Evolutionary analysis reveals the role of a non-catalytic domain of peptidyl arginine deiminase 2 in transcriptional regulation.

Peptidyl arginine deiminases (PADIs) catalyze protein citrullination, a post-translational conversion of arginine to citrulline. The most widely expressed member of this family, PADI2, regulates cellular processes that impact several diseases. We hypothesized that we could gain new insights into PADI2 function through a systematic evolutionary and structural analysis. Here, we identify 20 positively selected PADI2 residues, 16 of which are structurally exposed and maintain PADI2 interactions with cognate proteins. Many of these selected residues reside in non-catalytic regions of PADI2. We validate the importance of a prominent loop in the middle domain that encompasses PADI2 L162, a residue under positive selection. This site is essential for interaction with the transcription elongation factor (P-TEFb) and mediates the active transcription of the oncogenes c-MYC, and CCNB1, as well as impacting cellular proliferation. These insights could be key to understanding and addressing the role of the PADI2 c-MYC axis in cancer progression.

IQGAP1 mediates the communication between the nucleus and the mitochondria via NDUFS4 alternative splicing.

Constant communication between mitochondria and nucleus ensures cellular homeostasis and adaptation to mitochondrial stress. Anterograde regulatory pathways involving a large number of nuclear-encoded proteins control mitochondrial biogenesis and functions. Such functions are deregulated in cancer cells, resulting in proliferative advantages, aggressive disease and therapeutic resistance. Transcriptional networks controlling the nuclear-encoded mitochondrial genes are known, however alternative splicing (AS) regulation has not been implicated in this communication. Here, we show that IQGAP1, a scaffold protein regulating AS of distinct gene subsets in gastric cancer cells, participates in AS regulation that strongly affects mitochondrial respiration. Combined proteomic and RNA-seq analyses of IQGAP1KO and parental cells show that IQGAP1KO alters an AS event of the mitochondrial respiratory chain complex I (CI) subunit NDUFS4 and downregulates a subset of CI subunits. In IQGAP1KO cells, CI intermediates accumulate, resembling assembly deficiencies observed in patients with Leigh syndrome bearing NDUFS4 mutations. Mitochondrial CI activity is significantly lower in KO compared to parental cells, while exogenous expression of IQGAP1 reverses mitochondrial defects of IQGAP1KO cells. Our work sheds light to a novel facet of IQGAP1 in mitochondrial quality control that involves fine-tuning of CI activity through AS regulation in gastric cancer cells relying highly on mitochondrial respiration.

Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects.

The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches.

Rescue of common exon-skipping mutations in cystic fibrosis with modified U1 snRNAs.

In cystic fibrosis (CF), the correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on 10 common mutations/variants 711+3A>G/C, 711+5G>A, TG13T3, TG13T5, TG12T5, 1863C>T, 1898+3A>G, 2789+5G>A, and 3120G>A that induce skipping of the corresponding CFTR exons 5, 10, 13, 16, and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 small nuclear RNAs (snRNAs), named Exon Specific U1s (ExSpeU1s), that was engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all 10 splicing mutations analyzed are efficiently corrected by specific ExSpeU1s. Using complementary DNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants.

In cellular models we have demonstrated that a unique U1snRNA targeting an intronic region downstream of a defective exon (Exon-specific U1snRNA, ExSpeU1) can rescue multiple exon-skipping mutations, a relevant cause of genetic disease. Here, we explored in mice the ExSpeU1 U1fix9 toward two model Hemophilia B-causing mutations at the 5' (c.519A > G) or 3' (c.392-8T > G) splice sites of F9 exon 5. Hydrodynamic injection of wt-BALB/C mice with plasmids expressing the wt and mutant (hFIX-2G5'ss and hFIX-8G3'ss) splicing-competent human factor IX (hFIX) cassettes resulted in the expression of hFIX transcripts lacking exon 5 in liver, and in low plasma levels of inactive hFIX. Coinjection of U1fix9, but not of U1wt, restored exon inclusion of variants and in the intrinsically weak FIXwt context. This resulted in appreciable circulating hFIX levels (mean ± SD; hFIX-2G5'ss, 1.0 ± 0.5 µg/ml; hFIX-8G3'ss, 1.2 ± 0.3 µg/ml; and hFIXwt, 1.9 ± 0.6 µg/ml), leading to a striking shortening (from ~100 seconds of untreated mice to ~80 seconds) of FIX-dependent coagulation times, indicating a hFIX with normal specific activity. This is the first proof-of-concept in vivo that a unique ExSpeU1 can efficiently rescue gene expression impaired by distinct exon-skipping variants, which extends the applicability of ExSpeU1s to panels of mutations and thus cohort of patients.

