Evolution during millions of years in perpetual darkness leads to mutations in non-visual opsin genes (Melanopsin and TMT opsin) and an aberrant, blind circadian clock in cavefish.

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

SummaryThe ability to adapt growth and development to temperature variations is crucial to generate plant varieties resilient to predicted temperature changes. However, the mechanisms underlying plant response to progressive increases in temperature have just started to be elucidated. Here, we report that the cyclin‐dependent kinase G1 (CDKG1) is a central element in a thermo‐sensitive mRNA splicing cascade that transduces changes in ambient temperature into differential expression of the fundamental spliceosome component, ATU2AF65A. CDKG1 is alternatively spliced in a temperature‐dependent manner. We found that this process is partly dependent on both the cyclin‐dependent kinase G2 (CDKG2) and the interacting co‐factor CYCLIN L1 (CYCL1), resulting in two distinct messenger RNAs. The relative abundance of both CDKG1 transcripts correlates with ambient temperature and possibly with different expression levels of the associated protein isoforms. Both CDKG1 alternative transcripts are necessary to fully complement the expression of ATU2AF65A across the temperature range. Our data support a previously unidentified temperature‐dependent mechanism based on the alternative splicing (AS) of CDKG1 and regulated by CDKG2 and CYCL1. We propose that changes in ambient temperature affect the relative abundance of CDKG1 transcripts, and this in turn translates into differential CDKG1 protein expression coordinating the AS of ATU2AF65A.

Small nuclear U1-RNAs (snRNAs), the spliceosome components selectively recognizing donor splice sites (5ss), were engineered to restore correct mRNA processing in a cellular model of severe coagulation factor VII (FVII) deficiency, caused by the IVS7 9726 ؉ 5g/a change. Three U1-snRNAs, complementary to the mutated 5ss (U1 ؉ 5a) or to neighboring sequences were expressed with FVII minigenes in a hepatoma cell line. The U1-snRNAs reduced from 80% to 40% the exon 7 skipping, thus increasing exon definition. The U1 ؉ 5a construct also dramatically increased recognition of the correct 5ss over the 37-bp downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5ss, a frequent cause of severe defects. IntroductionChanges affecting mRNA processing represent a frequent cause of severe coagulation factor defects 1-6 and of all inherited human diseases. 7-9 Different from gene therapy approaches inserting exogenous sequences that drive the expression of the missing factor, the specific correction of the mRNA processing would maintain the proper transcriptional control at the natural chromosomal environment and restore gene expression.Modified small nuclear RNAs (snRNAs) have been shown to promote changes in mRNA splicing in cellular and animal models of human diseases. However, these approaches were mainly aimed at inducing skipping of defective exons, 10,11 an approach that would not produce functional coagulation proteins, or masking newly generated cryptic exons, 12-14 an uncommon event in coagulation factor defects. Few attempts 15,16 have been made to redirect recognition of mutated donor splice site (5Јss) consensus sequences, the most frequent target in human disease genes. 8 On the other hand, bleeding disorders would significantly benefit even from low production of the correct mRNA and protein.As a model to exploit snRNAs for restoration of correct splicing in coagulation factor deficiencies, we chose the 9726 ϩ 5g/a mutation 17 in FVII occurring in the donor splice site of intron 7 (IVS7) at the ϩ 5 position, whose substitution has been frequently found to be associated with human diseases. [7][8][9] The homozygous patients experienced life-threatening hemorrhagic symptoms and require replacement therapy. Interestingly, coinheritance of the FVLeiden allele in a 9726 ϩ 5g/a homozygote produced a small increase in factor Xa and thrombin generation, resulting in a mild bleeding phenotype. 18 Through expression of minigenes and use of U1-snRNAs designed to selectively target the mutated site, we provided evidence for partial restoration of factor VII (FVII) mRNA processing impaired by the 9726 ϩ 5g/a change. Methods Construction of plasmidsThe...

The mechanisms underlying the circadian control of gene expression in peripheral tissues and influencing many biological pathways are poorly defined. Factor VII (FVII), the protease triggering blood coagulation, represents a valuable model to address this issue in liver since its plasma levels oscillate in a circadian manner and its promoter contains E-boxes, which are putative DNA-binding sites for CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers and hallmarks of circadian regulation. The peaks of FVII mRNA levels in livers of wild-type mice preceded those in plasma, indicating a transcriptional regulation, and were abolished in Clock ؊/؊ ; Npas2 ؊/؊ mice, thus demonstrating a role for CLOCK and NPAS2 circadian transcription factors. The investigation of Npas2؊/؊ and Clock ⌬19/⌬19 mice, which express functionally defective heterodimers, revealed robust rhythms of FVII expression in both animal models, suggesting a redundant role for NPAS2 and CLOCK. The molecular bases of these observations were established through reporter gene assays. FVII transactivation activities of the NPAS2-BMAL1 and CLOCK-BMAL1 heterodimers were (i) comparable (a fourfold increase), (ii) dampened by the negative circadian regulators PER2 and CRY1, and (iii) abolished upon E-box mutagenesis. Our data provide the first evidence in peripheral oscillators for an overlapping role of CLOCK and NPAS2 in the regulation of circadianly controlled genes.Circadian clocks are endogenous oscillators that drive biochemical, physiological, and behavioral processes in organisms (36), and the alteration of which is associated with human diseases (17,26,42). It is well documented that the circadian rhythms of mammals are controlled by a master circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus (38) as well as by peripheral oscillators located in most tissues (43).The basic circadian molecular clockworks consist of interacting positive and negative transcriptional/translational feedback loops (11). The positive loop involves CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers, three basic helix-loop-helix transcriptional activators that bind to E-box elements (CA CGTG) located in the regulatory regions of the Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes (3, 13, 16, 24). CRY and PER proteins form oligomers that, once transported into the nucleus, repress their own transcription by inhibiting CLOCK-BMAL1 or NPAS2-BMAL1 (25). Furthermore, other transcription factors (CIPC, DEC1, DEC2, and Fbxl3) play important roles in the negative-feedback loop (15,19,44). The positive and negative limbs are interlaced: CLOCK-BMAL1 heterodimers indirectly regulate a rhythm in Bmal1 transcription; the nuclear orphan receptor genes Reverb␣ and Rora are coordinately activated by CLOCK-BMAL1 to produce proteins that compete for the same promoter element but have opposing actions on Bmal1 transcription (1, 32). Oscillations in gene expression resulting from this feedback loop have period lengths of approximately 24 h, thus giving ri...

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Mutations affecting specific splicing regulatory elements offer suitable models to better understand their interplay and to devise therapeutic strategies. Here we characterize a meaningful splicing model in which numerous Hemophilia B-causing mutations, either missense or at the donor splice site (5′ss) of coagulation F9 exon 2, promote aberrant splicing by inducing the usage of a strong exonic cryptic 5′ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5′ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding and the comparative analysis of the F9 sequence across species showing that the cryptic 5′ss is always paralleled by the conserved ESS support a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligoribonucleotides masking the cryptic 5′ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5′ss. On the other hand, we observed a very poor correction effect by small nuclear RNA U1 (U1snRNA) variants with increased or perfect complementarity to the defective 5′ss, a strategy previously exploited to rescue splicing. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts (from 0 to 20–40%) from several mutants of the exon 2 5′ss. Based on the evidence of an altered interplay among ESS, cryptic and the authentic 5′ss as a disease-causing mechanism, we provide novel experimental insights into the combinatorial correction activity of antisense molecules and compensatory U1snRNAs.