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Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.

Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from −10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.

Acceptor splice site recognition (3&prime, splice site: 3&prime, ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3&prime, ss, of which U2AF35 has a dual function: (i) It binds to the intron&ndash, exon border of some 3&prime, ss and (ii) mediates enhancer-binding splicing activators&rsquo, interactions with the spliceosome. Alternative mechanisms for 3&prime, ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3&prime, ss recognition where the intron&ndash, exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3&prime, ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3&prime, ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons&rsquo, inclusion. Most probably, both factors stochastically bind the 3&prime, ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3&prime, ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants&rsquo, effects on splicing.

Both variants affecting splice sites and those in splicing regulatory elements (SREs) can impair pre-mRNA splicing, eventually leading to severe diseases. Despite the availability of many prediction tools, prognosis of splicing affection is not trivial, especially when SREs are involved. Here, we present data on 92 in silico-/55 minigene-analysed variants detected in genes responsible for the primary immunodeficiencies development (namely BTK, CD40LG, IL2RG, SERPING1, STAT3, and WAS). Of 20 splicing-affecting variants, 16 affected splice site while 4 disrupted potential SRE. The presence or absence of splicing defects was confirmed in 30 of 32 blood-derived patients' RNAs. Testing prediction tools performance, splice site disruptions and creations were reliably predicted in contrast to SRE-affecting variants for which just ESRseq, ΔHZEI-scores and EX-SKIP predictions showed promising results. Next, we found an interesting pattern in cryptic splice site predictions. These results might help PID-diagnosticians and geneticists cope with potential splicing-affecting variants.

Three-amino-acid-loop-extension homeodomain proteins affect various aspects during root development, such as root skewing or lateral root initiation and growth. Crossed lines of the BP overexpressor in Arabidopsis thaliana (L.) Heynh. were used to elucidate the role of BREVIPEDICELLUS (BP) in root development. The potential heterodimerization partner of BP, BEL1-like homeodomain protein 6 (BLH6), could be an important modulator of BP function. BLH6 is a member of BEL-like family of homeodomain proteins consisting of all conserved domains and motifs. We identified a new BLH-specific TERM motif at the C terminus of BLH proteins. BLH6 was expressed in different root tissues, including the meristematic zone and bases of lateral roots, and overlap the BP expression zone, enabling coordinated function of these transcription factors. To elucidate potential interactions of BP and BLH6, crossed lines of BP overexpressor were analyzed. While other tested BLH mutations do not affect BP overexpression phenotype, knock-out mutation blh6-1 weakened 35S-BP influence on shoot and root development. We demonstrated that growth of lateral roots was modulated by BP overexpression and auxins, and that this effect was dependent on lateral root position along the primary root. In the upper part of the primary root, BP inhibited lateral root growth despite indol-3-acetic acid treatment and this effect correlated with predicted activity of BLH6 in crossed lines of 35S-BP and blh6 mutants.

Recent progress in plant hormone and macronutrient research is largely associated with characterising the molecular mechanisms of their actions. Quantitative reverse transcription-PCR is a powerful technique that enables elucidation of regulatory relationships through quantifying transcript levels, but its reliability requires precise normalization. We compared eight Arabidopsis thaliana housekeeping genes for expression stability after treatment with cytokinins, auxins, ethylene and after macronutrient level manipulation. The expression stability of housekeeping genes in cytokinin series was studied in wide spectrum of plants treated with different adenine- and phenylurea-type cytokinins as well as in plants with controlled overexpression of agrobacterial isopentenyltransferase. NormFinder and geNorm software were used to predict a unique set of genes as best for particular hormone groups. Generally, 18S rRNA, GAPC, and ACT were confirmed as less stable; and UBQ10, UBC, and EF-1α exhibited increased stability. Based on geNorm pairwise variation analysis, we confirmed accurate normalization required at least three reference genes.

