Complex RNA Secondary Structures Mediate Mutually Exclusive Splicing of Coleoptera Dscam1

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA–RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

ABSTRACTThe mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity–aγ-Pcdh hallmark–is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only γ-Pcdh that cannot independently engage in homophilic trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.

Crystal structure of a Y-box binding protein 1 (YB-1)–RNA complex reveals key features and residues interacting with RNA

The Y-box binding protein 1 (YB-1) is a member of the cold shock domain (CSD) protein family and is recognized as an oncogenic factor in several solid tumors. By binding to RNA, YB-1 participates in several steps of posttranscriptional regulation of gene expression, including mRNA splicing, stability, and translation; microRNA processing; and stress granule assembly. However, the mechanisms in YB-1–mediated regulation of RNAs are unclear. Previously, we used both systematic evolution of ligands by exponential enrichment (SELEX) and individual-nucleotide resolution UV cross-linking and immunoprecipitation coupled RNA-Seq (iCLIP-Seq) analyses, which defined the RNA-binding consensus sequence of YB-1 as CA(U/C)C. We also reported that through binding to its core motif CAUC in primary transcripts, YB-1 regulates the alternative splicing of a CD44 variable exon and the biogenesis of miR-29b-2 during both Drosha and Dicer steps. To elucidate the molecular basis of the YB-1–RNA interactions, we report high-resolution crystal structures of the YB-1 CSD in complex with different RNA oligos at 1.7 Å resolution. The structure revealed that CSD interacts with RNA mainly through π–π stacking interactions assembled by four highly conserved aromatic residues. Interestingly, YB-1 CSD forms a homodimer in solution, and we observed that two residues, Tyr-99 and Asp-105, at the dimer interface are important for YB-1 CSD dimerization. Substituting these two residues with Ala reduced CSD's RNA-binding activity and abrogated the splicing activation of YB-1 targets. The YB-1 CSD–RNA structures presented here at atomic resolution provide mechanistic insights into gene expression regulated by CSD-containing proteins.

Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing-mediated splicing bias in neuronal wiring

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can potentially generate 38,016 different isoforms through stochastic, yet highly biased, alternative splicing. Genetic studies demonstrated that stochastic expression of multiple Dscam1 isoforms provides each neuron with a unique identity for self/non-self-discrimination. However, due to technical obstacles, the functional significance of the highly specific bias in isoform expression remains entirely unknown. Here, we provide conclusive evidence that Dscam1 splicing bias is required for precise mushroom body (MB) axonal wiring in flies in a variable exon-specific manner. We showed that targeted deletion of the intronic docking site perturbed base pairing-mediated regulation of inclusion of variable exons. Unexpectedly, we generated mutant flies with normal overall Dscam1 protein levels and an identical number but global changes in exon 4 and exon 9 isoform bias (DscamΔ4D−/− and DscamΔ9D−/−), respectively. DscamΔ9D−/− mutant exhibited remarkable mushroom body defects, which were correlated with the extent of the disrupted isoform bias. By contrast, the DscamΔ4D−/− animals exhibited a much less severe defective phenotype than DscamΔ9D−/− animals, suggestive of a variable domain-specific requirement for isoform bias. Importantly, mosaic analysis revealed that changes in isoform bias caused axonal defects but did not influence the self-avoidance of axonal branches. We concluded that, in contrast to the Dscam1 isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a non-repulsive role in mushroom body axonal wiring.

Mechanisms of Drosophila Dscam mutually exclusive splicing regulation

Alternative splicing of pre-mRNA is a major mechanism to increase protein diversity in higher eukaryotes. Dscam, the Drosophila homologue of human DSCAM (Down's syndrome cell adhesion molecule), generates up to 38016 isoforms through mutually exclusive splicing in four variable exon clusters. This enormous molecular diversity is functionally important for wiring of the nervous system and phagocytosis of invading pathogens. Current models explaining this complex splicing regulation include a default repressed state of the variable exon clusters to prevent the splicing together of adjacent exons, the presence of RNA secondary structures important for the release of one specific variable exon from the repressed state and combinatorial interaction of RNA-binding proteins for choosing a specific exon.

Post-Transcriptional Regulation of Tissue Factor Expression in Human Monocytic Cells: Identification of Novel Exonic Splicing Enhancers for the Spliceosomal Protein SRp40 and Intronic Elements Critical for Exon 5 Definition

