Remodelling of the Host Cell RNA Splicing Machinery During an Adenovirus Infection

We have studied in HeLa cells at the electron microscope level the response to adenovirus infection of the RNA processing machinery. Components of the spliceosomes were localized by in situ hybridization with biotinylated U1 and U2 DNA probes and by immunolabeling with Y12 anti-Sm monoclonal antibody, whereas poly(A)+ RNAs were localized by specific binding of biotinylated poly(dT) probe. At early stages of nuclear transformation, the distribution of small nuclear RNPs was similar to that previously described in non-infected nuclei (Visa, N., Puvion-Dutilleul, F., Bachellerie, J.P. and Puvion, E., Eur. J. Cell Biol. 60, 308–321, 1993; Visa, N., Puvion-Dutilleul, F., Harper, F., Bachellerie, J. P. and Puvion, E., Exp. Cell Res. 208, 19–34, 1993). As the infection progresses, the large virus-induced inclusion body consists of a central storage site of functionally inactive viral genomes surrounded by a peripheral shell formed by clusters of interchromatin granules, compact rings and a fibrillogranular network in which are embedded the viral single-stranded DNA accumulation sites. Spliceosome components and poly(A)+ RNAs were then exclusively detected over the clusters of interchromatin granules and the fibrillogranular network whereas the viral single-stranded DNA accumulation sites and compact rings remained unlabeled, thus appearing to not be directly involved in splicing. Our data, therefore, suggest that the fibrillogranular network, in addition to being the site of viral transcription, is also a major site of viral RNA splicing. Like the clusters of interchromatin granules, which had been already involved in spliceosome assembly, they could also have a role in the sorting of viral spliced polyadenylated mRNAs before export to the cytoplasm. The compact rings, which contain non-polyadenylated viral RNA, might accumulate the non-used portions of the viral transcripts resulting from differential poly(A)+ site selection.

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In Silico Analysis of a Highly Mutated Gene in Cancer Provides Insight into Abnormal mRNA Splicing: Splicing Factor 3B Subunit 1K700E Mutant

Biomolecules ◽

10.3390/biom10050680 ◽

2020 ◽

Vol 10 (5) ◽

pp. 680 ◽

Cited By ~ 2

Author(s):

Asmaa Samy ◽

Baris Suzek ◽

Mehmet Ozdemir ◽

Ozge Sensoy

Keyword(s):

Rna Splicing ◽

In Silico Analysis ◽

Therapy Resistance ◽

Mrna Splicing ◽

Splicing Factor ◽

Cancer Types ◽

Dynamics Simulations ◽

Aberrant Rna ◽

The Impact ◽

Splicing Machinery

Cancer is the second leading cause of death worldwide. The etiology of the disease has remained elusive, but mutations causing aberrant RNA splicing have been considered one of the significant factors in various cancer types. The association of aberrant RNA splicing with drug/therapy resistance further increases the importance of these mutations. In this work, the impact of the splicing factor 3B subunit 1 (SF3B1) K700E mutation, a highly prevalent mutation in various cancer types, is investigated through molecular dynamics simulations. Based on our results, K700E mutation increases flexibility of the mutant SF3B1. Consequently, this mutation leads to i) disruption of interaction of pre-mRNA with SF3B1 and p14, thus preventing proper alignment of mRNA and causing usage of abnormal 3’ splice site, and ii) disruption of communication in critical regions participating in interactions with other proteins in pre-mRNA splicing machinery. We anticipate that this study enhances our understanding of the mechanism of functional abnormalities associated with splicing machinery, thereby, increasing possibility for designing effective therapies to combat cancer at an earlier stage.

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Abstract 5122: Integral view of copy number alteration and commonly targeted genes in MDS found a new aspect of correlation and interrelationship with mutated components of the RNA splicing machinery

10.1158/1538-7445.am2012-5122 ◽

2012 ◽

Author(s):

Yasunobu Nagata ◽

Masashi Sanada ◽

Kenichi Yoshida ◽

Ayana Kon ◽

Yusuke Sato ◽

...

Keyword(s):

Rna Splicing ◽

Copy Number ◽

Copy Number Alteration ◽

Splicing Machinery

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Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

Molecular Biology of the Cell ◽

10.1091/mbc.e05-09-0850 ◽

2006 ◽

Vol 17 (8) ◽

pp. 3557-3568 ◽

Cited By ~ 46

Author(s):

James C. Warren ◽

Adam Rutkowski ◽

Lynne Cassimeris

Keyword(s):

