Targeting of tail-anchored proteins to yeast mitochondria in vivo

AbstractTail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo

piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm

Piwi-interacting RNAs (piRNAs) and PIWI proteins play a crucial role in germ cells by repressing transposable elements and regulating gene expression. In Drosophila, maternal piRNAs are loaded into the embryo mostly bound to the PIWI protein Aubergine (Aub). Aub targets maternal mRNAs through incomplete base-pairing with piRNAs and can induce their destabilization in the somatic part of the embryo. Paradoxically, these Aub-dependent unstable mRNAs encode germ cell determinants that are selectively stabilized in the germ plasm. Here we show that piRNAs and Aub actively protect germ cell mRNAs in the germ plasm. Aub directly interacts with the germline-specific poly(A) polymerase Wispy, thus leading to mRNA polyadenylation and stabilization in the germ plasm. These results reveal a role for piRNAs in mRNA stabilization and identify Aub as an interactor of Wispy for mRNA polyadenylation. They further highlight the role of Aub and piRNAs in embryonic patterning through two opposite functions

Yeast hEST1A/B (SMG5/6)- Like proteins contribute to environment-sensing adaptive gene expression responses

During its natural life cycle, budding yeast (Saccharomyces cerevisiae) has to adapt to drastically changing environments, but how environmental-sensing pathways are linked to adaptive gene expression changes remains incompletely understood. Here, we des

Wirkung des Fusarientoxins Deoxynivalenol beim wachsenden Schwein in Abhängigkeit von der Darreichungsform