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

Pre-mRNA splicing is an essential step in gene expression, when introns are removed and exons joined by the complex of proteins called spliceosome. Correct splicing requires a precise exon/intron junction definition, which is determined by a consensual donor and acceptor splice site at the 5′ and 3′ end, respectively. An acceptor splice site (3′ss) consists of highly conserved AG nucleotides in positions E−2 and E−1. These nucleotides can appear in tandem, located 3 bp from each other. Then they are referred to as NAGNAG or tandem 3′ss, which can be alternatively spliced. NAG/TAG 3′ss motif abundance is extremely low and cannot be easily explained by just a nucleotide preference in this position. We tested artificial NAG/TAG motif’s potential negative effect on exon recognition using a minigene assay. Introducing the NAG/TAG motif into seven different exons revealed no general negative effect on exon recognition. The only observed effect was the partial use of the newly formed distal 3′ss. We can conclude that this motif’s extremely low preference in a natural 3′ss is not a consequence of the NAG/TAG motif’s negative effect on exon recognition, but more likely the result of other RNA processing aspects, such as an alternative 3′ss choice, decreased 3′ss strength, or incorporating an amber stop codon.

Mutations in the C1 inhibitor (C1INH) encoding gene, SERPING1, are associated with hereditary angioedema (HAE) which manifests as recurrent submucosal and subcutaneous edema episodes. The major C1INH function is the complement system inhibition, preventing its spontaneous activation. The presented study is focused on SERPING1 exon 3, an alternative and extraordinarily long exon (499 bp). Endogenous expression analysis performed in the HepG2, human liver, and human peripheral blood cells revealed several exon 3 splicing variants alongside exon inclusion: a highly prevalent exon skipping variant and less frequent +38 and -15 variants with alternative 3' splice sites (ss) located 38 and 15 nucleotides downstream and upstream from the authentic 3' ss, respectively. An exon skipping variant introducing a premature stop codon, represented nearly one third of all splicing variants and surprisingly appeared not to be degraded by NMD. The alternative -15 3' ss was used to a small extent, although predicted to be extremely weak. Its use was shown to be independent of its strength and highly sensitive to any changes in the surrounding sequence. -15 3' ss seems to be co-regulated with the authentic 3' ss, whose use is dependent mainly on its strength and less on the presence of intronic regulatory motifs. Subtle SERPING1 exon 3 splicing regulation can contribute to overall C1INH plasma levels and HAE pathogenesis.

Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired β-glucosidase activity. The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize β-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of β-glucosidases for either fundamental studies or potential biotechnological applications.

Plant hormones control plant development by modulating the expression of regulatory genes, including homeobox-containing KNOXI genes. However, much remains to be elucidated about the interactions involved. Therefore, hormonal regulation of KNOXI gene expression was investigated using hormone applications and an inducible transgenic ipt expression system to increase endogenous cytokinin (CK) levels. Treatments with auxin, abscisic acid (ABA), cytokinins, ethylene, and gibberellin (GA) did not result in ectopic expression of the BP (BREVIPEDICELLUS) gene. However, BP expression was strongly reduced by ABA, increased by auxin treatment (correlating with the initiation of lateral root meristems, which strongly express BP), and did not significantly respond to short-term treatments with the other hormones in whole seedlings. Following short-term ipt activation, organ-specific differential regulation of KNOXI gene expression was observed. While several KNOXI genes were transiently up-regulated to low levels, STM was selectively repressed (especially at low light) in hypocotyls. In cotyledons, activation of CK-responsive genes preceded ipt induction, suggesting that CKs are transported more rapidly than the inducing agent (dexamethasone). Long-term increases in CK levels induced raised levels of several KNOXI transcripts in hypocotyls, correlating with the radial expansion of vascular tissues, the main domains of KNOXI gene expression, suggesting that CKs had little effect on KNOXI promoter activity. No alterations in hormone sensitivity were observed in a bp null mutant. Constitutive BP overexpression caused reductions in the length and number of lateral roots, while the primary root remained unaffected. The transgenic seedlings displayed weak, but significant, alterations in sensitivity to ABA, CK, and 1-amino-cyclopropane-1-carboxylic acid.

