Widespread Isoform Research Articles

Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing.

SummaryAlternative splicing is a post-transcriptional regulatory mechanism producing distinct mRNA molecules from a single pre-mRNA with a prominent role in the development and function of the central nervous system. We used long-read isoform sequencing to generate full-length transcript sequences in the human and mouse cortex. We identify novel transcripts not present in existing genome annotations, including transcripts mapping to putative novel (unannotated) genes and fusion transcripts incorporating exons from multiple genes. Global patterns of transcript diversity are similar between human and mouse cortex, although certain genes are characterized by striking differences between species. We also identify developmental changes in alternative splicing, with differential transcript usage between human fetal and adult cortex. Our data confirm the importance of alternative splicing in the cortex, dramatically increasing transcriptional diversity and representing an important mechanism underpinning gene regulation in the brain. We provide transcript-level data for human and mouse cortex as a resource to the scientific community.

Splice-Junction-Based Mapping of Alternative Isoforms in the Human Proteome

SUMMARYThe protein-level translational status and function of many alternative splicing events remain poorly understood. We use an RNA sequencing (RNA-seq)-guided proteomics method to identify protein alternative splicing isoforms in the human proteome by constructing tissue-specific protein databases that prioritize transcript splice junction pairs with high translational potential. Using the custom databases to reanalyze ~80 million mass spectra in public proteomics datasets, we identify more than 1,500 noncanonical protein isoforms across 12 human tissues, including ~400 sequences undocumented on TrEMBL and RefSeq databases. We apply the method to original quantitative mass spectrometry experiments and observe widespread isoform regulation during human induced pluripotent stem cell cardiomyocyte differentiation. On a proteome scale, alternative isoform regions overlap frequently with disordered sequences and post-translational modification sites, suggesting that alternative splicing may regulate protein function through modulating intrinsically disordered regions. The described approach may help elucidate functional consequences of alternative splicing and expand the scope of proteomics investigations in various systems.

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with KIs in the range of 97.5 nM – 7.26 µM and 52.5 nM – 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (KIs of 4.75 – 8.87 µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (KIs > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (KIs of 59.8 nM – 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (KIs of 33.3 nM – 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KIs > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.

N-Substituted and ring opened saccharin derivatives selectively inhibit transmembrane, tumor-associated carbonic anhydrases IX and XII

A series of N-substituted saccharins incorporating aryl, alkyl and alkynyl moieties, as well as some ring opened derivatives were prepared and investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The widespread cytosolic isoforms CA I and II were not inhibited by these sulfonamides whereas transmembrane, tumor-associated ones were effectively inhibited, with KIs in the range of 22.1–481nM for CA IX and of 3.9–245nM for hCA XII. Although the inhibition mechanism of these tertiary/secondary sulfonamides is unknown for the moment, the good efficacy and especially selectivity for the inhibition of the tumor-associated over the cytosolic, widespread isoforms, make these derivatives of considerable interest as enzyme inhibitors with various pharmacologic applications.

7-Amino-3,4-dihydro-1H-quinolin-2-one, a compound similar to the substituted coumarins, inhibits α-carbonic anhydrases without hydrolysis of the lactam ring

7-Amino-3,4-dihydro-1H-quinolin-2-one, a compound structurally similar to coumarins, recently discovered class of inhibitors of the α-carbonic anhydrases (CAs, EC 4.2.1.1) was investigated for its interaction with all human (h) CA isoforms, hCA I-XIV. The compound was not an inhibitor of the cytosolic, widespread isoform hCA II (KI > 10 µM), was a weak inhibitor of hCA I, III, IV, VA, VI and XIII (KIs in the range of 0.90–9.5 µM) but effectively inhibited the cytosolic isoform hCA VII (KI of 480 nM) as well as the transmembrane isoforms hCA IX, XII and XIV (KIs in the range of 16.1–510 nM). Against many CA isoforms this lactam was a better inhibitor compared to the structurally similar 4-methyl-7-aminocoumarin, but unlike this compound, the lactam ring was not hydrolyzed and the inhibition was due to the intact bicyclic amino-quinolinone scaffold. Bicyclic lactams strucurally related to coumarins are thus a new class of CA inhibitors possessing however a distinct inhibition mechanism compared to the coumarins which undergo a hydrolysis of their lactone ring for generating the enzyme inhibitory species.