In der Literatur finden sich zahlreiche widersprüchliche Angaben zur Wirkung des Mykotoxins Deoxynivalenol (DON) bei Schweinen, wobei meist für natürlich mit DON kontaminiertes Futter (DONnat) stärkere Wirkungen beobachtet wurden als für künstlich mit DON-Reinsubstanz kontaminiertes Futter (DONrein). In dieser Arbeit wurde der Einfluß von Deoxynivalenol (DON) auf die Entwicklung wachsender Schweine untersucht. Von besonderem Interesse war hierbei die Frage, inwieweit für natürlich kontaminiertes Futter beobachtete Wirkungen (DONnat) auch durch Verfütterung einer mit DON-Reintoxin künstlich kontaminierten, getreidefreien Futtermatrix (DONrein) reproduziert werden können. Hierzu wurden männliche Läuferschweine einerseits mit einer natürlich kontaminierten Getreideration und andereseits mit einer getreidefreien Ration auf Kartoffelbasis unter Zusatz von Reintoxin gefüttert. Aufgrund der baulichen Gegebenheiten sowie der tierschutzrechtlichen Bestimmungen, wurde das Projekt in Teilabschnitten umgesetzt. Neben den Leistungsparametern Futteraufnahme und Gewichtsentwicklung wurden ferner Parameter wie Blut, Darmenzymatik, Gewebeveränderungen und DON-Metabolisierung im Kot untersucht. Zur Abschätzung der erforderlichen Toxingehalte für ein sicheres Auftreten eines Toxineffektes wurden in einem Vorversuch (Durchgang A) jeweils 5 Tiere parallel mit 2000 mg/kg und 4000 mg/kg DONnat bzw. DONrein belastet. Das Fütterungsregime entsprach einer restriktiven Futtervorlage, welche so bemessen war, dass sie einer ad libitum-Fütterung entsprechen sollte. Zu jeder Belastungsgruppe in jeder Futtervariante wurde eine Kontrollgruppe mitgeführt. Die Ergebnisse aus dem ersten Durchgang (A) zeigten lediglich Trends hinsichtlich einer möglichen Toxinwirkung auf. Insbesondere Tiere der natürlichen Belastungsgruppe wiesen Gewichtseinbußen auf. Demgegenüber waren in der Gruppe DONrein, trotz der hohen Toxinbelastung, keine Unterschiede der Leistungsparameter festzustellen. In einem zweiten Durchgang (B) wurde daraufhin jeweils 5 Tieren ausschließlich eine kontaminierte Weizenration mit einer DON-Belastung von 4000 mg/kg und 6000 mg/kg verabreicht, und im Anschluß daran in einem dritten Durchgang (C) wiederum jeweils 5 Tiere ausschließlich mit DONrein in Höhe von 4000 mg/kg und 6000 mg/kg in einer getreidefreien Futtermatrix belastet. Auch das Fütterungsregime wurde in diesen beiden Abschnitten an eine ad libitum-Fütterung adaptiert. Das variierte Versuchsverfahren in Durchgang B ließ signifikante Unterschiede in Gewichtsentwicklung und Futteraufnahme der Tiere erkennen, im gleichermaßen gestalteten Durchgang C konnte jedoch kein Einfluß des zugesetzten reinen DON in der getreidefreien Diät abgeleitet werden. Die Untersuchung der Blutparameter lieferte keinen Anhaltspunkt auf einen systemischen Toxineffekt. Veränderungen einzelner Parameter traten sporadisch und inkonstant auf. Die Thyroxingehalte stiegen nur in der Versuchsgruppe mit reinem Toxin regelmäßig in den Durchgängen A und C gegen Versuchsende an. In den Durchgängen A und B lagen die T4-Werte der getreidehaltig gefütterten Gruppen deutlich höher, als die der getreidefrei gefütterten Tiere, was allerdings der Diät zuzuschreiben war. In Versuchsdurchgang B fiel der Blut-Triglyceridgehalt mit einem signifikanten Anstieg auf, allerdings nur in der mittleren Belastungsgruppe 4000 mg/kg DONrein. Dagegen konnte in diesem Abschnitt ein signifikanter SDH-Anstieg in der Gruppe DONnat gefunden werden. Bezüglich der IgA-Gehalte im Serum waren zwischen den Behandlungen keine Unterschiede zu erkennen. Mit zunehmendem DON-Gehalt im Futter ließ sich lediglich ein Trend zu höheren IgA-Gehalten feststellen, der bei Verabreichung von DONnat deutlich ausgeprägter erschien. Die Fähigkeit der Darmmikroflora (aus dem Rektum), DON zu dem Metaboliten Deepoxy-Deoxynivalenol (DOM-1) zu transformieren war sowohl von der Darreichungsform und der Toxinmenge als auch vom Fütterungsregime abhängig. Der Anteil transformierender Mikroorganismen im Kot nahm unabhängig von der Darreichungsform mit steigender Toxinkonzentration im Futter zu. Bei den Kontrolltieren dagegen war kein einheitliches Muster abzuleiten. Ein Einfluß des Toxins auf den Proteingehalt der Darmmukosa sowie der ALT- und -KGDH-Aktivität der Enterozyten war nicht eindeutig zu bestimmen. Histologisch ließen sich vereinzelt deutlich Veränderungen der Mukosa von Magen und Darm finden, allerdings traten diese Veränderungen ebenfalls unabhängig von der Behandlung auf. Diese Arbeit zeigt den grundsätzlichen Unterschied bezüglich der Efffekte von DON als Reinsubstanz und als natürlich gebildetes Toxin in kontaminiertem Getreide auf. Die bislang festgestellten toxischen Wirkungen von DON sind allein durch Verabreichung der Reinsubstanz ohne natürliche Matrix nicht reproduzierbar. Das heißt, dass im natürlich kontaminierten Futter ein oder mehrere andere toxische Agentien zu den Vergiftungssymptomen beitragen oder diese sogar ausschließlich verursachen. Andererseits ist bei Vergiftungsfällen in der Praxis immer auch DON in entsprechenden Mengen nachzuweisen, DON könnte somit als Leitsubstanz benannt werden. Um die Zusammenhänge und auch um eine sichere Einschätzung der Gefährdung durch diese Substanz gewährleisten zu können, sind hierzu weitere Untersuchung erforderlich. Aber sowohl hinsichtlich der Kosten und des Aufwandes als auch unter Tierschutzaspekten sind die aufzustellenden Versuchskonzepte nur sehr schwer umsetzbar.Publications show a considerable amount of inconsistant information for effects of mycotoxin deoxynivalenol in pigs. Naturally contaminated feeds (DONnat) seem to cause more severe effects than pure DON in artificially contaminated feed (DONpure). This study examined the development of growing pigs under DON-influence. Most interestingly was the question, wether effects of DON-contaminated feed (DONnat) could be replicated using a grainless diet containing pure DON (DONpure). Therefore a group of male pigs were fed a diet containing naturally contaminated wheat and compared to another group fed a grainless diet based on potato supplemented with DONpure. Due to the building capacity and for reasons of animal welfare, the project had to be divided in several parts. Beside the performance parameters feed intake and weight development other parameters (blood, intestinal enzymes, tissue alterations and DON-metabolisation in feces) were examined. To estimate the required DON dose to provide certain toxic effects a preceding study (Part A) was drawn consisting of 4 groups with 5 animals each. The treatment was both naturally contaminated wheat diet and pure DON in grainless potato diet. The contents in both diets were 2000 mg/kg and 4000 mg/kg DONnat respectively DONpure. The amount of food was calculated corresponding to ad libitum feeding. Every treatment group was compared to a control group. The results of Part A only showed slight trends concerning a possible toxic effect. Especially the naturally contaminated group demonstrated weight loss. In contrast, there was no evidence of any toxic effect in the DONpure –group concerning performance. In a second study (Part B) 3 groups comprising 5 animals each received wheat diet, exclusively, containing 4000 mg/kg and 6000 mg/kg DONnat and control group, followed by Part C, altered by feeding grainless potato diet with corresponding amounts of DONpure. Also the feeding regime was changed to a real ad libitum feeding. The trial variation in Part B showed significant differences in weight gain and feed intake. These were not reproducible in Part C, no effect of admitted DONpure in grainless diet was derived. The examination of blood parameters gave no evidence of a systemic toxic effect. Alterations of single parameters were inconstant and intermittent. Only the thyroxin levels increased in the grainless group during Parts A and C at the end of each trial. In Part A and B the levels in the wheat diet groups increased, indicating an effect of the diet. In Part B, the blood triglycerides showed a significant rise, but only in the group with medium exposure of pure DON (4000 mg DONpure /kg). In contrast, a significant rise of SDH contents was found in the contaminated wheat diet group (DONnat). Regarding the serum IgA-levels no differences between the treatments could be diagnosed. With higher DON-levels in food a distinct trend to higher IgA-levels, esp. in the naturally contaminated group (DONnat), could be assessed. The ability of Intestinal flora (rectum) for DON-degradation (DOM-1) depended on both, sort of food (ingredients) and dosage and also the feeding regime. The fraction of transforming microorganisms in faeces rose with increasing toxin contents independent of diet. In contrast, the control animals showed no consistent pattern. The influence of protein content of intestinal mucosa and activity of ALT and -KGDH in enterocytes could not be identified clearly. Several mucosal variations of stomach and intestine were determined in histological examination. These changes also appeared independent of treatment. This study showed basic differences of pure DON and DON from a naturally contaminated source, referring to toxic effects. Only pure DON without natural material cannot bring out any toxic effect, which was described up to now. That means, there must be ne or more further agents in naturally contaminated material, supporting or just releasing an intoxication. On the other hand, in cases of intoxication DON is detected regularly. Therefore the conclusion for DON as leading substance may be established. For connections and a reliable estimation of the risks through this substance, further examinations are necessary. But expenses, complexity and also animal welfare reasons make the realisation of required trials very difficult

