Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila

Abstract The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.

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Loss of function of the RNA export factor, Nxt1, in Drosophila causes muscle degeneration and reduced expression of genes with long introns

10.1101/543942 ◽

2019 ◽

Author(s):

Kevin van der Graaf ◽

Helen White-Cooper

Keyword(s):

Gene Expression ◽

Protein Synthesis ◽

Mrna Export ◽

Circular Rnas ◽

Specific Gene ◽

Muscle Degeneration ◽

Loss Of Function ◽

Export Pathway ◽

Expression Of Genes ◽

Rna Export

AbstractThe RNA export pathway is essential for export-competent mRNAs to pass from the nucleus into the cytoplasm, and thus is essential for protein production and normal function of cells. Drosophila with partial loss of function of Nxt1, a core factor in the pathway, show reduced viability and male and female sterility. The male sterility has previously been shown to be caused by defects in testis-specific gene expression, particularly of genes without introns. Here we describe a specific defect in growth and maintenance of the larval muscles, leading to muscle degeneration in Nxt1 mutants. RNAseq revealed reduced expression of mRNAs of many genes in Nxt1 mutant muscles. Despite this, the degeneration was rescued by increased expression of a single gene, the costamere component tn (abba), in muscles. Genes under-expressed in the mutant typically have long introns, and most normally encode circular RNAs in addition to mRNAs. This is the first report of a specific role for the RNA export pathway gene Nxt1 in muscle integrity. Our data on Nxt1 links the mRNA export pathway to a global role in mRNA expression of genes that also produce circular RNAs, in vivo.Author summaryIn eukaryotic cells the DNA encoding instructions for protein synthesis is located in the nucleus, it is transcribed to generate pre-mRNA, which is processed at both ends and spliced to remove internal spacer regions (introns) to generate mRNA. This mRNA is then transported by the mRNA export pathway via nuclear pores to the cytoplasm for protein synthesis. We have previously shown that reduction in activity of a specific protein in the mRNA export pathway, Nxt1, has an additional role in testis-specific transcription. Here we describe a further role for this protein specifically in gene expression, particularly of genes with long introns, and in muscle maintenance. Drosophila larvae with reduced Nxt1 activity have normal muscle pattern when they are small, but show muscular atrophy and degeneration as they grow, resulting in significant defects in their movement speed. We discovered that expression of many genes is reduced in the mutant larvae, but that restoring the expression of just one of these, abba, the Drosophila homologue of Trim32 (a human gene involved in muscular dystrophy) is capable of preventing the muscle degeneration.

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Ras controls growth, survival and differentiation in the Drosophila eye by different thresholds of MAP kinase activity

Development ◽

10.1242/dev.128.9.1687 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1687-1696 ◽

Cited By ~ 3

Author(s):

K. Halfar ◽

C. Rommel ◽

H. Stocker ◽

E. Hafen

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Activity ◽

Photoreceptor Cells ◽

Loss Of Function ◽

Cellular Functions ◽

Partial Loss ◽

Cycle Progression ◽

Mapk Activity ◽

Dividing Cells

Ras mediates a plethora of cellular functions during development. In the developing eye of Drosophila, Ras performs three temporally separate functions. In dividing cells, it is required for growth but is not essential for cell cycle progression. In postmitotic cells, it promotes survival and subsequent differentiation of ommatidial cells. In the present paper, we have analyzed the different roles of Ras during eye development by using molecularly defined complete and partial loss-of-function mutations of Ras. We show that the three different functions of Ras are mediated by distinct thresholds of MAPK activity. Low MAPK activity prolongs cell survival and permits differentiation of R8 photoreceptor cells while high or persistent MAPK activity is sufficient to precociously induce R1-R7 photoreceptor differentiation in dividing cells.

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Genetic analysis of laminin A reveals diverse functions during morphogenesis in Drosophila

Development ◽

10.1242/dev.118.2.325 ◽

1993 ◽

Vol 118 (2) ◽

pp. 325-337 ◽

Cited By ~ 9

Author(s):

C. Henchcliffe ◽

L. Garcia-Alonso ◽

J. Tang ◽

C.S. Goodman

Keyword(s):

Cell Fate ◽

Genetic Characterization ◽

Complete Loss ◽

Loss Of Function ◽

Partial Loss ◽

Multidomain Structure ◽

A Subunit ◽

Wing Structure

In order to dissect the functions of laminin A in vivo, we have undertaken a molecular and genetic characterization of the laminin A subunit (lamA) gene in Drosophila. Sequence analysis predicts a multidomain structure similar to mammalian homologs. We generated a series of complete and partial loss-of-function mutant alleles of the lamA gene; complete loss-of-function mutations lead to late embryonic lethality. Certain combinations of partial loss-of-function lamA alleles give rise to escaper adults, which have rough eyes associated with changes in cell fate and pattern, misshapen legs and defects in wing structure. These phenotypes suggest that laminin A has diverse functions during morphogenesis in Drosophila.

