Alternative Cleavage Sites Research Articles

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base.

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral–gastro–duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20–30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment.

NLRP3 inflammasome triggers interleukin-37 release from human monocytes.

IL-37 is an anti-inflammatory member of the IL-1 family that dampens inflammation associated with many noncommunicable diseases. However, mechanisms of IL-37 regulation remain understudied. We aimed to investigate the enzymatic cleavage of IL-37 that potentiates extracellular signalling, as well as pathways of IL-37 secretion. In human monocytes, mature IL-37 (mIL-37) was released following canonical NLRP3 inflammasome activation. The release of IL-37 was blocked by inhibiting plasma membrane permeability and in gasdermin-D-deficient THP-1 cells. While the cleavage of IL-37 was found to be constitutive, the release of mIL-37 was blocked in NLRP3-deficient THP-1 cells and by NLRP3 inhibitor MCC950 in THP-1s and primary human monocytes. IL-37 secretion also occurred after 18-h exposure to LPS, independently of the alternative NLRP3 inflammasome. This LPS-dependent IL-37 secretion required plasma membrane permeability, but not conventional protein secretion apparatus. Mutagenesis of the suggested caspase-1 cleavage site (D20) or the proposed alternative cleavage site (V46) did not completely block IL-37 processing. Therefore, we propose a novel pathway in which IL-37 is cleaved by caspase-1-independent mechanisms and released following canonical and alternative NLRP3 inflammasome triggers by differential pathways.

Dynamic Variations of 3'UTR Length Reprogram the mRNA Regulatory Landscape.

This paper concerns 3′-untranslated regions (3′UTRs) of mRNAs, which are non-coding regulatory platforms that control stability, fate and the correct spatiotemporal translation of mRNAs. Many mRNAs have polymorphic 3′UTR regions. Controlling 3′UTR length and sequence facilitates the regulation of the accessibility of functional effectors (RNA binding proteins, miRNAs or other ncRNAs) to 3′UTR functional boxes and motifs and the establishment of different regulatory landscapes for mRNA function. In this context, shortening of 3′UTRs would loosen miRNA or protein-based mechanisms of mRNA degradation, while 3′UTR lengthening would strengthen accessibility to these effectors. Alterations in the mechanisms regulating 3′UTR length would result in widespread deregulation of gene expression that could eventually lead to diseases likely linked to the loss (or acquisition) of specific miRNA binding sites. Here, we will review the mechanisms that control 3′UTR length dynamics and their alterations in human disorders. We will discuss, from a mechanistic point of view centered on the molecular machineries involved, the generation of 3′UTR variability by the use of alternative polyadenylation and cleavage sites, of mutually exclusive terminal alternative exons (exon skipping) as well as by the process of exonization of Alu cassettes to generate new 3′UTRs with differential functional features.

The first Conus genome assembly reveals a primary genetic central dogma of conopeptides in C. betulinus

Although there are various Conus species with publicly available transcriptome and proteome data, no genome assembly has been reported yet. Here, using Chinese tubular cone snail (C. betulinus) as a representative, we sequenced and assembled the first Conus genome with original identification of 133 genome-widely distributed conopeptide genes. After integration of our genomics, transcriptomics, and peptidomics data in the same species, we established a primary genetic central dogma of diverse conopeptides, assuming a rough number ratio of ~1:1:1:10s for the total genes: transcripts: proteins: post-translationally modified peptides. This ratio may be special for this worm-hunting Conus species, due to the high diversity of various Conus genomes and the big number ranges of conopeptide genes, transcripts, and peptides in previous reports of diverse Conus species. Only a fraction (45.9%) of the identified conotopeptide genes from our achieved genome assembly are transcribed with transcriptomic evidence, and few genes individually correspond to multiple transcripts possibly due to intraspecies or mutation-based variances. Variable peptide processing at the proteomic level, generating a big diversity of venom conopeptides with alternative cleavage sites, post-translational modifications, and N-/C-terminal truncations, may explain how the 133 genes and ~123 transcripts can generate thousands of conopeptides in the venom of individual C. betulinus. We also predicted many conopeptides with high stereostructural similarities to the putative analgesic ω-MVIIA, addiction therapy AuIB and insecticide ImI, suggesting that our current genome assembly for C. betulinus is a valuable genetic resource for high-throughput prediction and development of potential pharmaceuticals.

