Getting to the heart of regeneration in zebrafish

A scientific and clinical prerogative of the 21st century is to stimulate the regenerative ability of the human heart. While the mammalian heart shows little or no natural regeneration in response to injury, certain non-mammalian vertebrates possess an elevated capacity for cardiac regeneration. Adult zebrafish restore ventricular muscle removed by surgical resection, events that involve little or no scarring. Recent studies have begun to reveal cellular and molecular mechanisms of this regenerative process that have exciting implications for human cardiac biology and disease.     

Getting to the heart of COX-2 inhibition

Aspirin protects against heart disease, while COX-2 Inhibitors appear to injure the heart. The studies by [this Issue of Cell Metabolism]) shed light on two distinct prostaglandin receptors contributing to these conflicting cardiovascular effects.     

Linking C5 deficiency to an exonic splicing enhancer mutation

As an important component of the innate immune system, complement provides the initial response to prevent infections by pathogenic microorganisms. Patients with dysfunction of C5 display a propensity for severe recurrent infections. In this study, we present a patient with C5 deficiency demonstrated by immunochemical and functional analyses. Direct sequencing of all C5 exons displayed no mutation of obvious functional significance, except for an A to G transition in exon 10 predicting an exchange from lysine to arginine. This sequence alteration was present in only one allele of family members with a reduced serum C5 concentration and in both alleles of the patient with almost complete C5 deficiency, suggesting that this alteration may be producing the phenotype. Recent findings indicate that distinct nucleotide sequences, termed exonic splicing enhancers (ESEs), influence the splicing process. cDNA from all family members harboring the mutated allele showed skipping of exon 10, which resulted in a premature STOP codon, explaining the lack of C5 in the propositus. Sequence analysis of the mutated region revealed the substitution to be located within an ESE, as predicted by the RESCUE-ESE program. The altered ESE sequence is located close to the 5' splicing site and also lowers the predicted strength of the splice site itself. This apparently inconsequential sequence alteration represents a noncanonical splicing mutation altering an ESE. Our finding sheds a new light on the role of putative silent/conservative mutations in disease-associated genes.     

Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1.

The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.     

Getting to the heart of planar cell polarity signaling

The genes that underpin normal heart development, and which can be disrupted to result in congenital structural malformations, are rapidly being uncovered. However, the specific cellular processes that lie downstream of these genetic cascades, accurately shaping tissues and complex structures within the heart, remain relatively unclear. The noncanonical Wnt planar cell polarity (PCP) signaling pathway is known to have a role in embryonic morphogenesis and as such is an important candidate pathway to carry out these roles in heart development. The pathway regulates the polarization of cells in a variety of contexts, allowing cells to change shape and position and to "know" their orientation within a mass of tissue. PCP signaling has also been shown recently to regulate the cellular position of the primary cilium. This organelle is known to be crucial for the establishment of left-right patterning in the early embryo and may also act as a signaling antenna for other developmental and regulatory pathways. It is not surprising that recent studies have also linked PCP to left-right patterning. In this review, we will examine the current evidence suggesting that PCP signaling has a central role in cardiac development and malformation.     

Polypurine sequences within a downstream exon function as a splicing enhancer.

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.     

Binding of PurH to a muscle-specific splicing enhancer functionally correlates with exon inclusion in vivo

Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote muscle-specific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.     

Specific binding of an exonic splicing enhancer by the pre-mRNA splicing factor SRp55.

Purine-rich exonic splicing enhancers (ESEs) have been identified in many alternatively spliced exons. Alternative splicing of several ESE-containing exons has been shown to depend on subsets of the SR protein family of pre-mRNA splicing factors. In this report, we show that purified SR protein family member SRp55 by itself binds a 30-nt ESE-containing exon, the alternatively spliced exon 5 of avian cardiac troponin T. We show that purified SRp55 binds specifically to this RNA sequence with an apparent Kd of 60 nM as assayed by gel mobility retardation experiments. Mutations in the exon 5 sequence that increase or decrease exon 5 inclusion in vivo and in vitro have correspondingly different affinities for SRp55 in our assays. The exon 5 sequence contains two purine-rich motifs, common to many ESEs, and both are required for SRp55 binding. Hill plot analysis of binding titration reactions indicates that there is a cooperative binding of at least two SRp55 proteins to the exon sequence. Chemical modification interference studies using kethoxal show that SRp55 binding to exon 5 requires the N1 and/or the N2 of almost every G residue in the exon. Dimethylsulfate modification interference studies indicate that none of the N1 positions of A residues in the exon are important for binding. We postulate that SRp55 may recognize both primary sequence and RNA secondary structural elements within pre-mRNA.     