Host Cell ◽

Dynamic Instability ◽

Infection Process ◽

Microtubule Dynamics ◽

Cell Periphery ◽

Adenovirus Infection ◽

Drug Induced ◽

Infected Cells ◽

The Stability ◽

Induced Changes

Adenovirus translocation to the nucleus occurs through a well characterized minus end-directed transport along microtubules. Here, we show that the adenovirus infection process has a significant impact on the stability and dynamic behavior of host cell microtubules. Adenovirus-infected cells had elevated levels of acetylated and detyrosinated microtubules compared with uninfected cells. The accumulation of modified microtubules within adenovirus-infected cells required active RhoA. Adenovirus-induced changes in microtubule dynamics were characterized at the centrosome and at the cell periphery in living cells. Adenovirus infection resulted in a transient enhancement of centrosomal microtubule nucleation frequency. At the periphery of adenovirus-infected cells, the dynamic instability of microtubules plus ends shifted toward net growth, compared with the nearly balanced growth and shortening observed in uninfected cells. In infected cells, microtubules spent more time in growth, less time in shortening, and underwent catastrophes less frequently compared with those in uninfected cells. Drug-induced inhibition of Rac1 prevented most of these virus-induced shifts in microtubule dynamic instability. These results demonstrate that adenovirus infection induces a significant stabilizing effect on host cell microtubule dynamics, which involve, but are not limited to, the activation of the RhoGTPases RhoA and Rac1.

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Mutational Spectrum Analysis of Interesting Correlation and Interrelationship Between RNA Splicing Pathway and Commonly Targeted Genes in Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.273.273 ◽

2011 ◽

Vol 118 (21) ◽

pp. 273-273 ◽

Cited By ~ 2

Author(s):

Yasunobu Nagata ◽

Masashi Sanada ◽

Ayana Kon ◽

Kenichi Yoshida ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Rna Splicing ◽

Genetic Basis ◽

De Novo ◽

Gene Mutations ◽

Myeloid Neoplasms ◽

Disease Subtypes ◽

The Difference ◽

De Novo Aml ◽

Insight Into ◽

Splicing Machinery

Abstract Abstract 273 Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms showing a frequent transition to acute myeloid leukemia. Although they are discriminated from de novo AML by the presence of a preleukemic period and dysplastic cell morphology, the difference in molecular genetics between both neoplasms has not been fully elucidated because of the similar spectrum of gene mutations. In this regards, the recent discovery of frequent pathway mutations (45∼90%) involving the RNA splicing machinery in MDS and related myeloid neoplasm with their rare mutation rate in de novo AML provided a novel insight into the distinct molecular pathogenesis of both neoplasms. Thus far, eight components of the RNA splicing machinery have been identified as the targets of gene mutations, among which U2AF35, SF3B1, SRSF2 and ZRSR2 show the highest mutation rates in MDS and CMML. Meanwhile, the frequency of mutations shows a substantial variation among disease subtypes, although the genetic/biological basis for these differences has not been clarified; SF3B1 mutations explain >90% of the spliceosome gene mutations in RARS and RCMD-RS, while mutations of U2AF35 and ZRSR2 are rare in these categories (< 5%) but common in CMML (16%) and MDS without increased ring sideroblasts (20%). On the other hand, SRSF2 mutations are most frequent in CMML (30%), compared with other subtypes (<10 %) (p<0.001) (Yoshida K, et al, unpublished data). So to obtain an insight into the genetic basis for these difference, we extensively explored spectrums of gene mutations in a set of 161 samples with MDS and related myeloid neoplasms, in which mutations of 10 genes thus far identified as major targets in MDS were examined and their frequencies were compared with regard to the species of mutated components of the splicing machinery. The mutation status of the 161 specimens was determined using the target exon enrichment followed by massively parallel sequencing. In total, 86 mutations were identified in 81(50%) in the 8 components of the splicing machinery. The mutations among 4 genes, U2AF35 (N = 20), SRSF2 (N = 31), SF3B1 (N = 15) and ZRSR2 (N = 10), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 3), PRPF40B (N = 3), U2AF65 (N = 3) and SF1 (N = 1). Conspicuously, higher frequency 4 components of the splicing machinery were mutated in 76 out of the 161 cases (47.2%) in a mutually exclusive manner. On the other hand, 172 mutations of the 10 common targets were identified among 117, including 41 TET2 (25%), 32 RUNX1 (20%), 26 ASXL1 (16%), 24 RAS (NRAS/KRAS) (15%), 22 TP53 (14%), 17 IDH1/2 (10%), 10 CBL (6%) and 10 EZH2 (6%) mutations. We examined the difference between the major spliceosome mutations in terms of the number of the accompanying mutations in the 10 common gene targets. The possible bias from the difference in disease subtypes was compensated by multiple regressions. The SRSF2 mutations are more frequently associated with accompanying gene mutations with a significantly higher number of those mutations (N=29; OR 6.2; 95%CI 1.1–35) compared with that of the U2AF35 mutations (N=14) (p=0.038). Commonly involving the E/A splicing complexes, these splicing pathway mutations lead to compromised 3' splice site recognition. However, individual mutations may still have different impacts on cell functions, which could contribute to the determination of discrete disease phenotypes. It was demonstrated that SRSF2 was involved in the regulation of DNA stability and that depletion of SRSF2 can lead to DNA hypermutability, which may explain the higher number of accompanying gene mutation in SRSF2-mutated cases than cases with other spliceosome gene mutations. In conclusion, it may help to disclosing the genetic basis of MDS and related myeloid neoplasms that highly paralleled resequencing was confirmed SRSF2 mutated case significantly overlapped common mutations. Disclosures: No relevant conflicts of interest to declare.

