expected of a factor that functionally interacts with the 3' splice site during the second step of splicing. Using antisera to various known splicing factors, we find that p70 corresponds to a previously reported 69 kDa protein of unknown function associated with the Sm core domain of spliceosomal small nuclear ribonucleoproteins.     

The C-terminal region but not the Arg-X-Pro repeat of Epstein-Barr virus protein EB2 is required for its effect on RNA splicing and transport

The Epstein-Barr virus BMLF1 gene product EB2 has been shown to efficiently transform immortalized Rat1 and NIH 3T3 cells, to bind RNA, and to shuttle from the nucleus to the cytoplasm. In transient-expression assays EB2 seems to affect mRNA nuclear export of intronless RNAs and pre-mRNA 3' processing, but no direct proof of EB2 being involved in RNA processing and transport has been provided, and no specific functional domain of EB2 has been mapped. Here we significantly extend these findings and directly demonstrate that (i) EB2 inhibits the cytoplasmic accumulation of mRNAs, but only if they are generated from precursors containing weak (cryptic) 5' splice sites, (ii) EB2 has no effect on the cytoplasmic accumulation of mRNA generated from precursors containing constitutive splice sites, and (iii) EB2 has no effect on the 3' processing of precursor RNAs containing canonical and noncanonical cleavage-polyadenylation signals. We also show that in the presence of EB2, intron-containing and intronless RNAs accumulate in the cytoplasm. EB2 contains an Arg-X-Pro tripeptide repeated eight times, similar to that described as an RNA-binding domain in the herpes simplex virus type 1 protein US11. As glutathione S-transferase fusion proteins, both EB2 and the Arg-X-Pro repeat bound RNA in vitro. However, by using EB2 deletion mutants, we demonstrated that the effect of EB2 on splicing and RNA transport requires the C-terminal half of the protein but not the Arg-X-Pro repeat.     

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.