Sample Processing Obscures Cancer-Specific Alterations in Leukemic Transcriptomes

Substantial effort is currently devoted to identifying cancer-associated alterations using genomics technologies. While the genome is inherently stable over short time frames, the transcriptome is dynamic and potentially susceptible to alteration as samples move from the patient to the lab bench. Here, we show that standard blood collection procedures rapidly change the transcriptional and post-transcriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways, up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms, and inhibition of RNA surveillance. These artifacts affect almost all published leukemia genomics studies, explaining up to 40% of putative cancer-associated differential expression and alternative splicing.To determine how standard blood collection procedures affect hematopoietic transcriptomes, we collected whole blood from four healthy donors in anticoagulant blood collection tubes. We then left this blood at room temperature for defined lengths of time (0-48h) in order to mimic the variable incubation that patient samples are typically subjected to during transfer from the primary treating physician to a research center, isolated peripheral blood mononuclear cells (PBMCs), and performed deep RNA-seq (Figure A). Contrary to the common assumption that RNA degrades during incubation, we observed no evidence of decreased RNA quality. Nonetheless, rapid and dramatic changes affected virtually every level of the gene expression process. The changes were highly biased; for example, pseudogenes and antisense RNAs were preferentially up-regulated, while cassette exons were preferentially repressed (Figure B).Incubation-induced changes in the transcriptome confound the identification of true cancer-specific alterations. Many genes affected by incubation participate in biological pathways of current interest in leukemia, including cytokine production, NF-κB signaling, chromatin modification, and RNA splicing. Furthermore, incubation for as little as 4 hours, our shortest time point, introduced dramatic changes in the post-transcriptional landscape. We observed widespread isoform switches, wherein isoforms that were rare or even undetectable at 0h became the major isoform after 8 to 24h of incubation, in genes such as NOTCH2, LEF1, and PHF20 that have been previously used as leukemic biomarkers (Figure C). Perhaps most surprisingly, incubation rapidly inhibited RNA surveillance, leading to genome-wide expression of normally degraded RNAs. This highly abnormal RNA surveillance inhibition was readily detectable in all published leukemia genomics datasets that we analyzed, with the exception of TCGA, and undetectable in any lymphoma or solid tumor dataset (Figure D).Together, our data show that incubation-induced changes in the transcriptional and post-transcriptional landscapes generate substantial artifacts that complicate the interpretation of leukemia genomics studies. To facilitate the incorporation of sample processing artifacts into downstream analysis, we provide biomarkers that detect prolonged incubation of individual samples. We furthermore show that the simple expedient of keeping blood on ice drastically reduces changes to the transcriptome. Our findings have important implications for the interpretation of published and ongoing leukemia genomics studies. Figure. (A) Leukemic samples are frequently subject to an ex vivo incubation period of variable length. (B) Ex vivo incubation causes biased up-regulation of pseudogenes and antisense RNAs and repression of cassette exons. (C) Incubation causes complete isoform switches in the LEF1 and PHF20 genes. (D) RNA surveillance is inhibited after only 4h of incubation. This abnormal inhibition of RNA surveillance is visible in all analyzed leukemia genomics datasets, with the exception of TCGA, and not in any lymphoma or solid tumor. Numbers above each x axis label indicate the number of samples in each dataset. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], is a potent inhibitor of human carbonic anhydrases

The boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]) was investigated as inhibitor of the zinc enzyme, carbonic anhydrase (CA, EC 4.2.1.1). Eleven human (h) CA isoforms, hCA I–IV, VA, VI, VII, IX and XII–XIV, were included in the investigations. The anion, similar to tetraborate or phenylboronic acid, inhibited most of them. hCA III was not inhibited by K2[B3O3F4OH], whereas hCA VA, hCA VI, hCA IX and hCA XIII were inhibited in the submillimolar range, with KIs of 0.31–0.63 mM. hCA I and II (cytosolic, widespread isoforms), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by this anion, with inhibition constants ranging from 25 to 93 µM. hCA VII, a cytosolic enzyme present in the brain and associated to oxidative stress, was very effectively inhibited by K2[B3O3F4OH], with a KI of 8.0 µM. We propose that K2[B3O3F4OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately through its B-OH functionality. We hypothesize that some of the beneficial antitumor effects reported for K2[B3O3F4OH] may be due to the inhibition of CAs present in skin tumors.

Desmoglein 2 contributes to cell cohesion dependent on the desmosomal cadherin expression pattern

Desmoglein (Dsg) 2 is the most widespread isoform of the desmosomal cadherin protein family, of which four desmoglein (Dsg1–4) and three desmocollin (Dsc1–3) isoforms are expressed in keratinocytes. In the blistering skin disease pemphigus vulgaris (PV), Dsg3 is a target of autoantibodies indicating a central role for Dsg3 in keratinocyte cohesion. In contrast, for Dsg2 the role for keratinocyte cohesion has not been defined yet. We demonstrated the importance of Dsg2 for intestinal epithelial cells by incubation of Caco2 cells with a monoclonal Dsg2 antibody (Dsg2 mAb) which reduced cell integrity in a dissociation assay. However, Dsg2 mAb, in contrast to AK23, a pathogenic PV antibody targeting Dsg3, did not reduce cell cohesion in keratinocytes (HaCaT). To confirm that both antibodies are potent to bind their target protein we detected both antibodies’ heavy chains by Western blot analysis. Additionally both antibodies led to a decrease in the number of binding events of their corresponding recombinant Dsg target protein probed by atomic force microscopy (AFM). In contrast to another colorectal adenocarcinoma cell line (HT‐29), siRNA‐mediated silencing of Dsg2 in HaCaT cells reduced cell cohesion only under conditions of increased shear. These results indicate that Dsg2 contributes differently to cell cohesion dependent on the expression pattern of desmosomal cadherin family isoforms. DFG SFB487, SP1300–1

Intramolecular proton shuttle supports not only catalytic but also noncatalytic function of carbonic anhydrase II

Carbonic anhydrases (CAs) catalyze the reversible hydration of CO(2) to HCO(3)(-) and H(+). The rate-limiting step in this reaction is the shuttle of protons between the catalytic center of the enzyme and the bulk solution. In carbonic anhydrase II (CAII), the fastest and most wide-spread isoform, this H(+) shuttle is facilitated by the side chain of His64, whereas CA isoforms such as carbonic anhydrase III (CAIII), which lack such a shuttle, have only low catalytic activity in vitro. By using heterologous protein expression in Xenopus oocytes, we tested the role of this intramolecular H(+) shuttle on CA activity in an intact cell. The data revealed that CAIII, shown in vitro to have ∼1,000-fold reduced activity as compared with CAII, displays significant catalytic activity in the intact cell. Furthermore, we tested the hypothesis that the H(+) shuttle in CAII itself can facilitate transport activity of the monocarboxylate transporters 1 and 4 (MCT1/4) independent of catalytic activity. Our results show that His64 is essential for the enhancement of lactate transport via MCT1/4, because a mutation of this residue to alanine (CAII-H64A) abolishes the CAII-induced increase in MCT1/4 activity. However, injection of 4-methylimidazole, which acts as an exogenous H(+) donor/acceptor, can restore the ability of CAII-H64A to enhance transport activity of MCT1/4. These findings support the hypothesis that the H(+) shuttle in CAII not only facilitates CAII catalytic activity but also can enhance activity of acid-/base-transporting proteins such as MCT1/4 in a direct, noncatalytic manner, possibly by acting as an "H(+)-collecting antenna."