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2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Daniel Crosby ◽  
Melissa R. Mikolaj ◽  
Sarah B. Nyenhuis ◽  
Samantha Bryce ◽  
Jenny E. Hinshaw ◽  
...  

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.


2021 ◽  
Author(s):  
Benjamin C Shaw ◽  
Henry C Snider ◽  
Andrew K Turner ◽  
Diana J Zajac ◽  
James F Simpson ◽  
...  

Background: Genetic variants in TREM2 are strongly associated with Alzheimer's Disease (AD) risk but alternative splicing in TREM2 transcripts has not been comprehensively described. Objective: Recognizing that alternative splice variants can result in reduced gene expression and/or altered function, we sought to fully characterize splice variation in TREM2. Methods: Human blood and anterior cingulate autopsy tissue from 61 donors were used for genotyping and cDNA synthesis followed by both end-point and quantitative PCR to identify and quantify novel TREM2 isoforms. Results: In addition to previously described transcripts lacking exon 3 or exon 4, or retaining part of intron 3, we identified novel isoforms lacking exon 2, along with isoforms lacking multiple exons. Isoforms lacking exon 2 were predominant at approximately 10% of TREM2 mRNA in the brain. Expression of TREM2 and frequency of exon 2 skipping did not differ between AD samples and non-AD controls (p = 0.1268 and p = 0.4909, respectively). Further, these novel splice isoforms were also observed across multiple tissues (brain, liver, lung, kidney, heart, aorta, skeletal muscle) with similar frequency (range 5.3 - 13.0%). Using ectopic expression, we found that the exon 2 skipped isoform D2-TREM2 is translated to protein and localizes similarly to full-length TREM2 protein, and that both D2-TREM2 and FL-TREM2 proteins are primarily retained in the Golgi complex. Conclusion: Since the TREM2 ligand binding domain is encoded by exon 2, and skipping this exon retains reading frame while conserving localization, we hypothesize that D2-TREM2 acts as an inhibitor of TREM2 and that targeting TREM2 splicing may be a novel therapeutic pathway for AD.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3321-3321
Author(s):  
Leslie A Crews ◽  
Phoebe Mondala ◽  
Cayla Mason ◽  
Larisa Balaian ◽  
Wenxue Ma ◽  
...  

Abstract Secondary acute myeloid leukemia (sAML) is the most therapeutically recalcitrant form of AML with a life expectancy of less than 12 months. Secondary AML evolves from relatively prevalent myeloproliferative neoplasms (MPNs), myelodysplastic syndrome (MDS), or after chemotherapy, radiation therapy, or hematopoietic cell transplantation (HCT) that together confer a 14% risk of sAML at 15 years. Cumulative sequencing studies show that human splicing factor mutations, epigenetic spliceosome deregulation, RNA editing-induced splicing alterations, and pro-survival splice isoform switching drive dormant leukemia stem cell (LSC) generation and sAML resistance to chemotherapy and molecularly targeted agents resulting in high rates of relapse. LSC are immunologically silent in part because they activate adenosine deaminase acting on dsRNA (ADAR1), which attenuates the innate immune response. In addition, therapeutic splicing modulation has the potential to induce neoepitope formation and augment checkpoint inhibitor therapy. Thus, there is a pressing need for clinical development of splicing modulatory agents that eradicate therapy resistant LSC and reduce sAML drug resistance and relapse. Rebecsinib (17 S-FD-895) is a pharmacologically stable, potent, and selective small molecule splicing modulator that targets the SF3B core of the spliceosome at the interface of SF3B1, SF3B3 and PHF5A. We previously showed that Rebecsinib inhibits human LSC maintenance in sAML models at doses that spare normal hematopoietic stem and progenitor cells (HSPCs). In IND-enabling studies, we now demonstrate that splicing modulation with this potent agent is a pre-clinical tox-proven strategy to eradicate LSC with the potential to overcome immune checkpoint resistance via inhibition of ADAR1 splicing and activity. We further describe targeted LSC eradication that correlates with detection of unique intron-retained and exon-skipped transcripts that can be quantified by splice isoform-specific qRT-PCR and RNA-sequencing analyses and can be used as predictive biomarkers to monitor molecular responses to Rebecsinib treatment. Mechanistically, the therapeutic effects were accompanied by on-target splicing modulatory effects, including reductions in pro-survival MCL1L transcripts and splicing factor gene products such as SF3B1 and SF3B3, which form part of the splicing modulator binding pocket as well as alterations in self-renewal promoting ADAR1 and STAT3beta transcripts. In multi-species toxicology and pharmacokinetic/pharmacodynamic studies, Rebecsinib induced splicing modulation and was well-tolerated over a broad range of doses. Because of disrupted spliceosome function, SF3B1 overexpression and increased dependence on pro-survival splice isoform expression, Rebecsinib-mediated induction of pro-survival to pro-apoptotic splice isoform switching inhibits sAML LSC survival and self-renewal at doses that spare normal HSPCs in vitro and in humanized mouse models commensurate with dose-dependent changes in splicing reporter exon skipping and SF3B1, MCL1, BCL2 and CD44 isoform levels. Together, this potent and selective agent along with biomarkers of response to splicing modulation provide a sensitive method of detecting activity and mechanism of action of Rebecsinib, and demonstrate its LSC selectivity in humanized stromal co-cultures and humanized mouse models, which will have utility in future clinical development of this novel therapeutic agent. Disclosures Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.