A KLHL40 3' UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure and neonatal death. Here, we describe a 26-year-old man with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles’ contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound from age 13 after prolonged immobilization. Muscle magnetic resonance imaging at age 13 indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein, respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G > T) in the 3′ untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G > T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G > T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3’ UTR variants in ClinVar suggests variants that introduce aberrant 3’ UTR splicing may be underrecognized in Mendelian disease. We encourage consideration of this mechanism during variant curation.LD is supported by an Australian Government Research Training Program (RTP) Scholarship. GR (Investigator Grant, APP2007769) and NGL (Fellowship APP1117510) are supported by the Australian National Health and Medical Research Council (NHMRC). This work is funded by NHMRC Ideas Grant (APP2002640). This work was supported by resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia.Peer reviewe

A KLHL40 3’ UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism

Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure, and neonatal death. Here, we describe a man in his 20s with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles’ contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound during adolescence after prolonged immobilisation. Muscle MRI during adolescence indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs, and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G>T) in the 3’ untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G>T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G>T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3’ UTR variants in ClinVar suggests SNPs that introduce aberrant 3’ UTR splicing may be underrecognised in Mendelian disease. We encourage consideration of this mechanism during variant curation.This study was funded by an Australian NHMRC Investigator Grant (APP2007769), Fellowship (APP1117510) and Ideas Grant (APP2002640). The study was supported by an Australian Government Research Training Program (RTP) Scholarship, as well as resources provided by the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia.N

microRNA-Mediated Messenger RNA Deadenylation Contributes to Translational Repression in Mammalian Cells

Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3′ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3′ poly(A) tail, which contributes to their mRNA decay promoting function. The relationship between miRNA-mediated deadenylation and translational repression has been less clear. Using transfection of reporter constructs carrying three imperfectly matching let-7 target sites in the 3′ UTR into mammalian cells we observe rapid target mRNA deadenylation that precedes measureable translational repression by endogenous let-7 miRNA. Depleting cells of the argonaute co-factors RCK or TNRC6A can impair let-7-mediated repression despite ongoing mRNA deadenylation, indicating that deadenylation alone is not sufficient to effect full repression. Nevertheless, the magnitude of translational repression by let-7 is diminished when the target reporter lacks a poly(A) tail. Employing an antisense strategy to block deadenylation of target mRNA with poly(A) tail also partially impairs translational repression. On the one hand, these experiments confirm that tail removal by deadenylation is not strictly required for translational repression. On the other hand they show directly that deadenylation can augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. Taken together with published work, these results suggest a dual role of deadenylation in miRNA function: it contributes to translational repression as well as mRNA decay and is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs

The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species

Polyadenylation state microarray (PASTA) analysis

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tai

Transcriptome-wide measurement of mRNA polyadenylation state

The 3′ poly(A) tail has important roles throughout the eukaryotic mRNA life cycle. A characteristic aspect of poly(A) tail function is furthermore that it can be modulated by changes in its length. This is in turn a well-recognised cellular means to re