2108-P: Complex Posttranslational Processing of Glucagon Prohormone in Human Pancreatic Islets

An array of signaling peptides and peptide hormones in pancreatic islets results from posttranslational processing of precursor proteins by well-characterized endopeptidases. While the encoded peptide complement can be predicted in silico from the prohormone sequence, actual peptide products often are more diverse due to additional chemical or enzymatic modifications of mature predicted peptides. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for comprehensive molecular characterization, we compiled an experimentally deduced database of endogenous peptides in human pancreatic islets and revealed an unexpected complex processing of several prohormones including proglucagon. Live pancreatic islets from several donors were provided by the Human Pancreas Analysis Program, University of Pennsylvania. Total of 2034 chemically unique peptide sequences were identified and matched to 477 proteins in the human protein database. Among interesting observations, we find differential cleavage of the signal peptide on the glucagon precursor protein, NP_002045.1, and unusual further processing of its encoded mature peptides. Alternate signal peptide cleavage sites are confirmed by detection of both classical GRPP, R[21]-D[50], and its novel variant S[18]-D[50]. An amidated IP-2 D[131]-L[142] and an isoform of MPGF D[131]-D[178], missing the GLP-1 sequence were detected. Previously unreported N-terminus acetylation of glucagon, GLP-1, GLP-1 (7-36), and oxyntomodulin was detected. Novel peptides potentially resulted from variable processing of mature glucagon, GRPP, and oxyntomodulin at internal convention cleavage sites were characterized. New chemically unique peptides originating from proglucagon and documented in this study indicate a more complex posttranslational processing of proglucagon in human islets than previously thought. The finding expands opportunity for mechanistic studies on pathology, progression, and treatments of diabetes. Disclosure E.V. Romanova: None. S. Rubakhin: None. J.V. Sweedler: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-18-VSN-19 to J.V.S.)

Cis autocatalytic cleavage of glycine-linked Zika virus NS2B-NS3 protease constructs.

The flaviviral heterodimeric serine protease NS2B-NS3, consisting of the NS3 protease domain and the NS2B co-factor, is essential for ZIKA virus maturation and replication in cells. For invitro studies a 'linked' construct, where a polyglycine linker connects NS2BCF and NS3pro ,is often used. This construct undergoes autocatalytic cleavage. Here, we show that linked ZIKV NS2BCF -NS3pro is cleaved in cis in the NS2BCF exclusively at position R95 and not at the previously proposed alternate cleavage site at residue R29 in the NS3pro . Cleavage neither affects protease stability nor activity, despite some observed differences in spectroscopic behavior. This minimally modified construct may thus be useful for future structural and functional studies of the flaviviral protease, for examplewhen testing new inhibitors.

Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3

Viral infection triggers host defenses through pattern-recognition receptor-mediated cytokine production, inflammasome activation, and apoptosis of the infected cells. Inflammasome-activated caspases are known to cleave cyclic GMP-AMP synthase (cGAS). Here, we found that apoptotic caspases are critically involved in regulating both DNA and RNA virus-triggered host defenses, in which activated caspase-3 cleaved cGAS, MAVS, and IRF3 to prevent cytokine overproduction. Caspase-3 was exclusively required in human cells, whereas caspase-7 was involved only in murine cells to inactivate cGAS, reflecting distinct regulatory mechanisms in different species. Caspase-mediated cGAS cleavage was enhanced in the presence of dsDNA. Alternative MAVS cleavage sites were used to ensure the inactivation of this critical protein. Elevated type I IFNs were detected in caspase-3-deficient cells without any infection. Casp3-/- mice consistently showed increased resistance to viral infection and experimental autoimmune encephalomyelitis. Our results demonstrate that apoptotic caspases control innate immunity and maintain immune homeostasis against viral infection.

Alternative cleavage and polyadenylation sites in colorectal cancer

Alternative cleavage and polyadenylation sites in colorectal cancer

Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3'-RACE and High-Throughput Sequencing.

Detection of complex splice sites (SSs) and polyadenylation sites (PASs) of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS) have revealed prevalent alternative splicing (AS) and alternative polyadenylation (APA) in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE) and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2) transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR) verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.

Alternative Selection of β-Site APP-Cleaving Enzyme 1 (BACE1) Cleavage Sites in Amyloid β-Protein Precursor (APP) Harboring Protective and Pathogenic Mutations within the Aβ Sequence