Background. Circulating monocytes play a critical role in the pathophysiology of many thrombotic disorders. These cells are known to exhibit complex post-transcriptional regulation of Tissue Factor (TF), the principal trigger of coagulation, which they express in two forms – full length TF(flTF), a highly thrombogenic integral membrane protein, and alternatively spliced TF (asTF), a secreted hypomorphic TF form. Biosynthesis of these two forms is achieved by inclusion or, alternatively, exclusion of TF exon 5 during pre-mRNA processing. Little is known about molecular mechanisms controlling alternative pre-mRNA splicing in human monocytes. We recently developed a mini-gene splicing reporter system pGL-hTF to evaluate the dynamics of exon 5 splicing, and determined that SR proteins ASF/SF2 and SRp55, which are abundantly expressed in human monocytes, take part in TF pre-mRNA processing by promoting exon 5 definition. Objective. To expand our exploration of cis-acting elements and spliceosomal proteins governing regulated biosynthesis of human TF in monocytic cells. Results. In silico analysis of exon 5 revealed six putative binding motifs termed exonic splicing enhancers (ESE) for SRp40, an SR protein whose expression pattern in human tissues is largely unknown. SRp40 mRNA and protein were detected in monocytic cell lines THP-1 and SC, as well as freshly isolated peripheral blood mononuclear cells (PBMC). Notably, the apparent molecular weight of the SRp40 protein in PBMC was substantially larger than that observed in monocytic cell lines, indicating that SRp40 in PBMC is likely to be hyperphosphorylated and may thus exhibit increased activity. To assess functionality of the putative SRp40 ESE in TF exon 5, base substitutions were designed to weaken each ESE and introduced into pGL-hTF by site directed mutagenesis. Resultant mutants were expressed in THP-1 cells, and the expression patterns analyzed by RT-PCR. Weakening of the ESE at position 44 potentiated inclusion of exon 5, while weakening of the ESE at position 86 resulted in increased exon 5 skipping. This suggests that SRp40 plays a complex, positional role in exon 5 definition. To further confirm functionality of the identified SRp40 ESE, combinatorial mutants were generated featuring weakened ESE for the SR proteins SRp55 (position 39, in close proximity to SRp40 ESE 44) and ASF/SF2 (positions 87–117): inactivation of these ESE was shown to promote skipping of exon 5. As expected, weakened SRp40 ESE 86, when combined with weakened SRp55 ESE, resulted in more severe skipping of exon 5. In contrast to that, weakened SRp40 ESE 44, when combined with weakened SRp55 ESE, potently restored inclusion of exon 5, whereas combining weakened SRp40 ESE 44 with weakened ASF/SF2 ESE yielded only a partial restoration of exon 5 inclusion. This indicates that SRp40 ESE 44 uniquely promotes production of the TF mRNA species encoding asTF, whereas SRp40 ESE 86 appears to behave similarly to classic ESE for spliceosomal proteins SRp55 and ASF/SF2. Using pGL-hTF, we also evaluated major regulatory elements of intron 4: this relatively short intron precedes the variable exon 5 and is thus likely to be critical for its spliceosomal definition. To identify a branchpoint (BP) adenosine (A), we replaced each A within the last 100 bp of the intron with a non-canonical BP base (G); surprisingly, these mutations did not affect the degree of exon 5 inclusion, indicating that intron 4 features a highly unusual BP motif. In contrast to that, mutations within the polypyrimidine tract (PT) of intron 4 revealed that the entire PT sequence is critical to exon 5 definition: compared to wild type pGL-hTF, a pGL-hTF mutant featuring extensive purine substitutions within the PT generated ~11.4 times fewer exon 5 containing amplicons (p=0.0001), as detected by our sensitive molecular beacon qPCR assay. Conclusions. We show for the first time that the spliceosomal protein SRp40 is expressed in human monocytes and participates in TF biosynthesis. Using our reporter system, we identified two functional ESE for SRp40 in the variable exon 5. SRp40 appears to play a complex, positional role in production of TF protein variants with a distinct coagulant potential. Furthering our knowledge about the post-transcriptional mechanisms of TF biosynthesis in circulating monocytes is very likely to facilitate development of qualitatively novel classes of anti-thrombotic therapeutic agents.

Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene ( Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.

Molecular Dissection of Human Tissue Factor Pre-mRNA Processing: Identification and Characterization of Functional Exonic Splicing Enhancers in Variable Exon 5.

Tissue Factor (TF), the regulatory co-factor of FVII/VIIa, is the physiologic trigger of blood coagulation. Two naturally occurring isoforms of TF differ in their structure: the full length TF (flTF) is a membrane-spanning molecule, whereas the recently discovered alternatively spliced TF (asTF), generated by exclusion of exon 5, lacks the transmembrane domain, and is soluble. Biosynthesis of the soluble TF variant appears to be evolutionarily advantageous, as it exists in mice. Both isoforms are present in plasma and produced predominantly by circulating monocytes. The biologic role of asTF is unknown; it has, however, been proposed that precise regulation of the TF isoform ratio in blood is critical to normal hemostasis. This study aims to identify molecular mechanisms governing the fate of variable exon 5 during the processing of the human TF pre-mRNA, and to apply these findings to generate mini-genes expressing altered human flTF/asTF isoform ratios in animal models to elucidate the role of TF isoforms in normal hemostasis and/or development of coagulopathies. In silico analysis of the human TF exon 5 revealed 14 putative exonic splicing enhancer (ESE) motifs for SF2/ASF and SC35, essential SR proteins involved in pre-mRNA splicing. A segment of the TF gene comprising the end of exon 4 through the beginning of exon 6 (including exon 5 and the entire introns 4 and 5) was inserted into the GFP open reading frame of the pGL expression vector. The resultant construct (pGL-hTF) was transfected into a human monocytic cell line (THP-1) and shown to faithfully recapitulate the pattern of TF mRNA isoform production. Site-directed mutagenesis of each putative ESE motif was then performed, and the mRNA isoform levels were assayed by RT-PCR. Removal of four ASF/SF2 sites at positions 100–117 favored exclusion of exon 5. In contrast, removal of either of the two SC35 motifs at positions 33 and 81 favored inclusion of the exon. A pSPL-3b-hTF expression vector, featuring the above mutations in the context of a different promoter and a smaller segment of the TF gene, was generated to confirm these findings: similar shifts in isoform ratio production were observed. Results were further validated by introducing both synergistic and antagonistic double mutations of the functional ESEs in pGL-hTF. Production of the two naturally occurring human TF isoforms appears to be regulated at the post-transcriptional level by SR proteins ASF/SF2 and SC35. Further elucidation of the molecular mechanisms involved in TF isoform production is very likely to provide effective means for creating state-of-the-art animal models to characterize the biologic importance of flTF/asTF ratio in circulating blood. In addition, this study is the first to show that SF2/ASF and SC35 can exert opposite effects on pre-mRNA splicing.