Molecular Basis and Therapeutic Strategies to Rescue Factor IX Variants That Affect Splicing and Protein Function.

Mutations that result in amino acid changes can affect both pre-mRNA splicing and protein function. Understanding the combined effect is essential for correct diagnosis and for establishing the most appropriate therapeutic strategy at the molecular level. We have identified a series of disease-causing splicing mutations in coagulation factor IX (FIX) exon 5 that are completely recovered by a modified U1snRNP particle, through an SRSF2-dependent enhancement mechanism. We discovered that synonymous mutations and missense substitutions associated to a partial FIX secretion defect represent targets for this therapy as the resulting spliced-corrected proteins maintains normal FIX coagulant specific activity. Thus, splicing and protein alterations contribute to define at the molecular level the disease-causing effect of a number of exonic mutations in coagulation FIX exon 5. In addition, our results have a significant impact in the development of splicing-switching therapies in particular for mutations that affect both splicing and protein function where increasing the amount of a correctly spliced protein can circumvent the basic functional defects.

Therapeutic activity of modified U1 core spliceosomal particles.

Modified U1 snRNAs bound to intronic sequences downstream of the 5' splice site correct exon skipping caused by different types of mutations. Here we evaluate the therapeutic activity and structural requirements of these exon-specific U1 snRNA (ExSpeU1) particles. In a severe spinal muscular atrophy, mouse model, ExSpeU1, introduced by germline transgenesis, increases SMN2 exon 7 inclusion, SMN protein production and extends life span. In vitro, RNA mutant analysis and silencing experiments show that while U1A protein is dispensable, the 70K and stem loop IV elements mediate most of the splicing rescue activity through improvement of exon and intron definition. Our findings indicate that precise engineering of the U1 core spliceosomal RNA particle has therapeutic potential in pathologies associated with exon-skipping mutations.

Improvement of SMN2 pre-mRNA processing mediated by exon-specific U1 small nuclear RNA.

Exon-specific U1 snRNAs (ExSpe U1s) are modified U1 snRNAs that interact with intronic sequences downstream of the 5' splice site (ss) by complementarity. This process restores exon skipping caused by different types of mutation. We have investigated the molecular mechanism and activity of these molecules in spinal muscular atrophy (SMA), a genetic neuromuscular disease where a silent exonic transition on the survival motor neuron 2 (SMN2) leads to exon 7 (E7) skipping. By using different cellular models, we show that a single chromosome-integrated copy of ExSpe U1 induced a significant correction of endogenous SMN2 E7 splicing and resulted in the restoration of the corresponding SMN protein levels. Interestingly, the analysis of pre-mRNA transcript abundance and decay showed that ExSpe U1s promote E7 inclusion and stabilizes the SMN pre-mRNA intermediate. This selective effect on pre-mRNA stability resulted in higher levels of SMN mRNAs in comparison with those after treatment with an antisense oligonucleotide (AON) that targets corresponding intronic sequences. In mice harboring the SMN2 transgene, AAV-mediated delivery of ExSpe U1 increased E7 inclusion in brain, heart, liver, kidney, and skeletal muscle. The positive effect of ExSpe U1s on SMN pre-mRNA processing highlights their therapeutic potential in SMA and in other pathologies caused by exon-skipping mutations.