A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2′-phosphotransferase to transfer the splice junction 2′-phosphate from ligated tRNA to NAD, producing ADP ribose 1′′–2′′ cyclic phosphate (Appr>p). We show here that functional 2′-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2′-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2′-phosphate. Analysis of the database shows that likely members of the 2′-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2′-phosphotransferase into Eubacteria, suggesting that the 2′-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2′-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2′-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1α and aggst1β) reported within are interrupted by introns. The gene aggst1α contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1α are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1β gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Splicing is required for rapid and efficient mRNA export in metazoans

Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (Δi-mRNAs) into Xenopus oocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical Δi-mRNAs. Moreover, competition studies using excess Δi-mRNA indicate that different factor(s) are involved in the inefficient export of Δi-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and Δi-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP) in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the Δi-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans.

Alternative splicing, muscle calcium sensitivity, and the modulation of dragonfly flight performance

Calcium sensitivity of myosin cross-bridge activation in striated muscles commonly varies during ontogeny and in response to alterations in muscle usage, but the consequences for whole-organism physiology are not well known. Here we show that the relative abundances of alternatively spliced transcripts of the calcium regulatory protein troponin T (TnT) vary widely in flight muscle of Libellula pulchella dragonflies, and that the mixture of TnT splice variants explains significant portions of the variation in muscle calcium sensitivity, wing-beat frequency, and an index of aerodynamic power output during free flight. Two size-distinguishable morphs differ in their maturational pattern of TnT splicing, yet they show the same relationship between TnT transcript mixture and calcium sensitivity and between calcium sensitivity and aerodynamic power output. This consistency of effect in different developmental and physiological contexts strengthens the hypothesis that TnT isoform variation modulates muscle calcium sensitivity and whole-organism locomotor performance. Modulating muscle power output appears to provide the ecologically important ability to operate at different points along a tradeoff between performance and energetic cost.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R −/− mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R −/− mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.

Hair cell-specific splicing of mRNA for the α1D subunit of voltage-gated Ca2+ channels in the chicken’s cochlea

The L-type voltage-gated Ca2+ channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca2+-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type α1D subunit that predominates in hair cells of the chicken’s cochlea. The α1D subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the α1D mRNA contributes to the unusual behavior of the hair cell’s voltage-gated Ca2+ channels.

Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Complex Patterns of Alternative Splicing Mediate the Spatial and Temporal Distribution of Perlecan/UNC-52 in Caenorhabditis elegans

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan.  Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

Endoplasmic Reticulum Stress-induced mRNA Splicing Permits Synthesis of Transcription Factor Hac1p/Ern4p That Activates the Unfolded Protein Response

An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions (“ER stress”). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin–proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.

The Interaction and Colocalization of Sam68 with the Splicing-associated Factor YT521-B in Nuclear Dots Is Regulated by the Src Family Kinase p59fyn

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5–20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein–protein interaction domain. The Src family kinase p59fyn-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59fyn dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

Aberrant B cell receptor signaling from B29 (Igβ, CD79b) gene mutations of chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igβ, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing μ, κ, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with μ or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

Role of adenine functional groups in the recognition of the 3′-splice-site AG during the second step of pre-mRNA splicing

The AG dinucleotide at the 3′ splice sites of metazoan nuclear pre-mRNAs plays a critical role in catalytic step II of the splicing reaction. Previous studies have shown that replacement of the guanine by adenine in the AG (AG → GG) inhibits this step. We find that the second step was even more severely inhibited by cytosine (AG → CG) or uracil (AG → UG) substitutions at this position. By contrast, a relatively moderate inhibition was observed with a hypoxanthine substitution (AG → HG). When adenine was replaced by a purine base (AG → PG) or by 7-deazaadenine (AG → c7AG), little effect on the second step was observed, suggesting that the 6-NH2 and N7 groups do not play a critical role in adenine recognition. Finally, replacement of adenine by 2-aminopurine (AG → 2-APG) had no effect on the second step. Taken together, our results suggest that the N1 group of adenine functions as an essential determinant in adenine recognition during the second step of pre-mRNA splicing.

The SRm160/300 splicing coactivator is required for exon-enhancer function

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing “coactivator,” SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound “activators” and the snRNP machinery of the spliceosome.