Polycistronic pre-mRNA processing in vitro: snRNP and pre-mRNA role reversal in trans-splicing.

Spliced leader (SL) trans-splicing in Caenorhabditis elegans attaches a 22-nucleotide (nt) exon onto the 5' end of many mRNAs. A particular class of SL, SL2, splices mRNAs of downstream operon genes. Here we use an embryonic extract-based in vitro splicing system to show that SL2 specificity information is encoded within the polycistronic pre-mRNA, and that trans-splicing specificity is recapitulated in vitro. We define an RNA sequence required for SL2 trans-splicing, the U-rich (Ur) element, through mutational analysis and bioinformatics as a short stem-loop followed by a sequence motif, UAYYUU, located approximately 50 nt upstream of the trans-splice site. Furthermore, this element is predicted in intercistronic regions of numerous operons of C. elegans and other species that use SL2 trans-splicing. We propose that the UAYYUU motif hybridizes with the 5' splice site on the SL2 RNA to recruit the SL to the pre-mRNA. In this way, the UAYYUU motif in the pre-mRNA would serve an analogous function to the similar sequence in the U1 snRNA, which binds to the 5' splice site of introns, effectively reversing the roles of snRNP and pre-mRNA in trans-splicing.

Vaccine adjuvant-elicited CD8+ T cell immunity is co-dependent on T-bet and FOXO1.

T-bet and FOXO1 are transcription factors canonically associated with effector and memory T cell fates, respectively. During an infectious response, these factors direct the development of CD8+ T cell fates, where T-bet deficiency leads to ablation of only short-lived effector cells, while FOXO1 deficiency results in selective loss of memory. In contrast, following adjuvanted subunit vaccination in mice, both effector- and memory-fated T cells are compromised in the absence of either T-bet or FOXO1. Thus, unlike responses to challenge with Listeria monocytogenes, productive CD8+ T cell responses to adjuvanted vaccination require coordinated regulation of FOXO1 and T-bet transcriptional programs. Single-cell RNA sequencing analysis confirms simultaneous T-bet, FOXO1, and TCF1 transcriptional activity in vaccine-elicited, but not infection-elicited, T cells undergoing clonal expansion. Collectively, our data show that subunit vaccine adjuvants elicit T cell responses dependent on transcription factors associated with effector and memory cell fates.

A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements.

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.

Alternative splicing in disease and therapy.

Alternative splicing is the major source of proteome diversity in humans and thus is highly relevant to disease and therapy. For example, recent work suggests that the long-sought-after target of the analgesic acetaminophen is a neural-specific, alternatively spliced isoform of cyclooxygenase 1 (COX-1). Several important diseases, such as cystic fibrosis, have been linked with mutations or variations in either cis-acting elements or trans-acting factors that lead to aberrant splicing and abnormal protein production. Correction of erroneous splicing is thus an important goal of molecular therapies. Recent experiments have used modified oligonucleotides to inhibit cryptic exons or to activate exons weakened by mutations, suggesting that these reagents could eventually lead to effective therapies.

Trans-splicing.

Trans-splicing is the joining together of portions of two separate pre-mRNA molecules. The two distinct categories of spliceosomal trans-splicing are genic trans-splicing, which joins exons of different pre-mRNA transcripts, and spliced leader (SL) trans-splicing, which involves an exon donated from a specialized SL RNA. Both depend primarily on the same signals and components as cis-splicing. Genic trans-splicing events producing protein-coding mRNAs have been described in a variety of organisms, including Caenorhabditis elegans and Drosophila. In mammalian cells, genic trans-splicing can be associated with cancers and translocations. SL trans-splicing has mainly been studied in nematodes and trypanosomes, but there are now numerous and diverse phyla (including primitive chordates) where this type of trans-splicing has been detected. Such diversity raises questions as to the evolutionary origin of the process. Another intriguing question concerns the function of trans-splicing, as operon resolution can only account for a small proportion of the total amount of SL trans-splicing.

Identification of an intronic splicing enhancer essential for the inclusion of FGFR2 exon IIIc.

The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human beta-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5'-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.

Embryonic and larval expression of zebrafish voltage-gated sodium channel alpha-subunit genes.

Whereas it is known that voltage-gated calcium channels play important roles during development, potential embryonic roles of voltage-gated sodium channels have received much less attention. Voltage-gated sodium channels consist of pore-forming alpha-subunits (Na(v)1) and auxiliary beta-subunits. Here, we report the embryonic and larval expression patterns for all eight members of the gene family (scna) coding for zebrafish Na(v)1 proteins. We find that each scna gene displays a distinct expression pattern that is temporally and spatially dynamic during embryonic and larval stages. Overall, our findings indicate that scna gene expression occurs sufficiently early during embryogenesis to play developmental roles for both muscle and nervous tissues.