β-Site APP-cleaving enzyme 1 (BACE1) cleaves amyloid β-protein precursor (APP) at the bond between Met671 and Asp672 (β-site) to generate the carboxyl-terminal fragment (CTFβ/C99). BACE1 also cleaves APP at another bond between Thr681 and Gln682 (β'-site), yielding CTFβ'/C89. Cleavage of CTFβ/C99 by γ-secretase generates Aβ(1-XX), whereas cleavage of CTFβ'/C89 generates Aβ(11-XX). Thus, β'-site cleavage by BACE1 is amyloidolytic rather than amyloidogenic. β' cleavage of mouse APP is more common than the corresponding cleavage of human APP. We found that the H684R substitution within human Aβ, which replaces the histidine in the human protein with the arginine found at the corresponding position in mouse, facilitated β' cleavage irrespective of the species origin of BACE1, thereby significantly increasing the level of Aβ(11-XX) and decreasing the level of Aβ(1-XX). Thus, amino acid substitutions within the Aβ sequence influenced the selectivity of alternative β- or β'-site cleavage of APP by BACE1. In familial Alzheimer's disease (FAD), the APP gene harbors pathogenic variations such as the Swedish (K670N/M671L), Leuven (E682K), and A673V mutations, all of which decrease Aβ(11-40) generation, whereas the protective Icelandic mutation (A673T) increases generation of Aβ(11-40). Thus, A673T promotes β' cleavage of APP and protects subjects against AD. In addition, CTFβ/C99 was cleaved by excess BACE1 activity to generate CTFβ'/C89, followed by Aβ(11-40), even if APP harbored pathogenic mutations. The resultant Aβ(11-40) was more metabolically labile in vivo than Aβ(1-40). Our analysis suggests that some FAD mutations in APP are amyloidogenic and/or amyloidolytic via selection of alternative BACE1 cleavage sites.

Sequence variation between 462 human individuals fine-tunes functional sites of RNA processing.

Recent advances in the cost-efficiency of sequencing technologies enabled the combined DNA- and RNA-sequencing of human individuals at the population-scale, making genome-wide investigations of the inter-individual genetic impact on gene expression viable. Employing mRNA-sequencing data from the Geuvadis Project and genome sequencing data from the 1000 Genomes Project we show that the computational analysis of DNA sequences around splice sites and poly-A signals is able to explain several observations in the phenotype data. In contrast to widespread assessments of statistically significant associations between DNA polymorphisms and quantitative traits, we developed a computational tool to pinpoint the molecular mechanisms by which genetic markers drive variation in RNA-processing, cataloguing and classifying alleles that change the affinity of core RNA elements to their recognizing factors. The in silico models we employ further suggest RNA editing can moonlight as a splicing-modulator, albeit less frequently than genomic sequence diversity. Beyond existing annotations, we demonstrate that the ultra-high resolution of RNA-Seq combined from 462 individuals also provides evidence for thousands of bona fide novel elements of RNA processing—alternative splice sites, introns, and cleavage sites—which are often rare and lowly expressed but in other characteristics similar to their annotated counterparts.

Intronic cleavage and polyadenylation regulates gene expression during DNA damage response through U1 snRNA.

The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis.

UPF2-Dependent Nonsense-Mediated mRNA Decay Pathway Is Essential for Spermatogenesis by Selectively Eliminating Longer 3'UTR Transcripts.

During transcription, most eukaryotic genes generate multiple alternative cleavage and polyadenylation (APA) sites, leading to the production of transcript isoforms with variable lengths in the 3’ untranslated region (3’UTR). In contrast to somatic cells, male germ cells, especially pachytene spermatocytes and round spermatids, express a distinct reservoir of mRNAs with shorter 3’UTRs that are essential for spermatogenesis and male fertility. However, the mechanisms underlying the enrichment of shorter 3’UTR transcripts in the developing male germ cells remain unknown. Here, we report that UPF2-mediated nonsense-mediated mRNA decay (NMD) plays an essential role in male germ cells by eliminating ubiquitous genes-derived, longer 3’UTR transcripts, and that this role is independent of its canonical role in degrading “premature termination codon” (PTC)-containing transcripts in somatic cell lineages. This report provides physiological evidence supporting a noncanonical role of the NMD pathway in achieving global 3’UTR shortening in the male germ cells during spermatogenesis.

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively). Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as ‘site 2’ cleavage). However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K) mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 (‘site 1’) or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C.

An Extra Arm May Impair the Open Hand.