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Splicing factor mutations in hematologic malignancies

Blood ◽

10.1182/blood.2019004260 ◽

2021 ◽

Author(s):

Sisi Chen ◽

Salima Benbarche ◽

Omar Abdel-Wahab

Keyword(s):

Rna Splicing ◽

Messenger Rna ◽

Hematologic Malignancies ◽

Splicing Factor ◽

Splicing Factors ◽

Loss Of Function ◽

Genes Encoding ◽

Precursor Rna ◽

Early Phase Clinical Trials ◽

Splicing Machinery

Mutations in genes encoding RNA splicing factors were discovered nearly ten years ago and are now understood to be amongst the most recurrent genetic abnormalities in patients with all forms of myeloid neoplasms and several types of lymphoproliferative disorders as well as subjects with clonal hematopoiesis. These discoveries implicate aberrant RNA splicing, the process by which precursor RNA is converted into mature messenger RNA, in the development of clonal hematopoietic conditions. Both the protein as well as the RNA components of the splicing machinery are affected by mutations at highly specific residues and a number of these mutations alter splicing in a manner distinct from loss of function. Importantly, cells bearing these mutations have now been shown to generate mRNA species with novel aberrant sequences, some of which may be critical to disease pathogenesis and/or novel targets for therapy. These findings have opened new avenues of research to understand biological pathways disrupted by altered splicing. In parallel, multiple studies have revealed that cells bearing change-of-function mutation in splicing factors are preferentially sensitized to any further genetic or chemical perturbations of the splicing machinery. These discoveries are now being pursued in several early phase clinical trials using molecules with diverse mechanisms of action. Here we review the molecular effects of splicing factor mutations on splicing, mechanisms by which these mutations drive clonal transformation of hematopoietic cells, and the development of new therapeutics targeting these genetic subsets of hematopoietic malignancies.

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A Master Autoantigen-ome Links Alternative Splicing, Female Predilection, and COVID-19 to Autoimmune Diseases

10.1101/2021.07.30.454526 ◽

2021 ◽

Author(s):

Julia Y. Wang ◽

Michael W. Roehrl ◽

Victor B. Roehrl ◽

Michael H. Roehrl

Keyword(s):

Alternative Splicing ◽

Autoimmune Diseases ◽

Host Cell ◽

Rna Splicing ◽

Viral Infections ◽

Dermatan Sulfate ◽

Apoptotic Cell ◽

Cell Types ◽

Altered Expression ◽

Molecular Alterations

Chronic and debilitating autoimmune sequelae pose a grave concern for the post-COVID-19 pandemic era. Based on our discovery that the glycosaminoglycan dermatan sulfate (DS) displays peculiar affinity to apoptotic cells and autoantigens (autoAgs) and that DS-autoAg complexes cooperatively stimulate autoreactive B1 cell responses, we compiled a database of 751 candidate autoAgs from six human cell types. At least 657 of these have been found to be affected by SARS-CoV-2 infection based on currently available multi-omic COVID data, and at least 400 are confirmed targets of autoantibodies in a wide array of autoimmune diseases and cancer. The autoantigen-ome is significantly associated with various processes in viral infections, such as translation, protein processing, and vesicle transport. Interestingly, the coding genes of autoAgs predominantly contain multiple exons with many possible alternative splicing variants, short transcripts, and short UTR lengths. These observations and the finding that numerous autoAgs involved in RNA-splicing showed altered expression in viral infections suggest that viruses exploit alternative splicing to reprogram host cell machinery to ensure viral replication and survival. While each cell type gives rise to a unique pool of autoAgs, 39 common autoAgs associated with cell stress and apoptosis were identified from all six cell types, with several being known markers of systemic autoimmune diseases. In particular, the common autoAg UBA1 that catalyzes the first step in ubiquitination is encoded by an X-chromosome escape gene. Given its essential function in apoptotic cell clearance and that X-inactivation escape tends to increase with aging, UBA1 dysfunction can therefore predispose aging women to autoimmune disorders. In summary, we propose a model of how viral infections lead to extensive molecular alterations and host cell death, autoimmune responses facilitated by autoAg-DS complexes, and ultimately autoimmune diseases. Overall, this master autoantigen-ome provides a molecular guide for investigating the myriad of autoimmune sequalae to COVID-19 and clues to the rare but reported adverse effects of the currently available COVID vaccines.