The dioecious white campion Silene latifolia (syn. Melandrium album) has heteromorphic sex chromosomes, XX in females and XY in males, that are larger than the autosomes and enable their separation by flow sorting. The group of MROS genes, the first male-specifically expressed genes in dioecious plants, was recently identified in S. latifolia. To localize the MROS genes, we used the flow-sorted X chromosomes and autosomes as a template for PCR with internal primers. Our results indicate that the MROS3 gene is located in at least two copies tandemly arranged on the X chromosome with additional copy(ies) on the autosome(s), while MROS1, MROS2, and MROS4 are exclusively autosomal. The specificity of PCR products was checked by digestion with a restriction enzyme or reamplification using nested primers. Homology search of databases has shown the presence of five MROS3 homologues in A. thaliana, four of them arranged in two tandems, each consisting of two copies. We conclude that MROS3 is a low-copy gene family, connected with the proper pollen development, which is present not only in dioecious but also in other dicot plant species.

The development and activity of the procambium and cambium, which ensure vascular tissue formation, is critical for overall plant architecture and growth. However, little is known about the molecular factors affecting the activity of vascular meristems and vascular tissue formation. Here, we show that the His kinase CYTOKININ-INDEPENDENT1 (CKI1) and the cytokinin receptors ARABIOPSIS HISTIDINE KINASE2 (AHK2) and AHK3 are important regulators of vascular tissue development in Arabidopsis thaliana shoots. Genetic modifications of CKI1 activity in Arabidopsis cause dysfunction of the two-component signaling pathway and defects in procambial cell maintenance. CKI1 overexpression in protoplasts leads to cytokinin-independent activation of the two-component phosphorelay, and intracellular domains are responsible for the cytokinin-independent activity of CKI1. CKI1 expression is observed in vascular tissues of inflorescence stems, and CKI1 forms homodimers both in vitro and in planta. Loss-of-function ahk2 and ahk3 mutants and plants with reduced levels of endogenous cytokinins show defects in procambium proliferation and an absence of secondary growth. CKI1 overexpression partially rescues ahk2 ahk3 phenotypes in vascular tissue, while the negative mutation CKI1H405Q further accentuates mutant phenotypes. These results indicate that the cytokinin-independent activity of CKI1 and cytokinin-induced AHK2 and AHK3 are important for vascular bundle formation in Arabidopsis.

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient’s fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient’s mutation induced any relevant changein surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. Key message: Novel exonic IDS gene mutation leads to a complex splicing pattern.Mutation activates multiple pseudoexons through a previously unreported mechanism.Multiple cryptic splice site (ss) activation results from a bypass of authentic ss.Authentic ss bypass is due to a competition between de novo and authentic ss.

Postembryonic de novo organogenesis represents an important competence evolved in plants that allows their physiological and developmental adaptation to changing environmental conditions. The phytohormones auxin and cytokinin (CK) are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their interaction(s) in control of plant organogenesis is largely unknown. Here, we show that CK modulates auxin-induced organogenesis (AIO) via regulation of the efflux-dependent intercellular auxin distribution. We used the hypocotyl explants-based in vitro system to study the mechanism underlying de novo organogenesis. We show that auxin, but not CK, is capable of triggering organogenesis in hypocotyl explants. The AIO is accompanied by endogenous CK production and tissue-specific activation of CK signaling. CK affects differential auxin distribution, and the CK-mediated modulation of organogenesis is simulated by inhibition of polar auxin transport. CK reduces auxin efflux from cultured tobacco cells and regulates expression of auxin efflux carriers from the PIN family in hypocotyl explants. Moreover, endogenous CK levels influence PIN transcription and are necessary to maintain intercellular auxin distribution in planta . Based on these findings, we propose a model in which auxin acts as a trigger of the organogenic processes, whose output is modulated by the endogenously produced CKs. We propose that an important mechanism of this CK action is its effect on auxin distribution via regulation of expression of auxin efflux carriers.