In the paper, “Inhibin Biosynthesis and Activity Are Limited by a Prodomain-Derived Peptide,” Walton et al (1) purport that processing of inhibin-α can result in a 43-amino acid pro-α peptide from an alternate cleavage site. This propeptide is projected to have an amphipathic α-helical structure that can wrap around the open hand or butterfly-shaped mature inhibin protein, thereby hindering inhibin association with β-glycan. The function of this proinhibin α-peptide is investigated as an inhibitor of mature inhibin action. The inhibins and activins are produced as prepro-proteins in which nonactive domains that are important to protein folding and dimerization of the molecules are cleaved, releasing the final active products. The β-subunits for inhibin and activin have only one classical cleavage site, but the α-inhibin subunit has another cleavage site between the pro-α-peptide and the N-peptide (Figure 1A). Uncleaved or incompletely cleaved inhibins have been described for many years as high-molecular-weight or big inhibins, with varying demonstrations of bioactivity (2,–4). A free α-subunit in association with the C portion, without the N portion, has also been described and is produced at high levels by a variety of normal and abnormal tissues (5, 6), but this is the first evaluation of a functional small prodomain cleavage peptide for inhibin. Figure 1. A, Inhibin precursor with the cleavage sites. Note the two cleavage sites on the α-subunit and the single cleavage site on the β-subunit. B, Inhibin A/B binding to β-glycan. C, Pro-α-peptide intercalating between inhibin ... The authors also noted some time ago that three hydrophobic amino acids in the prodomain of the inhibin α-subunit were critical to proper folding and secretion of mature inhibins (7). In that work they used mutagenesis evidence to propose a model of interaction of the N-terminal prodomain with a region near the C terminus of the proprotein to hold it in a coiled conformation that would allow the covalent bonds with the similarly held β-proprotein subunits and promote further processing. In that work they also proposed that the entire cleaved prodomain could remain associated with the active inhibin in a noncovalent way, such as the fact that the latency-associated peptide remains associated with TGFβ (8). In this work they have focused on the role of the small 43-amino acid propeptide. Not only do they demonstrate the necessity of cleavage of this promolecule from the N and C regions of inhibin for production of mature peptide to occur, but they also provide strong evidence for a role of this unassuming peptide in modulating the bioactivity of the mature inhibins. Yet it does not seem to interfere with activin activity. Thus, inhibin protein may come along with its own tiny inhibitor after processing, with the little associated prodomain peptide intercalating itself between the interface with inhibin A or B and the β-glycan receptor (Figure 1, B and C). Yet a function of β-glycan is to inhibit the activation of the activin receptor signaling cascade (9, 10). So the inhibitor is packaged with its own inhibitor, like a functional mini-me, providing direct modulation of inhibin function. This has profound implications biologically if this relationship is shown to occur in vivo. Activin action is already known to be tightly regulated on many levels, both directly and indirectly. From the expression of excess inhibin α-subunits (favoring inhibin production over activin), to follistatin or α-macroglobin binding, to production of inhibitory smads that can directly modulate the activin signaling cascade; the action of activin is tightly regulated in tissue-specific ways, and its effects are profoundly concentration and tissue specific (11, 12). Entry into this already complex system regulating activin, the action of a small amphipathic molecule that could be delivered to a specific site to block inhibin and thereby increase activin action and possibly result in increased unbound β-glycan may have great usefulness clinically and as a research tool across several overlapping fields of endocrinology.

Probing ADAMTS13 substrate specificity using phage display.

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73.

Analyses of disease-related GNPTAB mutations define a novel GlcNAc-1-phosphotransferase interaction domain and an alternative site-1 protease cleavage site.

Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/β-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/β-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/β-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive β-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype–phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity.

Expression of the myostatin gene in the adductor muscle of the Pacific lion-paw scallop Nodipecten subnodosus in association with growth and environmental conditions.

The cDNA sequence of the myostatin gene in the Pacific lion-paw Nodipecten subnodosus (Ns-mstn) was characterized, and the temporal expression during grow-out was analyzed for the first time in a scallop. Ns-mstn encodes a 459-amino-acid protein in which two propeptide proteolytic sites were identified, the previously recognized (RSKR) and a second one at position 266-269 aa (RRKR). The alternative furin cleavage site could be related with post-translational processing, or it could be a tissue-specific mechanism for signaling activity. The Ns-mstn transcript was located by in situ hybridization in sarcomeres and around the nucleus of muscle fibers. The temporal expression analysis by qPCR in the adductor muscle showed that Ns-mstn expression was significantly different (P < 0.05) between months during the grow-out period, increasing largely during the summer months when both biomass and muscle weight did not increase or even decreased; muscle fiber size and number were found to decrease significantly. Exogenous and endogenous factors such as high temperature and low food availability, as well as gametogenesis and reproduction, can be associated with the growth pattern and Ns-mstn expression changes. Our results indicate that MSTN is involved in adductor muscle growth regulation in N. subnodosus as it occurs in vertebrate skeletal muscle although Ns-mstn expression in non-muscle organs/tissues suggests additional functions.

Transcriptomic Messiness in the Venom Duct of Conus miles Contributes to Conotoxin Diversity

Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly.

The use of alternative polyadenylation sites renders integrin β1 (Itgb1) mRNA isoforms with differential stability during mammary gland development

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the β1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin β1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.