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Somatic Mutation of SF3B1, a Gene Encoding a Core Component of RNA Splicing Machinery, in Myelodysplasia with Ring Sideroblasts

Blood ◽

10.1182/blood.v118.21.3.3 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3-3 ◽

Cited By ~ 1

Author(s):

Luca Malcovati ◽

Elli Papaemmanuil ◽

Eva Hellström-Lindberg ◽

Jacqueline Boultwood ◽

David Bowen ◽

...

Keyword(s):

Somatic Mutation ◽

Rna Splicing ◽

Mutant Allele ◽

Somatic Mutations ◽

Point Mutations ◽

Refractory Anemia ◽

Core Component ◽

Ring Sideroblasts ◽

Risk Of Progression ◽

Splicing Machinery

Abstract Abstract 3 Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by dysplasia in one or more cell lines, ineffective hematopoiesis, and variable risk of progression to acute myeloid leukemia (AML). As any other neoplasm, MDS is expected to be driven by mutation, and its clonal evolution is likely a multistep process in which several genetic events occur. Somatic mutations of TET2 have been found in about 25% of MDS patients, while additional mutant genes (including ASXL1, ETV6, EZH2, IDH1, IDH2, RUNX1, and TP53) have been detected in smaller proportions of patients, particularly in those with poor prognosis. Refractory anemia with ring sideroblasts (RARS) is a phenotypically well-defined subtype of MDS, characterized by 15% or more ring sideroblasts (RS, erythroblasts with perinuclear iron-loaded mitochondria) in the bone marrow. We reasoned that the identification of recurrently mutated genes in RARS could provide novel insights into molecular pathogenesis of MDS, and used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 8 patients with RARS. We identified 62 point mutations across the 8 patients, and the mutation spectrum showed a predominance of transitions, especially C>T/G>A mutations. Within 5/8 patients studied, the observed proportion of reads reporting a mutant allele showed significantly greater variability than expected by chance, indicating that the population of malignant cells was genetically heterogeneous. In 6/8 RARS patients, we identified recurrent somatic mutations (found in granulocytes but not in T-lymphocytes) in a gene that encodes a core component of the RNA splicing machinery, SF3B1. Based on the proportion of reads reporting the mutant allele, the mutations all appeared to be heterozygous and present in the dominant clone of cells. To characterize the spectrum and frequency of SF3B1 mutations in greater detail, both in myeloid malignancies and other cancers, we undertook targeted resequencing of the gene. In particular, we studied patients with MDS, myelodysplastic/myeloproliferative neoplasm (MDS/MPN) or AML evolving from MDS. Somatic mutations of SF3B1 were found in 150/533 (28.1%) patients with MDS, 16/83 (19.3%) patients with MDS/MPN, and 2/38 (5.3%) patients with AML. The gene was also mutated in 1–5% of diverse other tumor types. All mutations appeared to be heterozygous substitutions, and we observed no frameshift indels, splice site mutations or nonsense substitutions. The mutations clustered in exons 12–15 of the gene, and K700E accounted for 97/168 (57.7%) of the variants observed. SF3B1 mutations were less deleterious than expected by chance, implying that the mutated protein retains structural integrity with altered function. Gene expression profiling revealed SF3B1 mutations are associated with down-regulation of key gene networks, including core mitochondrial pathways. Close relationships were found between mutant SF3B1 and presence of RS (P<.001), and between mutant allele burden and percentage of RS (P=.002). Overall, 83/105 (79%) of patients with RARS, 30/54 (57.7%) of those with refractory cytopenia with multilineage dysplasia and RS, and 12/18 (66.7%) of those with RARS associated with marked thrombocytosis (RARS-T) carried a somatic mutation of SF3B1. On the other hand, 97% of patients carrying a mutant SF3B1 had RS, and the mutant gene had a positive predictive value for RS of 97.7% (95% CI, 93.5–99.5%). We then studied the prognostic significance of the genetic lesion. In multivariable analysis including established risk factors, SF3B1 mutations were independently associated with better overall survival (HR=0.18, P=.028) and lower risk of progression to AML (HR=0.32, P=.048). In conclusion, mutations in SF3B1 implicate abnormalities of mRNA splicing, a pathway not previously known as a target for mutation, in the pathogenesis of MDS. The close relationship between this molecular lesion and RS is consistent with a causal relationship, and makes SF3B1 the first gene to be strongly associated with a specific morphological feature in MDS. Finally, SF3B1 mutations are independent predictors of favorable clinical outcome, and their detection may improve risk assessment in MDS. The first two authors equally contributed to this paper, which is on behalf of the International Cancer Genome Consortium Chronic Myeloid Disorders Working Group. Disclosures: No relevant conflicts of interest to declare.

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