A downstream splicing enhancer is essential for in vitro pre-mRNA splicing.

Splicing enhancers have previously been shown to promote processing of introns containing weak splicing signals. Here, we extend these studies by showing that also 'strong' constitutively active introns are absolutely dependent on a downstream splicing enhancer for activity in vitro. SR protein binding to exonic enhancer elements or U1 snRNP binding to a downstream 5' splice site serve redundant functions as activators of splicing. We further show that a 5' splice site is most effective as an enhancer of splicing. Thus, a 5' splice site is functional in S100 extracts, under conditions where a SR enhancer is nonfunctional. Also, splice site pairing occurs efficiently in the absence of exonic SR enhancers, emphasizing the significance of a downstream 5' splice site as the enhancer element in vertebrate splicing.     

Exonic splicing enhancer motif recognized by human SC35 under splicing conditions

Exonic splicing enhancers (ESEs) are important cis elements required for exon inclusion. Using an in vitro functional selection and amplification procedure, we have identified a novel ESE motif recognized by the human SR protein SC35 under splicing conditions. The selected sequences are functional and specific: they promote splicing in nuclear extract or in S100 extract complemented by SC35 but not by SF2/ASF. They can also function in a different exonic context from the one used for the selection procedure. The selected sequences share one or two close matches to a short and highly degenerate octamer consensus, GRYYcSYR. A score matrix was generated from the selected sequences according to the nucleotide frequency at each position of their best match to the consensus motif. The SC35 score matrix, along with our previously reported SF2/ASF score matrix, was used to search the sequences of two well-characterized splicing substrates derived from the mouse immunoglobulin M (IgM) and human immunodeficiency virus tat genes. Multiple SC35 high-score motifs, but only two widely separated SF2/ASF motifs, were found in the IgM C4 exon, which can be spliced in S100 extract complemented by SC35. In contrast, multiple high-score motifs for both SF2/ASF and SC35 were found in a variant of the Tat T3 exon (lacking an SC35-specific silencer) whose splicing can be complemented by either SF2/ASF or SC35. The motif score matrix can help locate SC35-specific enhancers in natural exon sequences.     

Binding of a candidate splice regulator to a calcitonin-specific splice enhancer regulates calcitonin/CGRP pre-mRNA splicing

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. A candidate calcitonin/CGRP splice regulator (CSR) isolated from rat brain was shown to inhibit calcitonin-specific splicing in vitro. CSR specifically binds to two regions in the calcitonin-specific exon 4 RNA previously demonstrated to function as a bipartate exonic splice enhancer (ESE). The two regions, A and B element, are necessary for inclusion of exon 4 into calcitonin mRNA. A novel RNA footprinting method based on the UV cross-linking assay was used to define the site of interaction between CSR and B element RNA. Base changes at the CSR binding site prevented CSR binding to B element RNA and CSR was unable to inhibit in vitro splicing of pre-mRNAs containing the mutated CSR binding site. When expressed in cells that normally produce predominantly CGRP mRNA, a calcitonin/CGRP gene containing the mutated CSR binding site expressed predominantly calcitonin mRNA. These observations demonstrate that CSR binding to the calcitonin-specific ESE regulates calcitonin/CGRP pre-mRNA splicing.     

An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.     

Getting a G--RRP on regulated exocytosis in the heart

A study by Rybkin et al. (see p. 527) substantially advances our understanding of regulated exocytois by specialized secretory cells, such as atrial myocytes. A second member of the Ras-related protein family, RRP17, was identified and shown to participate in regulating the secretion of the cardiac-derived peptide hormone, atrial natriuretic peptide. In addition to the heart, RRP17 was shown to be expressed in neuronal, pancreatic, and skeletal muscle cells, suggesting a widespread role in regulated secretion for this new protein.     

Regulation of splicing enhancer activities by RNA secondary structures

In this report, we studied the effect of RNA structures on the activity of exonic splicing enhancers on the SMN1 minigene model by engineering known ESEs into different positions of stable hairpins. We found that as short as 7-bp stem is sufficient to abolish the enhancer activity. When placing ESEs in the loop region, AG-rich ESEs are fully active, but a UCG-rich ESE is not because of additional structural constraints. ESEs placed adjacent to the 3′ end of the hairpin structure display high enhancer activity, regardless of their sequence identities. These rules explain the suppression of multiple ESEs by point mutations that result in a stable RNA structure, and provide an additional mechanism for the C6T mutation in SMN2.