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The prototype γ-2 herpesvirus nucleocytoplasmic shuttling protein, ORF 57, transports viral RNA through the cellular mRNA export pathway

Biochemical Journal ◽

10.1042/bj20041223 ◽

2005 ◽

Vol 387 (2) ◽

pp. 295-308 ◽

Cited By ~ 51

Author(s):

Ben J. L. WILLIAMS ◽

James R. BOYNE ◽

Delyth J. GOODWIN ◽

Louise ROADEN ◽

Guillaume M. HAUTBERGUE ◽

...

Keyword(s):

Nuclear Export ◽

Mammalian Cells ◽

Mrna Export ◽

Dominant Negative ◽

Viral Rna ◽

Exon Junction Complex ◽

Independent Manner ◽

Exon Junction ◽

Export Pathway ◽

Rna Export

HVS (herpesvirus saimiri) is the prototype γ-2 herpesvirus. This is a subfamily of herpesviruses gaining importance since the identification of the first human γ-2 herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS ORF 57 (open reading frame 57) protein is a multifunctional transregulatory protein homologous with genes identified in all classes of herpesviruses. Recent work has demonstrated that ORF 57 has the ability to bind viral RNA, shuttles between the nucleus and cytoplasm and promotes the nuclear export of viral transcripts. In the present study, we show that ORF 57 shuttles between the nucleus and cytoplasm in a CRM-1 (chromosomal region maintenance 1)-independent manner. ORF 57 interacts with the mRNA export factor REF (RNA export factor) and two other components of the exon junction complex, Y14 and Magoh. The association of ORF 57 with REF stimulates recruitment of the cellular mRNA export factor TAP (Tip-associated protein), and HVS infection triggers the relocalization of REF and TAP from the nuclear speckles to several large clumps within the cell. Using a dominant-negative form of TAP and RNA interference to deplete TAP, we show that it is essential for bulk mRNA export in mammalian cells and is required for ORF 57-mediated viral RNA export. Furthermore, we show that the disruption of TAP reduces viral replication. These results indicate that HVS utilizes ORF 57 to recruit components of the exon junction complex and subsequently TAP to promote viral RNA export through the cellular mRNA export pathway.

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Analysis of Small Critical Regions of Swi1 Conferring Prion Formation, Maintenance, and Transmission

Molecular and Cellular Biology ◽

10.1128/mcb.00206-17 ◽

2017 ◽

Vol 37 (20) ◽

Cited By ~ 5

Author(s):

Stephanie Valtierra ◽

Zhiqiang Du ◽

Liming Li

Keyword(s):

Amino Acids ◽

Nonsense Suppression ◽

Protein Conformations ◽

Loss Of Function ◽

Partial Loss ◽

Poor Growth ◽

Content Type ◽

A Subunit ◽

Critical Regions

ABSTRACT Saccharomyces cerevisiae contains several prion elements, which are epigenetically transmitted as self-perpetuating protein conformations. One such prion is [SWI + ], whose protein determinant is Swi1, a subunit of the SWI/SNF chromatin-remodeling complex. We previously reported that [SWI + ] formation results in a partial loss-of-function phenotype of poor growth in nonglucose medium and abolishment of multicellular features. We also showed that the first 38 amino acids of Swi1 propagated [SWI +]. We show here that a region as small as the first 32 amino acids of Swi1 (Swi11–32) can decorate [SWI +] aggregation and stably maintain and transmit [SWI +] independently of full-length Swi1. Regions smaller than Swi11–32 are either incapable of aggregation or unstably propagate [SWI +]. When fused to Sup35MC, the [PSI + ] determinant lacking its PrD, Swi11–31 and Swi11–32 can act as transferable prion domains (PrDs). The resulting fusions give rise to a novel chimeric prion, [SPS +], exhibiting [PSI +]-like nonsense suppression. Thus, an NH2-terminal region of ∼30 amino acids of Swi1 contains all the necessary information for in vivo prion formation, maintenance, and transmission. This PrD is unique in size and composition: glutamine free, asparagine rich, and the smallest defined to date. Our findings broaden our understanding of what features allow a protein region to serve as a PrD.

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The METTL3/MALAT1/PTBP1/USP8/TAK1 axis promotes pyroptosis and M1 polarization of macrophages and contributes to liver fibrosis

Cell Death Discovery ◽

10.1038/s41420-021-00756-x ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Bo Shu ◽

Ying-Xia Zhou ◽

Hao Li ◽

Rui-Zhi Zhang ◽

Chao He ◽

...