Insects ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 957
Author(s):  
Markus Friedrich

Together with sickle (skl), the Drosophila paralogs reaper (rpr), head involution defective (hid), and grim (RHG) control a critical switch in the induction of programmed cell death. RHG homologs have been identified in other dipteran and lepidopteran species but not beyond. Revisiting this issue with a “taxon hopping” BLAST search strategy in current genome and transcriptome resources, I detected high confidence RHG homologs in Coleoptera, Hymenoptera, Hemiptera, and Dictyoptera. Analyses of gene structure and protein sequence conservation revealed aconserved splicing pattern and highly conserved amino acid residues at both the N- and C-terminal ends that identify hid as the most ancestrally organized RHG gene family member in Drosophila. hid-like RHG homologs were also detected in mosquitoes, redefining their michelob_x (mx) genes as an expansion of derived RHG homologs. Only singleton homologs were detected in the large majority of other insect clades. Lepidopteran RHG homologs, however, stand out by producing an evolutionarily-derived splice isoform, identified in previous work, in addition to the newly detected hid-like isoform. Exceptional sequence diversification of select RHG homologs at the family- and genus-level explain their previous elusiveness in important insect genome model species like the red flour beetle Tribolium castaneum and the pea aphid Acyrthosiphon pisum. Combined, these findings expand the minimal age of the RHG gene family by about 100 million years and open new avenues for molecular cell death studies in insects.


2021 ◽  
Author(s):  
Kathrin Jansen ◽  
Noriko Shikama-Dorn ◽  
Moustafa Attar ◽  
Stefano Maio ◽  
Maria Lopopolo ◽  
...  

Thymic epithelial cells (TEC) control the selection of a T cell repertoire reactive to pathogens but tolerant of self. This process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire, but the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, remain largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific microexons in mTEC. We did not find unusual numbers of novel transcripts in TEC, and we show that Aire, the facilitator of promiscuous gene expression, promotes the generation of long “classical” transcripts (with 5′ and 3′ UTRs) but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of nonpromiscuously expressed SF genes, among which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss-of-function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the RBFOX family, to generate a broad but selective representation of the peripheral splice isoform repertoire.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Spencer D. Balay ◽  
Tobias Hochstoeger ◽  
Alexandra Vilceanu ◽  
E. Pascal Malkemper ◽  
William Snider ◽  
...  

AbstractCryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.


2021 ◽  
Vol 8 ◽  
Author(s):  
Ricardo De Paoli-Iseppi ◽  
Josie Gleeson ◽  
Michael B. Clark
Keyword(s):  

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Victoria Gudiño ◽  
Patrizia Cammareri ◽  
Caroline V. Billard ◽  
Kevin B. Myant

AbstractRAC1B is a tumour-related alternative splice isoform of the small GTPase RAC1, found overexpressed in a large number of tumour types. Building evidence suggests it promotes tumour progression but compelling in vivo evidence, demonstrating a role in driving tumour invasion, is currently lacking. In the present study, we have overexpressed RAC1B in a colorectal cancer mouse model with potential invasive properties. Interestingly, RAC1B overexpression did not trigger tumour invasion, rather it led to an acceleration of tumour initiation and reduced mouse survival. By modelling early stages of adenoma initiation we observed a reduced apoptotic rate in RAC1B overexpressing tumours, suggesting protection from apoptosis as a mediator of this phenotype. RAC1B overexpressing tumours displayed attenuated TGFβ signalling and functional analysis in ex vivo organoid cultures demonstrated that RAC1B negatively modulates TGFβ signalling and confers resistance to TGFβ-driven cell death. This work defines a novel mechanism by which early adenoma cells can overcome the cytostatic and cytotoxic effects of TGFβ signalling and characterises a new oncogenic function of RAC1B in vivo.


2021 ◽  
Vol 8 ◽  
Author(s):  
Ricardo De Paoli-Iseppi ◽  
Josie Gleeson ◽  
Michael B. Clark

Alternative splicing (AS) of RNA is a key mechanism that results in the expression of multiple transcript isoforms from single genes and leads to an increase in the complexity of both the transcriptome and proteome. Regulation of AS is critical for the correct functioning of many biological pathways, while disruption of AS can be directly pathogenic in diseases such as cancer or cause risk for complex disorders. Current short-read sequencing technologies achieve high read depth but are limited in their ability to resolve complex isoforms. In this review we examine how long-read sequencing (LRS) technologies can address this challenge by covering the entire RNA sequence in a single read and thereby distinguish isoform changes that could impact RNA regulation or protein function. Coupling LRS with technologies such as single cell sequencing, targeted sequencing and spatial transcriptomics is producing a rapidly expanding suite of technological approaches to profile alternative splicing at the isoform level with unprecedented detail. In addition, integrating LRS with genotype now allows the impact of genetic variation on isoform expression to be determined. Recent results demonstrate the potential of these techniques to elucidate the landscape of splicing, including in tissues such as the brain where AS is particularly prevalent. Finally, we also discuss how AS can impact protein function, potentially leading to novel therapeutic targets for a range of diseases.


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