Keyword(s):

Liver Fibrosis ◽

Western Blot ◽

Rna Stability ◽

Interferon Γ ◽

In Vivo Model ◽

Loss Of Function ◽

M1 Polarization ◽

The Impact

AbstractPro-inflammatory M1 macrophages, via activating hepatic stellate cells, contribute to liver fibrosis. In this study, we examined the mechanism and the significance of a signaling axis, METTL3/MALAT1/PTBP1/USP8/TAK1, in regulating pyroptosis and M1 polarization of hepatic macrophages. Liver fibrosis model was established in vivo by CCl4 treatment; M1 polarization was induced in vitro by treating macrophages with lipopolysaccharide or interferon γ. Expressions of METTL3, MALAT1, PTBP1, USP8, and TAK1 were measured by RT-PCR and/or Western blot in Kupffer cells (KCs) isolated from in vivo model or in vitro activated macrophages. Macrophage phenotypes including inflammation (RT-qPCR analysis of a panel of proinflammatory cytokines and ELISA on productions of interleukin (IL)−1β and IL-18) and pyroptosis (Western blot of NLRP3, Caspase-1, and GSDMD) were investigated. The impact of METTL3 on m6A methylation of MALAT1 was examined by methylated RNA immunoprecipitation (RIP), the interaction between PTBP1 and MALAT1 or USP8 mRNA by combining RNA pull-down, RIP, and RNA stability assays, and the crosstalk between USP8 and TAK1 by co-immunoprecipitation and protein degradation assays. Functional significance of individual component of METTL3/MALAT1/PTBP1/USP8/TAK1 axis was assessed by combining gain-of-function and loss-of-function approaches. In KCs isolated from in vivo liver fibrosis model or in vitro M1-polarized macrophages, METTL3 was up-regulated, and sequentially, it increased MALAT1 level via m6A methylation, which promoted USP8 mRNA degradation through the interaction with PTBP1. Reduced USP8 expression regulated the ubiquitination and protein stability of TAK1, which promoted pyroptosis and inflammation of macrophages. The signaling cascade METTL3/MALAT1/PTBP1/USP8/TAK1, by essentially stimulating pyroptosis and inflammation of macrophages, aggravates liver fibrosis. Therefore, targeting individual components of this axis may benefit the treatment of liver fibrosis.

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Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

10.1101/599266 ◽

2019 ◽

Cited By ~ 1

Author(s):

Rui Zhang ◽

Monica Varela ◽

Gabriel Forn-Cuni ◽

Vincenzo Torraca ◽

Michiel van der Vaart ◽

...

Keyword(s):

Cell Death ◽

Host Resistance ◽

Transmembrane Protein ◽

Mycobacterial Infection ◽

Loss Of Function ◽

Cellular Functions ◽

Zebrafish Larvae ◽

Cell Death Pathways ◽

New Evidence

AbstractDNA Damage Regulated Autophagy Modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, whereas only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic cell death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death.

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A heterochromatin-specific RNA export pathway facilitates piRNA production

10.1101/596171 ◽

2019 ◽

Cited By ~ 4

Author(s):

Mostafa F. ElMaghraby ◽

Peter Refsing Andersen ◽

Florian Pühringer ◽

Katharina Meixner ◽

Thomas Lendl ◽

...

Keyword(s):

Nuclear Export ◽

Mrna Export ◽

Surveillance Systems ◽

Rna Surveillance ◽

Export Pathway ◽

Transposon Silencing ◽

Nuclear Rna ◽

Rna Export ◽

Non Coding Rnas ◽

Pirna Pathway

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30nt piRNAs are processed in the cytoplasm from long non-coding RNAs. How piRNA precursors, which often lack RNA processing hallmarks of export-competent transcripts, achieve nuclear export is unknown. Here, we uncover the RNA export pathway specific for piRNA precursors in theDrosophilagermline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1, and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. Our findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to achieve export of heterochromatic, unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

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Mechanistic insights into global suppressors of protein folding defects

10.1101/2021.11.18.469098 ◽

2021 ◽

Author(s):

Gopinath Chattopadhyay ◽

Jayantika Bhowmick ◽

Kavyashree Manjunath ◽

Shahbaz Ahmed ◽

Parveen Goyal ◽

...

Keyword(s):

Bacterial Toxin ◽

Amino Acid Substitutions ◽

Receptor Binding Domain ◽

Loss Of Function ◽

Wild Type ◽

Partial Loss ◽

Suppressor Mutations ◽

Key Drivers

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined five global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. The suppressors both alone, and in conjunction with inactive mutants, stabilise the protein both thermodynamically and kinetically in-vitro, predominantly through acceleration of the refolding rate parameters. When coupled to inactive mutants they promote increased in-vivo solubilities as well as regain-of-function phenotypes. Our study also demonstrates that the global suppressor approach can be used to consistently stabilise wild-type proteins, including for downstream translational applications.

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The Yeast hnRNP-Like Proteins Yra1p and Yra2p Participate in mRNA Export through Interaction with Mex67p

Molecular and Cellular Biology ◽

10.1128/mcb.21.13.4219-4232.2001 ◽

2001 ◽

Vol 21 (13) ◽

pp. 4219-4232 ◽

Cited By ~ 98

Author(s):

Daniel Zenklusen ◽

Patrizia Vinciguerra ◽

Yvan Strahm ◽

Françoise Stutz

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Protein ◽

Rna Binding ◽

Mrna Export ◽

Deletion Mutants ◽

Variable Regions ◽

Evolutionarily Conserved ◽

Rna Export

ABSTRACT Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessentialSaccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)+ RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.

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